JP2006016362A - Nerve growth factor enhancer - Google Patents

Abstract

An agent for enhancing nerve growth factor action is provided.
The nerve growth factor action enhancer contains an isothiocyanate compound or a glycoside thereof. As the isothiocyanate compound, an isothiocyanate derived from a plant belonging to the Brassicaceae family can be used.
[Selection] Figure 1

Description

  The present invention relates to a nerve growth factor action enhancer that enhances the action of nerve growth factor and a nerve growth factor action enhancement composition containing the enhancer.

  In recent years, there has been a strong demand for the development of methods for preventing and treating so-called neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease. A neurodegenerative disease is a pathologically specific nerve cell degeneration of a specific group of nerve cells, and at the present time, etiology such as vascular disorder, infectivity, and metabolic disorder cannot be identified. The degenerative subjects of Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease are considered to be cerebral cortical neurons, spinal anterior horn motor neurons, and midbrain nigrodopamine neurons, respectively. Similarly to these neurodegenerative diseases, the development of methods for prevention and treatment is also desired for neurological diseases accompanied by degeneration and loss of nerves such as peripheral neuropathy caused by other diseases and aging.

Nerve Growth Factor (NGF) is one of several known neurotrophic factors and is a protein having a molecular weight of 130,000 and an a ₂ b ₂ subunit structure. Nerve growth factor is known to have various pharmacological actions such as maintenance, differentiation, maturation, and functional expression of nerve cells in the central nervous system and peripheral nervous system. It is considered to be an entity of neurite elongation action derived from neuron cells (Non-patent Document 1). Nerve growth factors are known to be associated with neurological diseases including various neurodegenerative diseases. Accordingly, development of methods for preventing and treating various neurological diseases using nerve growth factor is expected.

  As an attempt to achieve this, an attempt has been made to administer nerve growth factor to patients with neurodegenerative diseases and the like (Non-patent Document 2), but nerve growth factor is a protein and passes through the brain blood barrier. There are difficulties in targeting to target organs, such as inability to do so, so at this stage sufficient results have not been obtained. In addition, in order to overcome the targeting problem, low molecular weight compounds that exhibit nerve growth factor-like action have been searched, and staurosporine-like alkaloids, immunophilin ligands, cyclopentenone type prostaglandins, etc. have been found. (Non-Patent Document 1). However, its own hormone-like action, immunosuppressive action, and the like have hindered the establishment of effective treatments. Therefore, at present, an effective prevention / treatment method for these neurological diseases has not yet been found.

On the other hand, epidemiological and animal experiments have recently suggested that cancer extracts of cruciferous plant extracts such as cabbage, broccoli and wasabi, or isothiocyanate compounds contained as pungent components in these plants, have been shown to be cancerous. It is attracting attention as one of the most promising food factors. Examples of the isothiocyanate compound include allyl isothiocyanate and 6-methylsulfinylhexyl isothiocyanate contained in wasabi, benzyl isothiocyanate and phenethyl isothiocyanate contained in cabbage and watercress, and sulforaphane contained in broccoli. These isothiocyanate compounds have long been known to have antibacterial activity, but their cancer prevention effect is attributed to the metabolic activation of carcinogens or the detoxification of carcinogens produced as a result. Yes. The former is derived from the suppression of the phase I enzyme (cytochrome P450), and the latter is derived from the induction of the phase II enzyme (quinone reductase, glutathione-S-transferase; GST). The isothiocyanate compound has also been reported to have an apoptosis-inducing action as a physiological action related to suppression of carcinogenesis. However, there has been no report on the relationship between components derived from Brassicaceae plants and the effects of nerve growth factors such as neurite outgrowth, and there has been no suggestion about the relationship between the two.
Chogo Sato "Neurotrophic factor-like low molecular probe", Journal of the Japanese Pharmacological Society, 120, p.327-334 (2002) Yoshihiro Arakawa, Tatsuji Iga “Neurotrophic Factor Research for Drug Discovery”, Tissue Culture Engineering 27, p.506-510, New Science (2001)

  From the viewpoint of preventing, treating or ameliorating various neurological diseases, it is effective not only to express or reach an effective amount of nerve growth factor at the disease site, but also to effectively act at the disease site or other sites. Compounds that can be enhanced are desired. Moreover, it is preferable that such a compound is a compound which can be ingested continuously in a subject.

