JPWO2017104706A1 - Brain-derived neurotrophic factor expression inducer and composition - Google Patents

Abstract

An object of the present invention is to provide a pharmaceutical or a composition comprising a crude drug or extract having BDNF gene expression inducing activity as an active ingredient.
[Solution] Licorice, shredded sand, bonito, yellow cocoon, kudzu, yam, red cocoon, Daio, Chotou, bitter ginseng, cocoon, mao, proofed, Ryo and their extracts induce BDNF gene expression Kampo medicines that have activity and are combined alone or in combination with other herbal medicines are useful for improving brain function decline due to mental disorders such as depression and Alzheimer's disease and neurodegenerative diseases.
[Selection] Figure 2

Description

  The present invention relates to a crude drug having an activity of inducing expression of brain-derived neurotrophic factor.

In Japan, the rapid increase in nervous system diseases such as dementia is regarded as a problem due to the effects of an aging society.
The decline in brain function due to such diseases leads to a significant decline in the quality of life of the patient and the burden on the family involved in nursing care. Therefore, not only the development of therapeutic drugs for these diseases, but also from the viewpoint of prevention. Research and development is a very important initiative when considering mental health and social welfare.
Particularly in prevention, development of drugs that can be taken on a daily basis is required.
Attention is paid to traditional medicines represented by crude drugs.
While these traditional medicines have low hurdles to be overcome in terms of safety due to many years of use, there are many that lack scientific evidence, and there are many unexplained parts in particular regarding their effects on the central nervous system.
In addition, it is very difficult to search for a drug having a preventive effect on brain function deterioration from a large number of herbal medicines in terms of time, labor, and cost. For example, ginseng, psoriasis and yam Daikenchuto, a Chinese herbal medicine, enhances memory and learning in mice (Non-patent Document 1), neurotrophic factor consisting of apricot kernel, mahuang, katsushi, ginseng, Toki, Senkyu, psoriasis, licorice and gypsum Synthesis accelerators (Patent Document 1), nerve cell activators (Patent Document 2) in alcoholic extracts of medicinal carrots and the like have been reported.

Japanese Unexamined Patent Publication No. 7-25777 Japanese Unexamined Patent Publication No. 6-316527 J Nat Med (2006) 60: 64-67

Brain-derived neurotrophic factor (hereinafter referred to as BDNF) exists as a dimer of a polypeptide consisting of 119 amino acids, and is one of the proteins belonging to the neurotrophic factor family. In the brain / nervous system, the action is diverse, such as the survival of neurons, the formation of nerve networks, and the expression of higher brain functions such as memory fixation.
In addition, BDNF expression is decreased in various neurodegenerative diseases and psychiatric disorders, and a drug that induces BDNF expression or a composition containing the same has reduced brain function due to neurodegenerative diseases and psychiatric disorders. It is expected to improve.
On the other hand, safety has been ensured from long-term use results, and it is unknown how much of the herbal extracts that have BDNF gene expression inducing activity listed in the Japanese Pharmacopoeia.

  Under the circumstances described above, the inventors developed a measurement system using a primary culture system of cerebral cortical neurons derived from a genetically modified mouse that can measure changes in BDNF gene expression by intensity of light emission by firefly luminescence enzyme luciferase. The system succeeded in simply and rapidly screening for a herbal extract that acts on nerve cells to induce BDNF gene expression, leading to the completion of the present invention.

  The herbal medicines used in the present invention are licorice, shredded sand, bonito, yellow cocoon, kudzu, yam, red cocoon, dai yellow, choito tsubo, bitter ginseng, ginseng, mao, proof and good candy. The crude drugs are as follows.

  Licorice: Glycyrrhiza uralensis or Glycyrrhiza glabra Linne roots and strons, sometimes excluding pericytes (peeled licorice).

  Shukusha: A seed mass of the genus Amomum xanthioides.

  Kashuu: A tuberous root of the Polygonum multiflorum.

  Jaundice: The root of the leguminous genus Astragalus membranaceus or Astragalus mongholicus.

  Kakkon: This is the root excluding the pericarp of Pueraria lobata Ohwi.

  Salamander: Zanthoxylum piperitum mature fruit peel with as much seed as possible removed.

  Red peony: The root of the genus Paeonia lactiflora, with the outer skin still attached.

  Daihyo: Rheum palmatum, Rheum tanguticum, Rheum officinale, Rheum coreanum, or interspecific hybrids of rhododendrons.

