JP6033863B2 - Composition for improvement and / or treatment of central neurodegenerative diseases - Google Patents

Description

  The present invention relates to a composition for improving and / or treating central nervous degenerative diseases accompanied by learning / memory disorders.

  In the modern aging society, the number of patients with central neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease has increased and has become a social problem. Among them, Alzheimer's disease is a progressive central neurodegenerative disease accompanied by cognitive dysfunction, learning / memory impairment, etc. As a cause thereof, the function of N-methyl-D-aspartate (NMDA) receptor, which is important for learning / memory, is reduced. Neurodegeneration due to polymerization and accumulation of amyloid β peptide is considered (see Non-Patent Document 1).

  In general, memory is at least three processes: memory acquisition (hereinafter also referred to as “memorization”; encoding), retention and recall (hereinafter also referred to as “recollection”; recall). The mechanism of these three processes is considered to be different. In Alzheimer's disease, when neurodegeneration progresses, the ability to newly remember, that is, acquisition (or learning) or memorization of memory is hindered, and it is impossible to retain and recall memories acquired in the past. As a result, it is understood that the learning / memory disorder, which is the core symptom of the disease, becomes prominent.

で示されるノビレチン（Ｎｂ）が知られており（特許文献１または非特許文献１もしくは２を参照のこと）、ノビレチンはまた、神経細胞に対して神経突起伸長作用を有することも知られている（特許文献２を参照のこと）。
同様に、天然フラボノイド類の一つである、式（Ｉ−１）：

で示されるタンゲレチン（Ｓ１）には、神経細胞に対して神経突起伸長作用を有することが知られている（特許文献２を参照のこと）。Among these, as one of natural flavonoids having an action of improving memory retention and recall ability, the formula (I-5):

Is known (see Patent Document 1 or Non-Patent Document 1 or 2), and nobiletin is also known to have a neurite outgrowth action on nerve cells. (See Patent Document 2).
Similarly, one of natural flavonoids, formula (I-1):

It is known that tangeretin (S1) represented by the formula has a neurite outgrowth action on nerve cells (see Patent Document 2).

In the hippocampus of the neural circuit for learning and memory formation, cyclic adenosine 3 ′, 5′-1 phosphate (cAMP) / cAMP linked to the NMDA receptor is an important neuronal signaling pathway that controls learning and memory. A dependent protein kinase A (PKA) / ERK (extracellular signal-regulated kinase) / cAMP response element (CRE) binding protein (CREB) system has recently been revealed. When this intracellular signaling pathway (cAMP / PKA / ERK / CREB signaling pathway) is activated, the ability to learn and memory is enhanced, and at the same time, cAMP response element (CRE) -dependent transcriptional activity (hereinafter referred to as the present specification). (Also referred to as “CRE-dependent transcriptional activity”) has been shown to occur (see Non-Patent Document 3). Moreover, it is suggested that the function of cAMP / PKA / ERK / CREB signaling pathway is reduced in the brain of Alzheimer's disease patients (see Non-Patent Document 4). In fact, evidence supporting this has also been reported (see Non-Patent Document 5).
It has been found that this CRE-dependent transcriptional activity is related not only to the learning memory ability but also to the transcriptional regulation of tyrosine hydroxylase, a biosynthetic enzyme of dopamine (DA).

  The present inventors previously obtained an extract from a dried product of special citrus peels containing a high proportion of nobiletin (such as N-thinpi, citrus peel and large red citrus peel), and a conventional dried citrus peel (skin). It has been disclosed that it has a superior central nervous degenerative disease ameliorating effect compared with the extract extracted from, and exhibits CRE-dependent transcription activity and learning / memory impairment improving action (see Patent Document 3).

International Publication No. 2005/082351 JP 2002-60340 A International Publication 2011/105568

Journal of Pharmacology of Japan (Folia Pharmacol. Jpn.) 132, 155-159 (2008) The Journal of Pharmacology and Experimental Therapeutics, Vol. 321, no. 2, pp. 784-790 (2007) Nat Neurosci. 1,595-601 (1998) Trends in Pharmacological 27, 33-40 (2006) Brain Research 824, 300-303 (1999)

Under such circumstances, if a compound is developed that surpasses the anti-central neurodegenerative disease activity of nobiletin (Nb) and tangeretin (S1), particularly the improvement and / or therapeutic effect of Alzheimer's disease, a promising fundamental treatment in this disease Realization of drug development can be expected. This will be a major step toward overcoming intractable progressive neurological diseases such as dementia in the modern aging society.
In addition to improving memory retention and recall as well as learning and memory disorders like nobiletin, it also improves both memory acquisition and memory disorders in addition to memory retention and recall disorders. If a compound with an activity surpassing nobiletin is developed, it can be applied to therapeutic agents such as dementia with a new mechanism of action, particularly Alzheimer's disease, etc. The creation of useful drugs is expected.

  In the patent document 3, the present inventors have disclosed a nobiletin-rich skin (hereinafter, also referred to as “N-thinpi” in the present specification), tachibana peel, and Crimson tangerine pericarp in central nervous degenerative disease, particularly CRE-dependent transcription. It has been found that the accelerating activity shows a value exceeding the accelerating activity exhibited by the concentration of nobiletin contained in the peels of N Chinpi, Tachibana peel and Daikokukankan. From this, it is thought that the CRE-dependent transcription promoting activity of the N Chinpi, Tachibana peel and Daikokukankan peel is attributed to the cooperative action of Nobiletin and other components contained therein, earnestly, its activity We proceeded with research on the main body. As a result, in the present invention, it was found that a plurality of compounds having a specific structure as a kind of natural flavonoid or a specific combination thereof contribute to the activity of N-thinpy and the above two citrus peels. In particular, it has been found for the first time that one of these compounds, synencetin, contributes greatly to this activity, and its activity intensity far exceeds the CRE-dependent transcription promoting activity of nobiletin and tangeretin. Even more surprisingly, it was demonstrated in the present invention that sinensecetin improves not only memory retention and recall but also memory acquisition and memory impairment. The inventors of the present invention have reached the present invention based on the above facts that no one has ever found.