  Accordingly, an object of the present invention is to provide a nerve growth factor action potentiator and a nerve growth factor action potentiating composition containing the potentiator. Another object of the present invention is to provide a nerve growth factor action-enhancing agent derived from food factors and a nerve growth factor action-enhancing composition containing the potentiator.

  The inventors of the present invention have focused on cruciferous plants and studied the neurite outgrowth action of the cruciferous plant extracts. As a result, they found that this extract enhances neurite outgrowth of nerve growth factor. It was found that thiocyanate compounds contributed to the enhancing action, and the present invention was completed. That is, according to the present invention, the following means are provided.

  According to one form of this invention, the nerve growth factor action enhancer containing an isothiocyanate compound or its glycoside is provided. In this embodiment, the isothiocyanate compound is preferably one or more selected from allyl isothiocyanate, arylalkyl isothiocyanate and sulfinylalkyl isothiocyanate. The isothiocyanate compound is preferably a methylsulfinylalkyl group (wherein the alkyl group is a linear alkyl group having 4 to 8 carbon atoms), more preferably methylsulfinylhexyliso Thiocyanate. Furthermore, it is preferable that these NGF action enhancers contain one or more extracts selected from the group belonging to the Brassicaceae family, or a fraction obtained from the plant extract. It is.

  According to another aspect of the present invention, a nerve growth factor comprising one or more plant extracts selected from the group consisting of plants belonging to the Brassicaceae family or a fraction obtained from the extract An action enhancer is provided. In this embodiment, it is preferable that the plant belonging to the Brassicaceae family consists of cabbage, tincture, radish, watercress, broccoli, Chinese cabbage, komatsuna, mustard, mizuna, radish, cauliflower, Brussels sprouts and wasabi. More preferably, the plant consists of tangy vegetables, mizuna and wasabi. In this embodiment, the plant extract or the fraction preferably contains a methylsulfinylalkyl group (wherein the alkyl group is a linear alkyl group having 4 to 8 carbon atoms). More preferably methylsulfinylhexyl isothiocyanate.

  According to another aspect of the present invention, there is provided a nerve growth factor action-enhancing composition containing any of the above nerve growth factor action enhancers. This composition preferably contains nerve growth factor, and is preferably a food composition, and further comprises an effective amount of the nerve growth factor action potentiator, which enhances nerve growth factor action. A preferred embodiment is a pharmaceutical composition for treating or preventing a required disease.

  The NGF action enhancer of the present invention is characterized by containing an isothiocyanate compound or a glycoside thereof. An isothiocyanate compound is known as a pungent component peculiar to cruciferous plants. According to the present inventors, it has been found that in the presence of NGF, the neurite elongation action, which is a typical action of NGF, is enhanced. The isothiocyanate compound is greatly different in structure from a compound conventionally known as an NGF action enhancer. The isothiocyanate compound has a glutathione-S-transferase inducing action, but the NGF action in the present invention is completely different from these physiological activities. The mechanism of NGF action-enhancing effect by an isothiocyanate compound is not necessarily clear, but NGF action-enhancing effect has been found in plant-derived food factors. An action enhancer will be provided.

  Hereinafter, an NGF action potentiator which is an embodiment of the present invention will be described, and an NGF action enhancing composition containing the enhancer and its use will be described.

(Isothiocyanate compound)
The structure of the isothiocyanate compound in the present invention is not limited as long as it has an isothiocyanate group and has an NGF action enhancing effect. Preferably, alkyl isothiocyanate, alkenyl isothiocyanate, arylalkyl isothiocyanate, and methylsulfinyl alkyl isothiocyanate are used. Can be mentioned.

  Examples of the alkyl isothiocyanate include alkyl isothiocyanate having a linear alkyl group having 1 to 6 carbon atoms or a branched alkyl group as an alkyl group. Specifically, methyl isothiocyanate, ethyl isothiocyanate, n -Propyl isothiocyanate, isopropyl isothiocyanate, n-butyl isothiocyanate, isobutyl isothiocyanate, amyl isothiocyanate, isoamyl isothiocyanate, hexyl isothiocyanate.

  Examples of the alkenyl isothiocyanate include alkenyl isothiocyanate having a straight chain alkenyl group having 3 to 8 carbon atoms, and specifically include allyl isothiocyanate, 3-butenyl isothiocyanate, and 5-to. Examples include xenyl isothiocyanate, 4-pentenyl isothiocyanate, and 6-heptenyl isothiocyanate.