  Chotochou: A spine with a part of the stem branch of Uncaria rhynchophylla, Uncaria sinensis Haviland, or Uncaria macrophylla Wallich.

  Ginseng: The root of the leguminous family Clara (Sophora flavescens).

  Bamboo bud: A tuber of Aconitum carmichaeli or Aconitum japonicum is immersed in bittern for several days and heat-treated.

  Mao: The ground stalk of Ephedra sinica, Ephedra intermedia or Ephedra equisetina.

  Protected (bowie): A vine stem and rhizome of the genus Sinomenium acutum.

  Ryokyo: Rhizome of Alpinia officinarum.

In the present invention, the above-described herbal extract (hereinafter referred to as “extract”) can also be used.
As a method for producing the extract, the herbal medicine is extracted with, for example, water (hot water, hot water, etc.) or extracted with an organic solvent.
As the organic solvent, for example, alcohols such as methanol and ethanol, glycols such as polyethylene glycol, propylene glycol and butylene glycol, acetonitrile and the like can be used, and these organic solvents are used alone or in appropriate combination, and a certain ratio. Can be mixed and used.
Preferably, methanol, ethanol, water, butylene glycol, hydrous alcohol, acetonitrile-water mixed solvent, or the like is preferable.

As a method of water extraction or organic solvent extraction, a method used for normal herbal medicine extraction can be used. For example, 5 to 300 parts by weight of water and / or organic solvent is used for 1 part by weight of herbal medicine and stirred. However, it is desirable to heat and reflux at a temperature below the boiling point.
The extraction step is usually performed for 5 minutes to 7 days, preferably 10 minutes to 24 hours.

After completion of the extraction step, a crude extract can be obtained by separating insolubles from water and / or an organic solvent extract by an appropriate method such as filtration or centrifugation.
In addition to the hot water extract or organic solvent extract according to a conventional method, a purified extract obtained by further treating the crude extract with various organic solvents or adsorbents can also be used in the present invention.
These crude extracts and purified extracts that have been subjected to various purification treatments are prepared by pulverizing the extracted solution, the solvent concentrated, the solvent evaporated to dryness, the freeze-dried, and the dried solid. Any of powders and the like may be used.

The licorice, shredded sand, bonito, jade, katsune, yam, red cocoon, red sardine, Daio, Choto, ginseng, ginseng, mao, proofed, and potatoes used in the present invention are each independent. Or may be used in combination as a prescription.
As for the use alone, it is preferable to use a garbage, a yam, a red carp, a fishing wisteria or a good carp.
The prescription may include other herbal medicines such as carrots.
For example, prescriptions with ginseng extract include yam extract + carrot extract, gauze extract + carrot extract, ginseng extract + ginseng extract of Test Example 4 to be described later, and also used for Daikenchuyu. For example, a combination of carrots, yam, and dried ginger.

  The above simple extract and a combination of extracts may be formulated into tablets, powders, granules, capsules and the like and administered orally.

For oral preparations, tablets, powders, granules, and capsules can be produced by conventional methods by adding a binder, lubricant, disintegrant, coloring agent, corrigent and the like as necessary.
Moreover, antiseptic | preservative, antioxidant, a stabilizer, etc. can be added as needed.

  The dose of extract and the like varies depending on the disease, symptoms, age, therapeutic measures used in combination, etc., but when administered to humans as an oral preparation, 0.1 to 100 mg / kg as an extract once a day It may be administered in several divided doses.

An extract or the like can be made into a food or drink or a composition for food or drink containing the extract or the like.
Here, “food and drink” means food and drink in general, but in addition to general foods including so-called health foods, health foods such as foods for specified health use and nutritional function foods stipulated in the health functional food system of the Ministry of Health, Labor and Welfare. Functional foods are also included, and supplements, feeds, food additives and the like are also included in the food and drink of the present invention.

In order to use an extract or the like in a food or drink or a composition for food or drink, the food or drink or the composition for food or drink as it is or mixed with raw materials of food or drink such as processed meat, fruit juice, milk, soy milk together with various nutritional components Can be manufactured.
Moreover, when using an extract etc. as a health food, a nutritional supplement, etc., it can prepare in forms, such as a tablet, a capsule, a powder, a granule, a suspension agent, a syrup, using a conventional means, for example.

  The blending amount of the extract or the like in the above food or drink or the composition for food or drink varies depending on the addition form and dosage form and can be selected from a wide range, but it is usually desirable to blend 0.01 to 10% by weight.