において、
Ｒ_１、Ｒ_２、Ｒ_３およびＲ_４がメトキシ基であってＲ_５がＨである化合物Ｓ４（シネンセチン）、
Ｒ_２、Ｒ_４およびＲ_５がメトキシ基であってＲ_１およびＲ_３がＨである化合物Ｓ３（６−デメトキシタンゲレチン）、および
Ｒ_１、Ｒ_２、Ｒ_４およびＲ_５がメトキシ基であってＲ_３がＨである化合物Ｓ２（６−デメトキシノビレチン）
から選択される少なくとも１つの化合物を有効成分として含んでなる、中枢神経変性疾患の改善および／または治療用の組成物。That is, the present invention is as follows.
Item 1: General formula I:

In
Compound S4 (sinensetin) in which R ₁ , R ₂ , R ₃ and R ₄ are methoxy groups and R ₅ is H,
Compound S3 (6-demethoxytangerine) in which R ₂ , R ₄ and R ₅ are methoxy groups and R ₁ and R ₃ are H, and R ₁ , R ₂ , R ₄ and R ₅ are methoxy groups Compound S2 (6-demethoxynobiletin) in which R ₃ is H
A composition for improving and / or treating central neurodegenerative diseases, comprising as an active ingredient at least one compound selected from the group consisting of:

で表される化合物Ｓ４である、項１に記載の組成物。Item 2: The active ingredient is represented by formula I-4:

Item 6. The composition according to Item 1, which is a compound S4 represented by:

  Compound S4 (Sinensetin) is preferable in that the effect of increasing CRE-dependent transcription activity is extremely high.

で表される化合物Ｓ４（シネンセチン）と、式Ｉ−３：

で表される化合物Ｓ３（６−デメトキシタンゲレチン）および／または式Ｉ−２：

で表される化合物Ｓ２（６−デメトキシノビレチン）との混合物である、項１に記載の組成物。Item 3: The active ingredient is represented by formula I-4:

A compound S4 (sinensetin) represented by formula I-3:

Compound S3 (6-demethoxytangeretin) and / or Formula I-2:

Item 6. The composition according to Item 1, which is a mixture with the compound S2 (6-demethoxynobiletin) represented by:

  Item 4: The composition according to any one of Items 1 to 3, wherein the central neurodegenerative disease is Alzheimer's disease, learning disorder, memory disorder, Parkinson's disease, Pick's disease, or Huntington's disease.

  Item 5: The composition according to any one of Items 1 to 4, wherein the central neurodegenerative disease is a learning disorder or a memory disorder.

  Item 6: The composition according to Item 5, wherein the memory failure is memory acquisition failure, retention failure or recall failure, memorization failure, recall failure, memory enhancement failure, memory decline failure, or memory error.

  Item 7: The composition according to Item 6, wherein the memory disorder is a memory acquisition disorder or a memory disorder.

  Item 8: The composition according to Item 4, wherein the central neurodegenerative disease is Alzheimer's disease.

  Item 9: The composition according to Item 4, wherein the central neurodegenerative disease is Parkinson's disease.

  Item 10: Any one of Items 1 to 9, wherein the improvement and / or treatment of a central neurodegenerative disease is caused by an increase in cAMP responsive element-dependent transcription activity (CRE-dependent transcription activity) or an increase in memory formation function. A composition according to 1.

  Item 11: Improvement and / or treatment of a central neurodegenerative disease is caused by an increase in cAMP response element-dependent transcription activity (CRE-dependent transcription activity), or acquisition of memory, retention of memory, or increased ability of recall 10. The composition according to any one of 10 to 10.

  Item 12: The composition according to any one of Items 1 to 11, wherein the compounds S4, S3, and S2 are water-extracted from a dried citrus peel.

  Item 13: The composition according to Item 12, wherein the citrus fruit is Citrus reticulata Branco (Rutaceae), Tachibana or Great Red Tangerine.

  Item 14: Wei Sensen, glaze, Yongxiao, jaundice, yellow Gon, jade, distant, cuckold, kudzu root, psoriasis, licorice, bellflower, chrysanthemum, coconut, apricot kernel, cinnamon, safflower, garlic, kobetsu, Gyu-knee, Kureo, Gomi, Saiko, Yamiko, Ji-Huang, Glaze, Ginger, Sesame, Kamisu, Gypsum, River Kyu, Mae-hu, Kashiwa, Sugi, Soyo, Daihuang, Daigo, Great stomach, Samurai, Bamboo Jo, Mother, Tsutomu Tsutomu, Tenma, Tenmon Winter, Tokio, Taojin, Ginseng, Malt, Mumon Winter, Semi-Summer, White Shishi, White Birch, Lion, Samurai, Protected, Glass, Windbreak, Item 14. The item according to any one of Items 1 to 13, comprising a herbal medicine ingredient selected from the group consisting of peony skin, mahuang, woody, incense, benevolent herb, ryu gall, wagyu active and combinations of two or more thereof. Composition.

  Item 15: The composition according to any one of Items 1 to 14, which is an oral dosage form.

  Item 16: The composition according to Item 15, wherein the oral dosage form is in the form of fine granules, tea, decoction, capsule, tablet, granule, jelly, powder, liquid, syrup or extract.

  Item 17: The composition according to any one of Items 1 to 16, which is a pharmaceutical composition for treating a central neurodegenerative disease.

［式中、各記号は項１に記載のものと同義である］
で示される化合物から選択される少なくとも１つの化合物を有効量にて治療を必要とする対象に投与することを特徴とする、中枢神経変性疾患の治療方法。Item 18: General formula I:

[Wherein each symbol has the same meaning as described in item 1]
A method for treating a central neurodegenerative disease, comprising administering at least one compound selected from the compounds represented by the above to a subject in need thereof in an effective amount.

［式中、各記号は項１に記載のものと同義である］
で示される化合物から選択される少なくとも１つの化合物の使用。Item 19: General Formula I for Improvement and / or Treatment of Central Neurodegenerative Diseases:

[Wherein each symbol has the same meaning as described in item 1]
Use of at least one compound selected from the compounds represented by:

において、
Ｒ_１、Ｒ_２、Ｒ_３およびＲ_４がメトキシ基であってＲ_５がＨである化合物Ｓ４、
Ｒ_２、Ｒ_４およびＲ_５がメトキシ基であってＲ_１およびＲ_３がＨである化合物Ｓ３、および
Ｒ_１、Ｒ_２、Ｒ_４およびＲ_５がメトキシ基であってＲ_３がＨである化合物Ｓ２
から選択される少なくとも１つの化合物、および添加成分を含む食品。Item 20: Formula I:

In
Compound S4 wherein R ₁ , R ₂ , R ₃ and R ₄ are methoxy groups and R ₅ is H,
Compound S3 wherein R ₂ , R ₄ and R ₅ are methoxy groups and R ₁ and R ₃ are H, and R ₁ , R ₂ , R ₄ and R ₅ are methoxy groups and R ₃ is H Compound S2
A food comprising at least one compound selected from: and an additive component.