  Examples of the arylalkyl isothiocyanate include benzyl isothiocyanate, phenethyl isothiocyanate, and p-hydroxybenzyl isothiocyanate. Furthermore, examples of the methylsulfinylalkyl isothiocyanate include those having an alkyl group having 4 to 8 carbon atoms, specifically, 4-methylsulfinylbutyl isothiocyanate, 5-methylsulfinylpentyl isothiocyanate, 6 -Methylsulfinylhexyl isothiocyanate, 7-methylsulfinyl heptyl isothiocyanate, 8-methylsulfinyl octyl isothiocyanate.

  Among these isothiocyanate compounds, methylsulfinylalkyl isothiocyanate is preferable. In methylsulfinylalkyl isothiocyanate, it is 6-methylsulfinylhexyl isothiocyanate.

  Further, as the isothiocyanate compound, derivatives of these various isothiocyanates can also be preferably used. As derivatives, in these various isothiocyanate compounds, the hydrogen atom is a hydrocarbon group such as an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an arylalkyl group, a hydroxyl group, a hydroxyalkyl group, an alkyl ether group. The thing substituted by substituents, such as these can be mentioned. The hydrocarbon group is preferably a linear or branched hydrocarbon group having about 1 to 5 carbon atoms.

  As the isothiocyanate compound in this enhancer, these isothiocyanates can be used alone or in combination of two or more.

  At least a part of such an isothiocyanate compound is an isothiocyanate compound derived from a cruciferous plant. Many of these isothiocyanate compounds are derivatives thereof. The isothiocyanate compounds derived from Brassicaceae include allyl isothiocyanate, benzyl isothiocyanate, phenethyl isothiocyanate, 4-methylsulfinyl butyl isothiocyanate, 5-methylsulfinyl pentyl isothiocyanate, 6-methylsulfinyl hexyl isothiocyanate, 7-methyl. Examples thereof include sulfinyl heptyl isothiocyanate and 8-methylsulfinyl octyl isothiocyanate. Although the isothiocyanate compounds derived from the Brassicaceae plants have different structures, all have an NGF action enhancing effect. Therefore, it is good also as what contains the isothiocyanate compound derived from a Brassicaceae plant as one form of the enhancer of this invention. Here, the isothiocyanate compound derived from the Brassicaceae plant means that it can be obtained from the Brassicaceae plant.

  In Brassicaceae plants, isothiocyanate compounds are glycosides (glucosinolates) such as sinigrin (a glycoside of allyl isothiocyanate in horseradish) and sinalbin (a glycoside of parahydroxyisothiocyanate in pear). ) Is present in the cell. An isothiocyanate compound is produced when a glycoside comes into contact with an enzyme such as myrosinase by cell destruction. Such glycosides are known to be converted to isothiocyanate compounds in the intestines of animals including humans. Therefore, the enhancer of the present invention can contain a glycoside of an isothiocyanate compound instead of or together with the isothiocyanate compound. Glycosides of isothiocyanate compounds can be extracted from cruciferous plants. Glycosides of isothiocyanate compounds do not have the unique hotness of isothiocyanate compounds. Therefore, it is useful when this enhancer is used for food, feed or pharmaceutical use.

  Therefore, as one form of the present enhancing agent, the present enhancing agent can be used for the whole plant of the cruciferous plant, or the fruit, seed, seed coat, flower, stem, leaf, root and / or rhizome which is a part thereof, Or the processed material which gave the process of pulverizing, drying, etc. can be contained. Furthermore, the whole cruciferous plant, a part thereof, or a cruciferous plant extract obtained from a processed product as a raw material or a fraction obtained from the extract can be used. In addition, even cruciferous plants that are supplied as so-called vegetables, in addition to edible parts that are generally used for food, non-edible parts, parts processed, and extracts thereof, It can be included in the enhancer.

  Examples of the cruciferous plants include turnips, cabbage, Chinese cabbage, radish, watercress, broccoli, Chinese cabbage, komatsuna, mustard, mikuna, radish, cauliflower, Brussels sprouts and wasabi. As a raw material of this enhancer, it can be obtained from one or more cruciferous plants. Among them, turnip, cabbage, tangy vegetables, radish, watercress, rape blossoms, broccoli, mustard, mizuna, radish, cauliflower, and wasabi are preferably used, and more preferred are tangy vegetables, mizuna and wasabi.

  Moreover, the enhancer mainly containing allyl isothiocyanate as an isothiocyanate compound can be made from cruciferous plants such as wasabi, cabbage, and mustard. The enhancer mainly containing arylalkyl isothiocyanate can be made from cabbage, watercress, turnip and the like. Furthermore, the enhancer mainly containing rufinyl alkyl isothiocyanate can be made from wasabi, broccoli, cauliflower and the like.