  Licorice, shredded sand, bonito, yellow cocoon, kudzu, yam, red fox, Daio, Chotou, bitter melon, cocoon, mao, banned, Ryoji and their extracts have BDNF gene expression inducing activity Kampo medicines, alone or in combination with other herbal medicines, can improve or prevent brain function deterioration due to diseases such as mild cognitive impairment (MCI) and dementia, mental disorders such as depression and Alzheimer's disease Useful for improving brain function due to neurodegenerative diseases.

It is a figure which shows the outline | summary of a screening. It is a figure which shows endogenous BDNF gene expression induction activity. BDNF mRNA expression level was corrected with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression level, which is one of the housekeeping genes, and the change relative to BDNF mRNA expression level before adding herbal extract (0 hour) is shown as the induction factor. It was. It is a figure which shows endogenous BDNF gene expression induction activity similarly to FIG. It is a figure which shows endogenous BDNF gene expression induction activity in the combination of crude drugs. It is a figure which shows the BDNF gene expression induction activity in Daikenchuto extract.

Hereinafter, although this invention is demonstrated by a test example, this invention is not limited to these.
The herbal extract used in the test example is obtained by decocting herbal medicine with pure water for 50 minutes, filtering, and then dissolving the freeze-dried extract powder in MilliQ water (10 mg / mL).

Test example 1
<BDNF gene expression inducing activity>
The BDNF gene expression inducing activity was examined using a genetically modified mouse (BDNF-Luc mouse) capable of measuring changes in BDNF gene expression based on luminescence intensity.
The cerebral cortex of embryonic day 16.5 day old BDNF-Luc mice was isolated, treated with enzymes, and the resulting cells were cultured on a 96-well culture plate for 13 days.
After the culture, the herbal extract was added to the culture solution so that the final concentration was 0.5 mg / mL, and the culture was performed again.
As an experimental control group, only MilliQ water as a solvent was added. Luminescence of each well was measured using a luciferase activity measurement kit (Promega) 6 hours, 24 hours and 48 hours after the addition of MilliQ water or herbal extract. Based on the luciferase activity when MilliQ water was added at each time, the induction ratio of the luciferase activity of the herbal extract was calculated.
Table 1 shows the induction ratio of the luciferase activity of the crude drug extract.

From the results shown in Table 1, those in which the induction ratio of luciferase activity was 10 times or more were: Kashou (What's Necklace), Salamander (Yamabuchi), Sekishaku (Akaban), Butterflyfish (Tsujido), Bowie (Present) ), And Ryokyo.
Among them, cashew, salamander and butterfly were more than 15 times.
The maximum is 21.29 cultivated butterfly.
Moreover, the thing which showed the value of 5 times-10 times was Daio (large yellow), Maou (mao yellow), and the carrot (carrot).

Test example 2
<Endogenous BDNF gene expression inducing activity>
Endogenous BDNF gene (BDNF gene actually expressed in cerebral cortical neurons) expression-inducing activity was examined by measuring changes in the BDNF mRNA expression level by RT-qPCR method.
The cerebral cortex of a 17-day-old rat was isolated and then subjected to enzyme treatment, and the resulting cells were cultured on a 6-well culture plate for 13 days.
After the culture, each herbal extract was added to the culture solution so that the final concentration was 0.5 mg / mL, and the culture was performed again.
As an experimental control group, only MilliQ water as a solvent was added.
After the addition of each herbal extract, after 0 hours, 0.5 hours, 1 hour, 3 hours, 6 hours, 9 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, RNA extraction kit (Qiagen) RNA was extracted from the cells, and a reverse transcription reaction was performed using Superscript II Reverse Transcriptase (Invitrogen).
Using the DNA product obtained after the reverse transcription reaction and SYBR Select Master Mix (Applied Biosystems), changes in the BDNF mRNA expression level after the addition of each herbal extract were analyzed.
The results are shown in FIG.
From the graph shown in FIG. 2, the ginseng extract, the salamander extract, and the Choito extract all showed higher endogenous BDNF gene expression-inducing activity than the control (solvent).
In particular, Choito extract showed a high value.

Test example 3
By using the same koji extract, large yellow extract, hemp yellow extract, bean extract, and anti-prevented extract, the endogenous BDNF gene expression inducing activity was examined in the same manner as in Test Example 2. The results are shown in FIG.
From the graph of FIG. 3, all showed high endogenous BDNF gene expression-inducing activity, and especially the large yellow extract, the maize extract and the bean extract showed high values.