で表される化合物Ｓ４を含む、項２０に記載の食品。Item 21: Formula I-4:

Item 20. The food according to Item 20, comprising the compound S4 represented by:

  Item 22: The food according to any one of Items 20 or 21, which is a food for improving central neurodegenerative diseases.

  S4 (sinensetin) according to the present invention has a CRE-dependent transcription enhancing activity far exceeding the CRE-dependent transcriptional activity promoting effects of nobiletin (Nb) and tangeretin (S1), which are conventionally known. Furthermore, since S4 has an effect of improving not only memory retention and recall but also memory acquisition and memory disorder not found in nobiletin, it is a marked improvement in central neurodegenerative diseases, particularly Alzheimer's disease. And / or indicate a therapeutic effect. On the other hand, S3 (6-demethoxytangeretin) and S2 (6-demethoxynobiletin) are Fr. Central nervous system degeneration such as dementia due to unique activity that works to increase CRE-dependent transcriptional activity in cooperation with S4 (sinensetin) and S1 (tangeretin), which are components of 2-2 (non-nobiletin fraction) Excellent effect in disease improvement and / or treatment.

  S4 (sinensetin), S3 (6-demethoxytangeretin), and S2 (6-demethoxynobiletin) according to the present invention also activate PKA / ERK / CREB signaling and are one of the symptoms of Alzheimer's disease It has the effect of improving and / or treating certain learning and memory disorders. S4 (sinensetin), S3 (6-demethoxytangeretin), and S2 (6-demethoxynobiletin) according to the present invention, among other effects of improving learning / memory impairment, are the ability to acquire, retain and recall memory and Demonstrate the effect of enhancing the ability of meditation and recollection. More preferably, it is an effect of enhancing the ability to acquire memory or the ability to memorize. Since CRE-dependent transcriptional activity is also closely related to increased transcription of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, S4 (sinensetin), S3 (6-demethoxytangerine) according to the present invention, And S2 (6-demethoxynobiletin) also show improvement and / or therapeutic effect on Parkinson's disease by dopamine (DA) synthesis / promoting action or dopamine synthesis / secretion promoting action.

FIG. 1 shows the first part of the separation step of the flavonoid compounds (S4 (sinensetin), S3 (6-demethoxytangeretin), and S2 (6-demethoxynobiletin)) according to the present invention from N-thinpi extract. Show. FIG. 2 shows the chemical structures of S4, S3 and S2 and comparative compounds (nobiletin (Nb) and tangeretin (S1)) according to the present invention. FIG. 3 shows CRE-dependent transcriptional activity of S4, S3 and S2 and comparative compounds (Nb and S1) according to the present invention in hippocampal neurons. In the figure, * p <0.05, ** p <0.01, *** p <0.001 is significant difference (test result) from the control (saline); §§ p <0.01 is A significant difference from Nb is shown. FIG. 4 shows the concentration-dependent dependence of CRE-dependent transcriptional activity of synensetin (S4) and comparative compound nobiletin (Nb) according to the present invention in hippocampal neurons. In the figure, ** p <0.01 and *** p <0.001 indicate a significant difference (test result) from the control (saline). FIG. 5 shows polymethoxyflavonoid fractions (Fr.2, Fr.2-1 and Fr.2-2; each concentration corresponds to the content ratio in N-thin pin), Fr. 2-1 and Fr. Mixed fraction reconstituted in 2-2 (Fr.2-1 + Fr.2-2; each concentration corresponds to the content in N-thinpi), reconstituted methoxyflavonoids (REC; sinensetin (0.222 μg / mL) + 6-demethoxytangeretin (0.135 μg / mL) + 6-demethoxynobiletin (0.177 mg / mL) + tangeretin (0.585 mg / mL) + nobiletin (1.89 μg / mL, 5.0 μM); Each concentration corresponds to the content ratio in N Chinpi) and shows the CRE-dependent transcriptional activity of the control (saline). In the figure, ** p <0.01 and *** p <0.001 indicate a significant difference from the control (test result). FIG. 6 shows CRE dependence of polymethoxyflavonoid fractions (Fr.2, Fr.2-1 and Fr.2-2) and nobiletin and control (saline) with a concentration of 30 μM in hippocampal neurons. Shows transcriptional activity. In the figure, ** p <0.01 and *** p <0.001 indicate a significant difference from the control (test result). FIG. 7 shows the improvement effect of sinensetin on MK801-induced memory acquisition impairment. The vertical axis shows freezing behavior (%), and the horizontal axis shows shock events (number of times). In the figure, *** p <0.001 and ****** p <0.0001 are significant differences (test results) from the control (saline), §§ p <0.01, §§§ p < 0.001 indicates a significant difference from “MK801”. FIG. 8 shows the effect of sinensetin on MK801-induced memory retention and recall disorders. The vertical axis shows freezing behavior (%). In the figure, *** p <0.0001 indicates a significant difference (test result) from the control (saline), and §§§ p <0.001 indicates a significant difference from “MK801”. FIG. 9 shows the effect of improving fraction 2 (Fr.2) of MK801-induced memory acquisition disorder. In the figure, *** p <0.001 indicates a significant difference (test result) from the control (saline), and ## p <0.01 indicates a significant difference from “MK801”. FIG. 10 shows the effect of fraction 2 (Fr.2) on MK801-induced memory retention and recall disorders. In the figure, *** p <0.001 indicates a significant difference (test result) from the control (saline), and ## p <0.001 indicates a significant difference from “MK801”. FIG. 11 shows the results of learning trials with nobiletin administration. FIG. 12 shows the results of confirmation trials with nobiletin administration. In the figure, * indicates a significant difference (test result) from the control (saline), and # indicates a significant difference from “MK801”.