  In addition, the extraction of the isothiocyanate compound from the cruciferous plant may be performed, for example, after pretreatment such as formation of the isothiocyanate compound in the cruciferous plant, for example, by crushing or crushing or shredding by grated or the like Thus, the extraction can be carried out with a suitable solvent after the lapse of time sufficient to produce the isothiocyanate compound. Depending on the type of cruciferous plant, the extraction solvent may be water, alcohols such as chloroform, methanol, ethanol, isopropyl alcohol, n-butyl alcohol, tert-butyl alcohol, amyl alcohol, or a mixture of alcohol and water. Further, ketones such as acetone and methyl ethyl ketone, esters such as methyl acetate and ethyl acetate, and solvents such as acetone can be used. The extraction solvent is preferably ethyl acetate. The ratio of the extraction solvent to the extraction raw material can be appropriately determined without any particular limitation. Moreover, extraction time, temperature, etc. can be determined suitably.

  If necessary, the extract is dehydrated using a dehydrating agent such as anhydrous sodium sulfate and the solvent is distilled off to extract a cruciferous plant extract, that is, a cruciferous plant-derived isothiocyanate compound (however, In many cases, it is obtained in the form of a mixture, that is, it contains at least one or two or more isothiocyanates, but may also contain other extraction components). The cruciferous plant extract thus obtained can be further fractionated and fractionated appropriately by column chromatography such as liquid chromatography, extraction, fractional precipitation and the like. The fraction of the obtained extract can be used instead of the extract. The fraction does not necessarily contain a single isothiocyanate compound. The fraction may be further purified and isolated by any method such as liquid-liquid distribution method, chromatography method, molecular distillation, rectification and the like.

The NGF action-enhancing effect was found by the present inventors in various isothiocyanate compounds derived from Brassicaceae plants, and is considered to depend on the isothiocyanate group. Therefore, it is possible to easily obtain an isothiocyanate compound having an NGF action enhancing effect by searching various isothiocyanate compounds using the NGF action enhancing effect as an index. The isothiocyanate compound can be obtained by chemical synthesis, or can be extracted from a cruciferous plant based on a conventional method as described above. As an example of the chemical synthesis method, the method of Kiaer et al. (Kiaer et al. Acta chem. Scand, 11, 1298, 1957) can be mentioned. In this method, methylsulfinyl isothiocyanate can be obtained by the following operation. First, ω-chloroalkenol is used as a starting material and refluxed with CH ₃ -SNa to obtain ω-methylthioalkenol, and SOCl ₂ is allowed to act on this to obtain ω-chloroalkenol methylsulfide. Next, an amino group is introduced using the Gabriel method to produce N- (ω-methylthioalkyl) -phthalimide, and hydrazine hydrate is added to the resultant to reflux to obtain ω-methylthioalkylamine. Further, according to the method of Li et al. (Lietal. J. Org. Chem., 62, 4539, 1997), ω-methylthioalkylisothiocyanate obtained via thiuram disulfide was oxidized with mCPBA to oxidize the methylthio group. -A methylsulfinylalkyl isothiocyanate can be obtained.

  For the various isothiocyanate compounds thus obtained, the NGF action enhancing effect is confirmed using an assay system capable of confirming the action of NGF such as PC12 cells. The NGF action enhancing effect can be evaluated by using dimethyl sulfoxide (DMSO) or the like as a control for the NGF action enhancing effect of an isothiocyanate compound in the presence of NGF. When it has a higher NGF action enhancing effect than DMSO, it can be evaluated as an isothiocyanate compound used as an NGF action enhancing agent of the present invention.

  The enhancer contains an isothiocyanate compound having an NGF action enhancing effect in the various forms described above. Therefore, this enhancer can enhance the action of NGF on nerve cells at the site where NGF and nerve cells exist. In particular, the neurite outgrowth action can be enhanced among various actions of NGF. Such neurite outgrowth action is related to nerve cell differentiation and is an important action in the treatment, amelioration, and prevention of various neurological diseases. In addition, the enhancer exhibits an NGF action enhancing effect in the presence of endogenous NGF or administered or ingested exogenous NGF at a site reached in vivo. For this reason, various methods can be provided in the expression method for the NGF action enhancing effect. Furthermore, since this enhancer is a low molecular weight compound different from NGF, unlike the case of NGF, there is no great difficulty in the technique for reaching the brain. For this reason, it is preferable for expressing the NGF action in the brain.