Test example 4
Endogenous BDNF gene expression inducing activity when ginseng extract, yaoi extract, or Choto extract was mixed was examined by measuring changes in BDNF mRNA expression level by RT-qPCR method.
The cerebral cortex of a 17-day-old rat was isolated and then subjected to enzyme treatment, and the resulting cells were cultured on a 6-well culture plate for 13 days.
After the culture, each herbal extract was added to the culture solution so that the final concentration was 0.5 mg / mL, and the culture was performed again.
In the case of the combination of ginseng extract and yam extract, and in the case of the combination of ginseng extract and anti-prevented extract, the cells are extracted using an RNA extraction kit (Qiagen Co., Ltd.) 3 hours after addition, and in the case of the combination of ginseng extract and Choito extract, 24 hours after addition. RNA was extracted from the DNA, and reverse transcription reaction was performed using Superscript II Reverse Transcriptase (Invitrogen).
Using the DNA product obtained after the reverse transcription reaction and SYBR Select Master Mix (Applied Biosystems), changes in the BDNF mRNA expression level after the addition of each herbal extract were analyzed.
The results are shown in FIG.
From the graph shown in FIG. 4, it was revealed that the combination with carrot extract showed higher inductive activity than each herbal medicine alone.

Test Example 5
<BDNF gene expression inducing activity of Daikenchuto extract>
The BDNF gene expression inducing activity was examined using a genetically modified mouse (BDNF-Luc mouse) capable of measuring changes in BDNF gene expression based on luminescence intensity.
The cerebral cortex of embryonic day 16.5 day old BDNF-Luc mice was isolated, treated with enzymes, and the resulting cells were cultured on a 96-well culture plate for 13 days.
After the culture, Daikenchuto extract was added to the culture solution so that the final concentration was 0.5 mg / mL, and the culture was performed again.
Luminescence of each well was measured using a luciferase activity measurement kit (Promega) 6 hours and 24 hours after the addition of Daikenchuto extract.
The results are shown in FIG.
From the graph shown in FIG. 5, it was revealed that Daikenchuyu, that is, carrot, yam, and dried rice cake also showed high activity.

Licorice, shredded sand, bonito, yellow cocoon, kudzu, yam, red cocoon, Daihuang, Chotou, ginseng, 炮 子, 麻黄, 防 已, 良姜 and their extracts have BDNF gene expression inducing activity Kampo medicines, alone or in combination with other herbal medicines, can improve and prevent brain function deterioration due to diseases such as dementia, brain function due to mental disorders such as depression and Alzheimer's disease, and neurodegenerative diseases. Useful for improving the decline.
In addition, the extract of licorice, shredded sand, bonito, yellow cocoon, kudzu, yam, red cocoon, Daihuang, Chotou, bitter ginseng, garlic, mao, proofed and / or Ryokan extract, alone, mixed or other Combining herbal medicines, it can be used as a food or beverage that improves or prevents brain function decline due to diseases such as dementia, and improves brain function decline due to mental or neurodegenerative diseases such as depression or Alzheimer's disease. .

Claims (6)

  As an active ingredient, one or more selected from licorice, shredded sand, bonito, yellow potato, kudzu, yam, red cocoon, Daihuang, Chotou, bitter ginseng, cocoon, mao, fenestrated, good bud, or extracts thereof An expression inducer for brain-derived neurotrophic factor, comprising:   An expression inducer for brain-derived neurotrophic factor, comprising as an active ingredient at least one selected from garlic, yam, red cormorant, cormorant fish, cormorant, rhubarb, mao or extract thereof.   The brain-derived neurotrophic factor expression inducer according to claim 1 or 2, wherein ginseng or an extract thereof is added as an active ingredient.   As an active ingredient, one or more selected from licorice, shredded sand, bonito, yellow potato, kudzu, yam, red cocoon, Daihuang, Chotou, bitter ginseng, cocoon, mao, fenestrated, good bud, or extracts thereof A composition for inducing the expression of brain-derived neurotrophic factor, comprising: What is claimed is: 1. A composition for inducing expression of brain-derived neurotrophic factor, comprising as an active ingredient at least one selected from garlic, yam, red sea bream, sea bream, good coral, large yellow, mao or extracts thereof .   6. The composition for inducing the expression of brain-derived neurotrophic factor according to claim 4 or 5, wherein ginseng or an extract thereof is added as an active ingredient.

Images