で表され、
Ｓ３（６−デメトキシタンゲレチン）は、式Ｉ−３：

で表され、および
Ｓ２（６−デメトキシノビレチン）は、式Ｉ−２：

で表される化合物である。Hereinafter, embodiments for carrying out the present invention will be described in detail.
The polymethoxyflavonoid compound used as an active ingredient in the present invention is at least one compound selected from the three types of S4 (sinensetin), S3 (6-demethoxytangeretin) and S2 (6-demethoxynobiletin). The active ingredient may be a mixture of any two compounds selected from the group consisting of S4, S3 and S2, or may be a mixture of these three compounds.
Of these, S4 (sinensetin) is represented by formula I-4:

Represented by
S3 (6-demethoxytangeretin) is represented by formula I-3:

And S2 (6-demethoxynobiletin) is represented by formula I-2:

It is a compound represented by these.

The polymethoxyflavonoids S4 (sinensetin), S3 (6-demethoxytangeretin) and S2 (6-demethoxynobiletin) according to the present invention are generally compounds obtained from the skin of citrus plants. Citrus fruits include Japanese Tachibana, C. nipponokoreana, flower buds, four seasons Tachibana, citrus fruit, orange (Citrus aurantium), Mediterranean mandarin (Citrus deliciosa), Dancy tangerine, citrus aurium (C. erythrosa), Nucleated Kishu, Fukurai mandarin orange, Cappuchi, Ota mandarin orange, Citrus kinokuni, Sankitsu, Cleopatra, Citrus (Citrus leiocarpa), Giri Mikan, Citrus citrus citrus Markovich), Citrus reticulata Branco (Rutaceae), Sikhwa Sir (Citrus depressa), Citrus junos, Bundan, Natsu Hinata, Citrus tangerina, Citrus natsudaidai, Navel orange, Citrus hassakuu, a and odor A plant selected from the group consisting of:
Citrus peels are widely distributed in the market as herbal medicines such as Chen and Tachibana.
Chen refers to the mature pericarp of Citrus unshiu Marcowicz or Citrus reticulata Blanco (Rutaceae), for example.

  Crustacea used as a raw material in the present invention (that is, N Chinpi, Tachibana bark or large red tangerine pericarp) has a yield of 20% to 50% in the yield of the above citrus pericarp (mass after drying / mass before drying). It means a substance dried under such conditions. As heat drying conditions, 1-3 hours are mentioned at the temperature of 50-100 degreeC, Preferably it is 2 hours at the temperature of 60 degreeC.

  The compound according to the present invention is based on an extract from the citrus peel (that is, an extract from N Chinpi, Tachibana peel or large red citrus peel), preferably an extract obtained by water extraction. It is a thing. The preferable water extraction in this specification is extraction with water at 60 to 100 ° C.

  The polymethoxyflavonoids S4 (sinensetin), S3 (6-demethoxytangeretin) and S2 (6-demethoxynobiletin) according to the present invention can be separated from the above extract by a conventional fractionation means such as column chromatography. It can be isolated via.

<General production method>
A non-limiting example of a separation method using N Chinpi extract as a starting material is shown in FIG. Based on this figure, the outline of the separation method of S4, S3 and S2 will be described.
That is, a solid phase in which a predetermined amount of N Chinpi extract is suspended in a polar solvent (for example, 30% acetonitrile / 70% purified water) and filled with surface-treated silica gel (for example, octadecylsilylated silica gel (ODS)). Pass through the extraction column. Analyze the composition of the effluent as appropriate (eg, thin layer chromatography, etc.), confirm the separability of the components, and pass a polar solvent (eg, 30% acetonitrile / 70% purified water, etc.) through the column. By concentration under reduced pressure, Fr. 1 is obtained. Further, using a hydrophobic elution solvent (for example, methanol), the next elution fraction was obtained, and when the solvent was distilled off under reduced pressure, Fr. 2 is obtained.
Next, Fr. 2 is subjected to ODS column chromatography using a polar solvent (for example, 40% acetonitrile / 60% purified water) as an eluent. By changing the eluent polarity (for example, combinations of acetone / hexane (1: 1) and ethyl acetate / hexane (4: 1), etc.) and repeating silica gel column chromatography, Fr. 2-1. On the other hand, the fraction containing nobiletin is Fr. 2-2.
The Fr. 2-1 is a nobiletin fraction consisting of high-purity nobiletin (Nb) whose presence of nobiletin was confirmed by thin layer chromatography (TLC). 2-2 is Fr. 2 to Fr. It is a non-nobiletin fraction excluding 2-1.

Next, the Fr. 2-2 (non-nobiletin fraction) is repeatedly separated and purified by ODS column chromatography using an eluent (eg, 60% methanol / 40% purified water) whose hydrophilicity is adjusted. After concentrating the obtained eluent and purifying it by recrystallization or the like, four kinds of crystalline substances can be isolated, and these are referred to as S1 to S4. Their structures can be determined by analyzing S1-S4 by IR, UV, ¹ H NMR, ¹³ C NMR and ESI-MS.

Next, the pharmacological activity of the composition according to the present invention will be described.
The composition comprising at least one of the polymethoxyflavonoids S4 (sinensetin), S3 (6-demethoxytangeretin) and S2 (6-demethoxynobiletin) according to the present invention is used to improve central neurodegenerative diseases and / or Or it can be used for treatment.

  Here, in an embodiment of the present invention, the improvement and / or treatment of central nervous degenerative disease is caused by an increase in cAMP response element-dependent transcription activity (CRE-dependent transcription activity), and further to the increase in the activity. It is brought about by an increase in memory formation functions such as memory acquisition, memory retention and recall ability. Therefore, an increase in CRE-dependent transcriptional activity contributes to improvement of memory formation dysfunction, and contributes not only to learning and memory ability but also to improvement of dopamine (DA) synthesis and secretion and accompanying Parkinson's disease.

  In some embodiments, the improvement and / or treatment of central neurodegenerative diseases is brought about by CRE-dependent transcriptional activity promotion.

  In one embodiment, the central neurodegenerative disease is a disease related to a learning and / or memory disorder, preferably a learning disorder or a memory disorder. The central neurodegenerative disease is more preferably memory acquisition, retention and / or recall disorder, memory disorder, recall disorder, memory enhancement disorder, memory decline disorder or memory error disorder, particularly preferably memory acquisition disorder Or it is a memorization trouble.

  In some embodiments, the central neurodegenerative disease is Alzheimer's disease.

  In one embodiment, the central neurodegenerative disease is Parkinson's disease, and its improvement and / or treatment is brought about by an action of promoting the ability to synthesize dopamine.

As used herein, the term “central neurodegenerative disease” includes Alzheimer's disease, particularly learning / memory impairment, Parkinson's disease, Pick's disease, Huntington's disease and the like.
As used herein, the term “memory disorder” includes memory acquisition, retention and / or recall disorder, memorization disorder, recall disorder, memory enhancement disorder, memory decline disorder, memory error disorder, and the like.