  In addition, the enhancer contains a food factor itself, a food factor-derived compound, or a derivative of a compound obtained from the food factor. Therefore, it is preferably used for applications in which NGF is related and continuously administered or ingested for the purpose of preventing or improving the symptoms of nervous system diseases in humans or non-human animals, for which NGF action enhancement is effective. . In addition, it is preferably used for applications that are ingested continuously for the purpose of maintaining or improving the health of humans or non-human animals. From the above, the enhancer is useful as a pharmaceutical, a quasi-drug, a food, a beverage, a dietary supplement (supplement), a feed and a veterinary drug, or a raw material thereof. In addition, when this enhancer contains a glycoside, the enzyme which produces | generates an isothiocyanate compound from an isothiocyanate compound glycoside, such as myrosinase, can be included in this composition. In addition, it is preferable that an enzyme such as myrosinase acts on a glycoside when the composition is ingested or after administration. For this purpose, before the composition is ingested or administered, it is necessary that water is excluded so that the enzyme does not act or both are isolated in a pharmaceutical form by microcapsules or the like.

  The enhancer can have various forms. Although it does not specifically limit as a form of this enhancer, Any form of powder form, solid form, liquid form, and paste form may be sufficient. The composition is appropriately selected depending on the form of utilization of the composition for enhancing NGF action using the enhancer as an active ingredient. The enhancer is also used in various aspects. That is, it can be used for the above-mentioned purposes, and can also be used as a research or test reagent.

(Pharmaceutical composition)
According to this enhancer, a pharmaceutical composition is provided. According to the pharmaceutical composition containing the potentiator, senile dementia such as Alzheimer's disease and peripheral neuropathy, cerebrovascular disorder, brain tumor, brain apex, head It is useful for the treatment or prevention of various nervous system diseases that require repair and regeneration of nerve function due to traumatic degenerative diseases, anesthetic drug poisoning, and the like. It is also useful for the treatment or prevention of amyotrophic lateral sclerosis, drug-induced peripheral neuropathy, diabetic peripheral neuropathy, Parkinson's disease, sensory neuropathy, pigmented retinopathy, macular degeneration, and the like.

  The pharmaceutical composition can contain NGF in addition to the potentiator. In particular, when the pharmaceutical composition is used for a neurological disease at a site where NGF target site delivery does not hinder, for example, a neurological disease in the peripheral nervous system such as the spinal cord and extremities, it is ensured by containing NGF at the same time. The effect of this pharmaceutical composition can be acquired.

  The pharmaceutical composition can be obtained by combining the enhancer with a known pharmaceutical carrier. For formulation, this enhancer is blended with a pharmaceutically acceptable liquid or solid carrier, and if necessary, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant. It can be achieved by adding an agent or the like to form a solid agent such as a tablet, granule, powder, powder, capsule or the like, or a normal solution, suspension, emulsion or the like. Further, it can be made into a solid product that is soluble at the time of use and can be made liquid by appropriate addition at the time of use, or it can be made into an external preparation.

  The pharmaceutical carrier can be selected according to the administration form and dosage form of the pharmaceutical composition. In the case of an oral preparation, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salt, etc. are used. In preparation of the oral preparation, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance and the like can be further added. On the other hand, in the case of parenteral preparations, in accordance with conventional methods, distilled water for injection, physiological saline, glucose aqueous solution, injectable vegetable oil, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol are used as a diluent. It can be prepared by dissolving or suspending in polyethylene glycol or the like and adding a bactericidal agent, stabilizer, tonicity agent, soothing agent, etc., if necessary. Alternatively, it can also be provided as a parenteral preparation such as an injection dissolved at the time of use that can be dissolved by these diluents at the time of use.

  The pharmaceutical composition of the present invention is administered in an appropriate dosage form such as internal use, external use, injection, drip, infusion, aspiration, etc., depending on the dosage form. Injections can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, and external preparations include suppositories. In addition, for example, the composition can be administered by being directly applied to a subcutaneous disease or degeneration site by application, spraying, injection or the like. In addition, at the time of such administration, nerve cells and neural stem cells can be simultaneously supplied to a disease site or the like. At this time, the nerve cells and the neural stem cells may be the same type, different types, or other types of cells, but preferably used in-house. Furthermore, at the time of administration, NGF may also be administered separately from the pharmaceutical composition at the disease site or the like at the same time or before and after administration of the pharmaceutical composition.