Examples of the anti-Alzheimer's disease activity and / or anti-Parkinson's disease activity of the compounds (S4, S3 and S2) according to the present invention include improvement of learning / memory disorders involved in the onset mechanism of central neurodegenerative diseases or promotion of dopamine synthesis. It is done. Specifically, CRE (cAMP responsive element) -dependent transcription activity, dopamine content increasing activity, dopamine secretion promoting activity, and the like.
More specifically, the composition for improving and / or treating a central neurodegenerative disease of the present invention has an activity depending on the content of the active ingredient (S4, S3, S2 or a combination thereof) and the ratio between each ingredient. May indicate the degree. For example, when the total content of the three compounds is the same, a composition having a high ratio of S4 (sinensetin) having the highest CRE-dependent transcription activity exhibits a high CRE-dependent transcription activity as a whole.

  As used herein, a learning / memory disorder is a disorder that reduces the following three abilities in the memory process: memory acquisition / encoding, retention, and recall / recall ability. In this specification, the terms “acquisition” and “memorandum” are to capture information as a memory, and “hold” is to save or maintain the captured information, and “recollection” and “recollection”. Means recalling stored or maintained information.

  The composition of the present invention can be produced by further appropriately blending herbal ingredients and / or additive components into the compounds (S4, S3 and S2) according to the present invention. In the present specification, the composition of the present invention may be a therapeutic agent for a central nervous degenerative disease, and the therapeutic agent includes an anti-Alzheimer's disease agent and / or an anti-Parkinson's disease agent, ie, an anti-Alzheimer's disease activity and / or Examples include pharmaceuticals having anti-Parkinson disease activity.

  As used herein, the term “pharmaceutical” means a substance for diagnosis, treatment and / or prevention of diseases in humans and animals, and includes pharmaceuticals including herbal medicines, such as herbal medicines and traditional Chinese medicines. A preferred pharmaceutical is an oral dosage form, more preferably a fine granule, tea, decoction, capsule, tablet, granule, jelly, powder, liquid, syrup or extract.

  In this specification, the term “food” refers to so-called health foods and health functional foods such as foods for specified health use and functional nutritional foods that are distinguished by the presence or absence of permission from the authorities and the purpose and function of the food. Including. Preferred food forms herein are fine granules, jelly sauce, peels, jams or tea, more preferably fine granules or tea.

  The content of S4, S3 and S2 as active ingredients in the composition for improving and / or treating central nervous degenerative disease in the present invention (also referred to as “effective amount” in the present specification) depends on the intended use of the subject ( Pharmaceutical or food), sex, symptoms, etc., can be adjusted as appropriate, but usually about 0.001 to about 5% by weight with respect to 100% by weight of the composition, preferably about 0.01 to about It is in the range of 3% by weight, more preferably about 0.02 to about 1.2% by weight.

The herbal medicine ingredients in this specification include, for example, mausoleum, glaze, Yongxiao, jaundice, yellow gon, jaundice, ambition, cuckoo (ko), kudzu root, psoriasis, licorice, bellflower, chrysanthemum, coconut, Apricot kernel, cinnamon bark, safflower, katsuki, kobuk, ox knee, wushu, goji, shiba, yamako, ground yellow, glaze, ginger, ramie, shrine, plaster, river cucumber, mae, cucumber, Soki, Soyo, Daio, Daigo, Daigo, Daiboshi, Sawaso, Bamboo (Chikujojo), My mother, Satoshi Choto, Tenma, Tenmon Winter, Toki, Tomono, Ginseng, Malt, Mumon Winter, Semi-Summer , White peony, white cocoon, coconut, cocoon, proofed, glazed, wind peony, peony skin, mahuang, woody, woody incense, beneficial mother grass, ryokuri, Japanese cypress, and combinations of two or more thereof.
The compounds (S4, S3 and S2) according to the present invention may be blended in the composition of the present invention as herbal medicine components.

  The additive component in the present specification is not particularly limited as long as it is usually used in the art, and for example, ascorbic acid, aspartame, apple flavor, orange flavor, carrageenan, caramel, carnauba wax, carmellose, carmellose calcium, reduced maltose Liquid sugar, reduced maltose syrup, hydrous silicon dioxide, xylitol, citric acid, trisodium citrate, granulated sugar, light anhydrous silicic acid, gelling agent (FG-2266, Nitta Gelatin Co., Ltd.), synthetic aluminum silicate hydroxy Propyl starch / crystalline cellulose, honey beeswax, titanium oxide, salt, sucrose fatty acid ester, magnesium stearate, cellulose, talc, dextrin, corn starch, lactose, honey, hydroxypropyl methylcellulose, two fine granules Of silicon, pullulan, pectin, maltose, magnesium aluminometasilicate, methylcellulose, lecithin, and locust bean gum and combinations thereof.

  The additive component may be blended at a blending ratio that is usually used in the art, and is typically 0.0 to 70.0% by weight, preferably 5.0 to 50%, based on 100% by weight of the composition of the present invention. 0.0 wt%, more preferably 10.0 to 40.0 wt%.

  In the following examples, N Chinpi used as a starting material for extracting compounds S4, S3 and S2 is available from Kotaro Kampo Pharmaceutical Co., Ltd.

<Production Example 1> (Production of N Chinpi extract)
The starting material N Chinpi used in the present invention was produced as follows.
N chimpi was prepared by drying the skin of Citrus reticulata Branco (Rutaceae) containing nobiletin in a high proportion (0.44% by weight), sun-drying or heat drying. Heat drying was performed until the yield (ratio of mass after drying / mass before drying) reached 20 to 50% with respect to the skin, and heat-dried at a temperature of 60 ° C. for 2 hours. 400 mL of pure water was added to about 10 g of the N-thin pin obtained in this manner and heated. After the mixture boiled, it was extracted at 100 ° C. for 1 hour. Next, the mixture was filtered through gauze (two sheets), and the filtrate was lyophilized to obtain an N thimpy extract. The yield of N Chinpi extract was 3.5 g, and the yield was 35%.

<Example 1>
The N Chinpi extract obtained as in Production Example 1 was used in a dry weight of 81.11 g and separated according to the following procedure.
81.11 g of N Chinpi extract was suspended in 360 mL of 30% acetonitrile / 70% purified water in a flask and passed through Varian Bond Elut C18 (10 g). When 100 mL of 30% acetonitrile / 70% purified water was passed through the column and the eluate was concentrated under reduced pressure, Fr. 1 was obtained (79.95 g). Further, elution was performed with 50 mL of methanol. 2 was obtained (1.16 g). Fr. 2 (0.505 g) was subjected to ODS column chromatography using 40% acetonitrile / 60% purified water as an eluent, and further eluted with acetone / hexane (1: 1) and ethyl acetate / hexane (4: 1). The silica gel column chromatography was repeated, and the fractions containing nobiletin were combined and Fr. 2-1 was obtained (0.224 g). In addition, the fractions containing nobiletin were combined with Fr. 2-2 (0.314 g). FIG. 1 shows a separation operation such as column chromatography of N-thinpi extract-containing components.
In this way, Fr. 1 (79.95 g), and Fr. 2-1 (0.224 g) and Fr. 2-2 (0.314 g) was obtained. Fr. 2-1 was identified as nobiletin (Nb) as described in the analysis section below.
Fr. As for 2-2 (non-nobiletin fraction), as a result of analysis by thin layer chromatography, it was found that it contains a plurality of components. Therefore, separation by column chromatography was further performed according to the procedure shown in Example 2 below. Proceeded.

<Example 2> (Separation of Fr.2-2 by column chromatography)
Fr. 2-2 (200 mg) was subjected to ODS column chromatography and fractionated using 40% acetonitrile / 60% purified water as an elution solvent. Further, purification and recrystallization were performed by ODS column chromatography using 60% methanol / 40% purified water as an elution solvent to obtain S1 (20 mg), S2 (11 mg), S3 (12 mg) and S4 (14 mg).
S4, S3, S2 and S1, respectively, are identified as sinensetin (S4), demethoxytangeretin (S3), demethoxynobiletin (S2) and tangeretin (S1), as described in the analysis section below. It was.

  The results obtained in Examples 1 and 2 are shown in FIG.

<Example of analysis>
Each fraction component obtained in Examples 1 and 2 was analyzed by IR, UV, ¹ H-NMR, ¹³ C-NMR and ESI-MS to determine their structures. The obtained results are shown below.

<Fr. Analysis results of 2-1>
Fr. 2-1 was identified as nobiletin by TLC and HPLC comparison with the following data and commercial standards.
Colorless needles; UV: λ _max (MeOH) nm (log ε): 248 (4.23), 269 (4.19), 331 (4.35); Positive-ion ESI-MS m / z 403 ([ M + H] ⁺ ).

<Fr. Analysis Results of Compounds S1 to S4 Isolated from 2-2>
[S1]: Tangeretin (S1) was identified by the following data.
Colorless needles; mp 150-151 ° C .; IR _max (KBr) cm ⁻¹ : 1650, 1608, 1514, 1465, 1408, 1364, 1266, 1182, 1110, 1074, 1019, 970, 830; UV: λ _max (MeOH) nm (log [epsilon]): 323 (4.46), 270 (4.31); Positive-ion ESI-MS m / z 373 ([M + H] ^< +>).

[S2]: The following data identified 6-demethoxynobiletin (S2).
Colorless needles; mp 199-200 ° C .; IR λ _max (KBr) cm ⁻¹ : 1640, 1601, 1514, 1427, 1327, 1259, 1234, 1124, 1048, 1021, 843; UV λ _max (MeOH) nm (Log ε): 338 (4.31), 269 (4.29), 248 (4.27); ¹ H NMR (CDCl ₃ ): δ 7.57 (1H, dd, J = 10.7, 2.8 Hz) ), 7.40 (1H, d, J = 2.8 Hz), 6.97 (1H, d, J = 10.7 Hz), 6.60 (1H, s), 6.42 (1H, s), 3.99 (3H, s), 3.97 (3H, s), 3.95 (3H, s), 3.94 (3H × 2, s); ¹³ C NMR (CDCl ₃ ): δ 177.8 160.5, 156.4, 156.3, 151.9 151.7, 149.2, 130.7, 124.0, 119.5, 111.1, 108.9, 108.5, 107.1, 92.4, 61.5, 56.5, 56. 3, 56.0, 55.9; Positive-ion ESI-MS m / z 373 ([M + H] ⁺ ).

[S3] The following data identified 6-demethoxytangeretin (S3).
Mp 213-214 ° C .; IR λ _max (KBr) cm ⁻¹ : 1638, 1598, 1510, 1342, 1249, 1211, 1184, 1112, 1048, 841, 802; UV λ _max (MeOH) nm (Log ε); 310 (4.29), 269 (4.37); ¹ H NMR (CDCl ₃ ): δ 7.86 (2H, d, J = 11.3 Hz), 7.00 (2H, d, J = 11.3 Hz), 6.58 (1H, s), 6.41 (1H, s), 3.98 (3H, s), 3.96 (3H, s), 3.93 (3H, s) ), 3.86 (3H, s); ¹³ C NMR (CDCl ₃ ): δ 177.8, 162.1, 160.6, 156.4, 156.2, 151.9, 130.7, 127. 6, 123.8, 114.4, 109.0, 106.9, 92.5, 61.5, 56.6, 56.2, 55.4; Positive-ion ESI-MS m / z 343 ([M + H] ⁺ ).

[S4] Based on the following data, it was identified as sinensetin (S4).
Colorless needles; mp 175-176 ° C .; IR λ _max (KBr) cm ⁻¹ 1636, 1601, 1516, 1421, 1326, 1253, 1121, 1022, 843; UV λ _max (MeOH) nm (log ε): 329 (4.39), 239 (4.30); ¹ H NMR (CDCl ₃ ): δ 7.50 (1H, dd, J = 10.7, 2.5 Hz), 7.31 (1H, d, J = 2.5 Hz), 6.95 (1H, d, J = 10.7 Hz), 6.78 (1H, s), 6.59 (1H, s), 3.98 (3H, s), 3. 97 (3H, s), 3.96 (3H, s), 3.94 (3H, s), 3.90 (3H, s); ¹³ C NMR (CDCl ₃ ): δ 177.2, 161.1 157.7, 154.5, 152.6, 151.8, 149.3 140.4, 124.1, 119.6, 112.9, 111.1, 108.7, 107.4, 96.2, 62.2, 61.5, 56.3, 56.1, 56 0; Positive-ion ESI-MS m / z 373 ([M + H] ⁺ ).