  The dosage when the pharmaceutical composition is administered for therapeutic or prophylactic purposes is appropriately set according to the formulation form, administration method, purpose of use and age, weight, and symptoms of the patient applied thereto, and is not constant. In general, the amount of the enhancer used in the present invention contained in the preparation is preferably 0.001 to 2000 mg / kg body weight, more preferably 0.01 to 200 mg / kg body weight per day for an adult. In addition, since the dosage varies depending on various conditions in addition to the type of enhancer to be used, an amount smaller than the above dosage may be sufficient or may be necessary beyond the range.

  These forms of the pharmaceutical composition are applied to various forms of quasi drugs including external preparations, oral preparations for beverages, and the like.

(Food composition)
The enhancer can be a food composition for consumption as a food or nutritional supplement. According to the present food composition, the enhancer can be easily and continuously ingested. Therefore, for patients with neurological diseases related to NGF, suppression of progression of symptoms of the nervous system diseases, Effective for improvement and improvement of QOL. Moreover, it is effective for prevention of a neurological disease, health maintenance, or improvement with respect to the person who is not a patient of a neurological disease, such as a healthy person. In addition, the nervous system disease here includes various nervous system diseases similar to those described in the pharmaceutical composition.

  The food composition can take various forms. That is, it can take the form of conventionally known foods, beverages, seasonings, etc., or the form of its raw materials, and is mainly an oral nutrient such as powders, granules, tablets, capsules, extracts, liquids (including beverages). It is possible to take the supplement form or the raw material thereof, the tube feeding nutrient form supplied to the stomach, intestine or blood vessel through the tube or the like or the raw material thereof. These food compositions can also contain NGF.

  Although content of this enhancer in this food composition is not specifically limited, Preferably it is 0.000001 weight% or more, More preferably, it can be 0.00001-100 weight%. In addition, the present food composition preferably has an active ingredient contained therein, for example, preferably 0.0001 to 100 mg / kg body weight, more preferably 0.001 to 10 mg / kg body weight per day for an adult. It can be taken to be 0.01-1 mg / kg body weight. In addition, since the intake amount varies depending on various conditions in addition to the type of enhancer to be used, an amount smaller than the above intake amount may be sufficient or may be necessary beyond the above range.

(Feed composition and veterinary pharmaceutical composition)
This potentiator is used for livestock such as cattle, pigs and sheep, poultry such as chickens, turkeys and quails, aquaculture fish such as yellowtail, yellowtail, carp and eel, shellfish such as shrimps and crabs, shellfish such as scallops and oysters, It can be used as a feed composition or animal pharmaceutical composition for pets such as dogs and cats, and research animals such as rats, mice and dogs. According to these compositions, since this enhancer can be ingested easily and continuously in livestock and the like, NGF-related diseases are treated, symptoms are improved, progression is suppressed and prevented, and health is maintained or improved. It is effective for. Moreover, as a result of these, in addition to improving yield, quality such as meat quality and flavor is improved. The NGF-related diseases mentioned here may include various diseases equivalent to those described in the pharmaceutical composition when these compositions are intended for mammals. In addition, when these compositions are intended for animals other than mammals such as fish, diseases that may occur in connection with NGF are included. In addition, a drink is also included as feed.

  The composition for feed can take various forms. The form of the feed additive containing this enhancer can also be taken, and the form of the feed containing this enhancer can also be taken. Moreover, as this animal pharmaceutical composition, various forms, such as an injection, an injection, a powder, a tablet, a liquid agent, a solubilizing agent, an immersion agent, can be taken according to the animal to be made and an administration form. Note that these compositions can also contain NGF in the target organism.

  In the present potentiator used in these compositions, the active ingredient is usually ingested or administered in an amount of 0.01 kg / kg, preferably 0.01 to 2000 mg / day of the target organism. The intake or dosage varies depending on various conditions in addition to the type of enhancer to be used, so an amount smaller than the above amount may be sufficient, or may exceed the above range.