<Pharmacological activity test example>
The following in vitro and in vivo tests were conducted on the compounds S1 to S4, nobiletin (Nb) and polymethoxyflavonoid fractions (Fr.2, Fr.2-1, Fr.2-2) obtained in the above examples. .

[General test procedure 1]
The general test procedure used in the following Test Examples 1 and 2 is as follows.
(Culture of rat fetal primary hippocampal neurons)
Pregnant Sprague-Dawley (SD) rats were bred by feeding and watering in a 12 hour light / dark cycle. The uterus was aseptically removed from the rat on day 18 of gestation (E18) by a midline abdominal incision under isoflurane deep anesthesia. Under a stereomicroscope, the fetal hippocampus was removed in ice-cold phosphate buffered saline (PBS), the tissue was dispersed with a nerve cell dispersion (Sumitomo Bakelite), and centrifuged at 1000 rpm for 4 minutes. Qing was removed. Next, the cell pellet is dispersed in a dispersion liquid (Sumitomo Bakelite), and then the removal liquid (Sumitomo Bakelite) is added to the cells sufficiently dispersed by pipetting, followed by centrifugation at 900 rpm for 5 minutes, and then the supernatant is removed. did.
Next, the pellet was suspended using Neurobasal Medium (Neurobasal Medium 500 mL / Phenol Red-free, 50-fold B27 Supplement 10 mL, 0.5 mM L-glutamine, 0.005% penicillin-streptomycin), and poly-L-lysine Seeded on a dish or plate coated with. After 1 day of culture, the medium was changed, and then half of the medium was changed every 3 to 4 days. The medium was cultured in a medium containing 10 μM Ara-C at 37 ° C. in a 5% CO ₂ incubator for 14 days.
In addition, Neurobasal Medium without Ara-C was used as a test medium for drug treatment experiments.

(Measurement of CRE-dependent transcriptional activity)
After primary culture of rat hippocampal neurons, reporter gene assay was performed. Hippocampal neurons were seeded in a 48-well plate at 8 × 10 ⁴ cells / well and cultured for 10-14 days in Neurobasal Medium. Reporter plasmid (0.1 μg / well) and Renilla phRG-TK plasmid (0.01 μg / well) were transfected by the lipofection method and cultured for 16 hours. Diluted with Neurobasal Medium without Ara-C (containing B-27 Supplement, L-glutamine, penicillin-streptomycin), and subjects were treated for 8 hours.
Transcriptional activity was measured using Promega's Dual-Luciferase (registered trademark) Reporter Assay System. Hippocampal neurons were solubilized with Passive Lysis Buffer (Promega), then Luciferase Assay Reagent II (Promega) and Stop & Glo (registered trademark) Reagent (Promega) were mixed, and the fluorescence value was measured with a luminometer.

(Statistical analysis 1)
The experimental results were evaluated using one-way ANOVA (Tukey). The significance level was tested at 5% on both sides, and p <0.05 was considered significant.

[Test Example 1] (CRE-dependent transcriptional activity of compounds (S4, S3, S2) and comparative compounds (Nb and S1) according to the present invention in hippocampal neurons (in vitro test))
In this test example, the CRE-dependent transcription activity of the compounds (S4, S3, S2) according to the present invention was compared with the CRE-dependent transcription activity of the comparative compounds (Nb and S1).

As described in Test Procedure 1 above, the compounds according to the present invention (S4, S3, S2) at a concentration of 30 μM and comparative compounds (Nb and S1) at the same concentration were treated.
The obtained results are shown in FIG. One-way ANOVA (post Tukey) test in all combinations in the figure. Values are mean ± standard error; number of specimens n = 4; * p <0.05, ** p <0.01, *** p <0.001 is significant difference from test (saline) (test) Results); §§ p <0.01 indicates a significant difference from Nb, respectively.
(Discussion 1)
As shown in FIG. 3, sinensetin (S4) exhibited CRE-dependent transcription activity that greatly exceeded the CRE-dependent transcription activity of nobiletin (Nb). In addition, 6-demethoxytangeretin (S3) and 6-demethoxynobiletin (S2) also showed equivalent CRE-dependent transcriptional activity compared to nobiletin (Nb) and tangeretin (S1).
These results indicate that synencetin (S4), 6-demethoxytangeretin (S3) and 6-demethoxynobiletin (S2) according to the present invention have an action of increasing memory formation function. It suggests that it can lead to treatment of Alzheimer's disease.

[Test Example 2] (Concentration dependence of CRE-dependent transcriptional activity of synencetin (S4) and comparative compound nobiletin (Nb) according to the present invention in hippocampal neurons (in vitro test))
In this test example, the concentration dependence of CRE-dependent transcriptional activity of the synencetin (S4) and the comparative compound nobiletin (Nb) according to the present invention was compared.
As described in Test Procedure 1 above, in order to confirm the concentration dependence of the drug efficacy of S4, S4 and a comparative compound Nb having a concentration of 1 to 30 μM were used.
The obtained results are shown in FIG. One-way ANOVA (post Tukey) test in all combinations in the figure. Values are mean ± standard error; number of specimens n = 4; ** p <0.01, *** p <0.001 indicates a significant difference (test result) from the control (saline).

(Discussion 2)
As shown in FIG. 4, synencetin (S4) was found to have a very strong CRE-dependent promoter activity. That is, nobiletin (Nb) shows maximum activity at 30 μM, but at a concentration of 1/30, that is, 1 μM, synencetin (S4) shows equivalent activity, and its maximum activity is 2 times that of nobiletin (Nb). It was twice as strong. Thus, the Cinencetin (S4) according to the present invention has a CRE-dependent transcriptional activity much stronger than that of the conventional nobiletin (Nb), surpassing nobiletin (Nb). Therefore, sinensetin (S4), which can exert an effect at a lower dose than nobiletin (Nb), is highly safe, exhibits a learning / memory impairment improving action that surpasses nobiletin (Nb), and more effectively recognizes Alzheimer's disease. It was suggested to improve dysfunction.