(Expression method, screening method, treatment method, prevention method, production method of domestic animals, rearing method, etc.)
Based on the usage forms of the enhancer, the pharmaceutical composition, the food composition, the feed composition, and the animal pharmaceutical composition described above, as another aspect of the present invention, the enhancer, the pharmaceutical composition, and Provided is a method for enhancing NGF action or a method for expressing NGF action, comprising a supply step for administering or ingesting any of the food compositions to a human, and a method for treating or preventing a neurological disease comprising the supply step Is done. Further, there is provided a method for enhancing NGF action or a method for expressing enhanced NGF action, comprising a supply step of administering or feeding any one of the enhancer, feed composition and veterinary pharmaceutical composition to a non-human animal, A method for treating or preventing a neurological disease of a non-human animal provided with a supply step is provided, and further, a method for raising a non-human animal such as livestock or the like, or a method for producing livestock or the like provided with the supply step. In addition, a method for screening for a therapeutic drug for NGF using NGF is also provided.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to these Examples at all.

(Evaluation of neurite outgrowth action by cruciferous plant extracts)
In this example, focusing on cruciferous plants, neurite outgrowth activity was evaluated using PC12 cells, which are cell lines derived from rat adrenal medulla cell types, with respect to ethyl acetate extracts of 14 cruciferous plants.

  About 500 g of each of the edible parts of 14 kinds of cruciferous plants shown in Table 1 were weighed, sufficiently stirred and ground with a homogenizer, and then added with 1 L of ethyl acetate, stirred, and subjected to immersion extraction overnight. After extraction, if necessary, the mixture was separated into an upper layer (ethyl acetate) and a lower layer (residue and aqueous layer) by cooling centrifugation (4 ° C., 3000 × g, 10 minutes), and the upper layer was collected. To this lower layer, about 1 L of ethyl acetate was added again and stirred, and immersion extraction was further performed overnight, and the upper layer (ethyl acetate) was collected by operating in the same manner as above. The collected ethyl acetate layers were combined and dehydrated and dried overnight over anhydrous sodium sulfate. After filtration, it was concentrated to the extent that a small amount of ethyl acetate remained in an evaporator, and the entire amount was transferred to a sample bottle, and then the remaining ethyl acetate was removed by gently blowing a nitrogen gas stream to obtain an ethyl acetate extract. . The amount of the extract was weighed, dissolved in DMSO, and stored frozen.

PC12 cells were seeded in a 24-well plate at 2 × 10 ⁴ cells / well and cultured overnight at 37 ° C. in a CO ₂ incubator using DMEM medium containing 10% horse serum and 5% FBS. The next day, each extract and NGF were administered to each well at 5 μg / ml and 1.5 ng / ml using 0.5% FBS-containing DMEM medium. 72 hours after administration, the inside of each well was observed using a microscope, about 100 PC12 cells were selected, the number of cells having protrusions longer than the major axis of the cells was counted, and neurites in the total number of observed cells The percentage (%) of the cells in which the elongation of 2 was observed was determined. Such observation was performed at three locations per well. As a control, DMSO, which is a solvent for dissolving the ethyl acetate extract, was used at the same time. The results are shown in FIG.

  As shown in FIG. 1, the neurite outgrowth action was recognized also about any cruciferous plant extract. Among them, a high neurite outgrowth effect was observed in chingensai, mizuna, wasabi and the like.

(Evaluation of neurite outgrowth action of isothiocyanate compounds)
In this example, allyl isothiocyanate (AITC), benzyl isothiocyanate (BITC), phenylethyl isothiocyanate (PEITC), 6-methylsulfinyl hexyl isotope, which are abundant in the ethyl acetate extract of cruciferous plants. Thiocyanate (6-HITC) and sulforaphane (SUL) were evaluated for neurite outgrowth action. The neurite outgrowth action was evaluated in the same manner as in Example 1 except that each isothiocyanate was administered at 1 μM and NGF 1.5 ng / ml. The results are shown in FIG.

    As shown in FIG. 2, 6-HITC had the highest elongation effect, followed by BITC, SUL, PEITC and AITC.

(Evaluation of neurite outgrowth action of 6-HITC)
6-HITC, which had the highest neurite outgrowth effect in Example 2, was evaluated for neurite outgrowth under each condition in the presence and absence of NGF. Furthermore, the concentration of 6-HITC was changed in the presence of NGF to evaluate the neurite elongation action. The evaluation in the presence of NGF was carried out in the same manner as in Example 1 except that 6-HITC was used at a predetermined concentration in place of the cruciferous plant extract. The evaluation in the absence of NGF was further performed using NGF. The procedure was the same as Example 1 except that no addition was made. The results are shown in FIGS.

  As shown in FIGS. 3 and 4, the neurite outgrowth action of 6-HITC was observed in the presence of NGF, but the neurite outgrowth action was not observed only in the absence of NGF. Further, as shown in FIGS. 5 and 6, in the presence of NGF, the process of extending the protrusion was enhanced depending on the concentration of 6-HITC. In particular, 10 μM showed an effect of enhancing the action by about 70% (see FIG. 5). From these results, it was found that 6-HITC itself does not show neurite elongation action but has an action to enhance neurite extension action of NGF.

  In this example, the relationship between the enhancement of neurite outgrowth by 6-HITC and the expression levels of NF-L and b-tubulin, which are cytoskeletal proteins serving as differentiation indicators, was confirmed. The neurite outgrowth action was evaluated in the same manner as in Example 1 except that each condition of 6-HITC only, NGF only, and NGF + 6-HITC was set, and the antibodies of the above two proteins against the obtained cultured cells Were subjected to immunostaining and observed with a microscope and image analysis. The results are shown in FIG. In addition, the same neurite outgrowth evaluation experiment was conducted, and after 8 hours, 24 hours, 48 hours, and 72 hours, the protein was extracted from the culture and subjected to Western blotting to change the expression levels of the two proteins. It was confirmed. The results are shown in FIGS.

  As shown in FIG. 7, a slight increase in the expression level could be confirmed for the sample administered with 6-HITC alone or NGF alone compared to the control without addition, but NGF and 6-HITC coadministration were confirmed. In some cases, a significant increase in the expression level of these proteins was observed. In addition, as shown in FIGS. 8 and 9, no significant increase in these proteins was observed in the absence of NGF, whereas a sustained expression level was observed up to 8-72 hours in the presence of NGF. It was.

The graph which shows the neurite extension effect | action of the cruciferous plant extract. The graph which shows the neurite extension effect | action of the isothiocyanate compound derived from a Brassicaceae plant. The graph which shows the neurite expansion | extension effect | action of 6-HITC in NGF presence and absence. The micrograph figure which shows the neurite expansion | extension effect | action of 6-HITC in NGF presence and absence. The graph which shows the relationship between the density | concentration change of 6-HITC in the presence of NGF, and a neurite extension effect | action. The microscope picture figure which shows the relationship of the density | concentration change of 6-HITC and the neurite extension effect | action in the presence or absence of NGF. The immuno-staining figure which shows the relationship between 6-HITC administration and the expression level of cytoskeletal protein. Western blotting diagram showing the relationship between the dose of 6-HITC and the expression level of cytoskeletal proteins. Western blotting diagram showing the relationship between 6-HITC administration time and cytoskeletal protein expression level.

Claims (12)

  A nerve growth factor action potentiator comprising an isothiocyanate compound or a glycoside thereof.   The nerve growth factor action enhancer according to claim 1, wherein the isothiocyanate compound includes an isothiocyanate compound derived from a cruciferous plant.   The nerve growth factor action enhancer according to claim 1 or 2, wherein the isothiocyanate compound is one or more selected from allyl isothiocyanate, arylalkyl isothiocyanate and sulfinylalkyl isothiocyanate.   The nerve growth factor action enhancer according to claim 3, wherein the isothiocyanate compound is a methylsulfinylalkyl group (wherein the alkyl group is a linear alkyl group having 4 to 8 carbon atoms).   The enhancement of nerve growth factor action according to any one of claims 1 to 4, comprising an extract of one or more plants selected from cruciferous plants or a fraction obtained from the plant extract. Agent.   A nerve growth factor action enhancer comprising an extract of one or more plants selected from plants belonging to the Brassicaceae family, or a fraction obtained from the extract.   7. The nerve growth factor action potentiator according to claim 6, wherein the plant belonging to the Brassicaceae family consists of cabbage, Chinese cabbage, radish, watercress, broccoli, Chinese cabbage, komatsuna, mustard, mikuna, radish, cauliflower, Brussels sprouts and wasabi.   The nerve growth factor action enhancer according to claim 7, wherein the plant belonging to the Brassicaceae family consists of tangy vegetables, mizuna and wasabi.   A nerve growth factor action-enhancing composition comprising the nerve growth factor action potentiator according to any one of claims 1 to 8 as an active ingredient.   The nerve growth factor effect | action enhancement composition of Claim 9 containing a nerve growth factor.   The composition for enhancing nerve growth factor action according to claim 9 or 10, which is a food composition.   The nerve growth factor action according to claim 9 or 10, which is a pharmaceutical composition for treating or preventing a disease requiring enhancement of nerve growth factor action, containing an effective amount of the nerve growth factor action enhancer. Enhancement composition.