[Test Example 3] (CRE-dependent transcriptional activity of N-thinpi-derived fraction in hippocampal neurons (in vitro test))
In this test example, the CRE-dependent transcriptional activity of the N-thinpi-derived fraction according to the present invention was compared with the CRE-dependent transcriptional activity of the comparative compounds (Nb and S1).
As described in Test Procedure 1 above, the polymethoxyflavonoid fraction (Fr.2), nobiletin fraction (Fr.2-1), and non-nobiletin fraction (Fr.2-2) obtained in Example 1 were used. Fr. 2-1 and Fr. Mixed fraction reconstituted in 2-2 (Fr.2-1 + Fr.2-2) and reconstituted methoxyflavonoids (REC; sinensetin 0.222 μg / mL + 6-demethoxytangeretin 0.135 μg / mL + 6-demethoxy Nobiletin 0.177 mg / mL + tangeretin 0.585 mg / mL + nobiletin 1.89 μg / mL (5.0 μM)) was tested for CRE-dependent transcriptional activity.
The obtained results are shown in FIG. The sample concentration is Fr. 2.29 μg / mL for Fr. 2-1 for 1.89 μg / mL, Fr. 2.67 μg / mL for 2-2, Fr. 2-1 + Fr. The sample amount was 4.56 μg / mL for 2-2 and 3.009 μg / mL for REC, and the sample amount was compared to the fraction amount in N Chinpi. One-way ANOVA (post Tukey) test in all combinations in the figure. Values are mean ± standard error; number of specimens n = 4; ** p <0.01, *** p <0.001 indicates a significant difference (test result) from the control (saline).
Moreover, Fr. was determined based on 12 μg / mL corresponding to a nobiletin concentration of 30 μM. 2, Fr. 2-1 and Fr. The data with 2-2 as the sample concentration of 12 μg / mL is shown in FIG. One-way ANOVA (post Tukey) test in all combinations in the figure. Values are mean ± standard error; number of specimens n = 4; ** p <0.01, *** p <0.001 indicates a significant difference (test result) from the control (saline).

(Discussion 3)
As shown in FIG. 5, in the CRE-dependent transcriptional activity related to the conversion from short-term memory to long-term memory in hippocampal neurons, a reconstituted mixed fraction containing only 5 μM nobiletin (Fr.2-1 + Fr.2- In the sample of polymethoxyflavonoids (REC) reconstituted with 2), an activity stronger than that of the nobiletin fraction (Fr. 2-1) was confirmed. In addition, these two kinds of reconstituted samples have the same activity, and the addition of 6-demethoxytangeretin and 6-demethoxynobiletin to synencetin works to increase CRE-dependent transcriptional activity, such as dementia It shows excellent effects in the improvement and / or treatment of CNS degenerative diseases.
As shown in FIG. 6, Fr. which is a non-nobiletin fraction. Although 2-2 did not contain nobiletin, it was confirmed that the activity was higher than that of nobiletin at the same concentration, and the effects of sinensetin and the like shown in FIG. 3 were reconfirmed.
Further, considering the result obtained in Test Example 1, the CRE-dependent transcriptional activity of the reconstituted methoxyflavonoids (REC) means that 6-demethoxytangeretin (S3) and 6-demethoxy Nobiletin (S2) expresses CRE-dependent transcriptional activity of REC by synergistic action with synencetin (S4) and tangeretin (S1). It can also be said that this is a unique activity that contributes to the expression of the activity of 2.

[Test Example 4] (Sinencetin (S4) according to the present invention improves learning / memory impairment (in vivo test))
In the in vivo test of this test example, the effect of the synencetin (S4) of the present invention on behavioral pharmacological learning / memory impairment was examined using MK801-induced memory impairment model mice.
Specifically, the effect of chronic administration of sinensetin on MK801-induced learning / memory impairment was examined. Mice were divided into 3 groups, and sinensetin 25 mg / kg was orally administered to group 1 and physiological saline to the remaining 2 groups for 7 days. 90 minutes after administration of these on day 7, MK801 (80 μg / kg), which is one of glutamate receptor subtypes, was intraperitoneally administered to 2 groups and physiological saline was administered to the control group. Thirty minutes later, a fear conditioned learning (memory acquisition) test was conducted.
In the horror learning test, mice were placed in a transparent box and allowed to explore freely for 2 minutes before giving an electrical stimulus of 0.7 mA, 2 s (shock event). This was repeated three times, and learning trials were performed to evaluate memory acquisition and memorization ability (that is, a stopped state for 1 minute (Freezing%) after energization).
The obtained results are shown in FIG. In the figure, the vertical axis indicates freezing behavior (%), and the horizontal axis indicates shock events (number of times). “Control” means a group to which only physiological saline was administered, “MK801” means a group to which physiological saline and MK801 (80 μg / kg) were administered, and “S25 + MK801” represents sinensetin (25 mg / kg) And MK801 (80 μg / kg). One-way ANOVA (post Tukey) test for all combinations. *** p <0.001, ****** p <0.0001 is significant difference (test result) from control (saline), §§ p <0.01, §§§ p <0.001 Indicates a significant difference from “MK801”.

After 24 hours of learning (acquisition of memory), the mouse is placed in a transparent box again, and the confirmation (memory retention and recall) trial is performed using the mouse's freezing action, that is, all movements other than breathing as an index. As a learning / memory action, memory retention, recall ability and recall ability were evaluated by measurement for 5 minutes (that is, 5 minutes after energization (freezing%)).
The obtained result is shown in FIG. In the figure, the vertical axis represents freezing behavior (%). One-way ANOVA (post Tukey) test for all combinations. *** p <0.0001 indicates a significant difference (test result) from the control (saline), and §§§ p <0.001 indicates a significant difference from “MK801”.

(Discussion 4)
As shown in FIG. 7 and FIG. 8, the MK801 treatment significantly reduced the freezing behavior observed as a result of learning and memory, but the decrease was observed in both the learning trial and the confirmation trial by administration of sinensetin. Improvement effect was observed. In particular, in a learning trial, mice administered with the sinencetin of the present invention significantly reduced the decrease in freezing behavior, despite the fact that the learning / memory function was inhibited by MK801 treatment, making it easy to forget. It was done.

[Test Example 5] (Improvement of learning / memory impairment by Fr.2 (in vivo test))
In Test Example 4 above, Fr. 2 (92.8 mg / kg) was orally administered. The result of the learning trial obtained is shown in FIG. In the figure, *** p <0.001 indicates a significant difference (test result) from the control (saline), and ## p <0.01 indicates a significant difference from “MK801”.
Moreover, the result of the obtained confirmation trial is shown in FIG. In the figure, *** p <0.001 indicates a significant difference from the control (test result), and ## p <0.001 indicates a significant difference from “MK801”.

(Discussion 5)
As shown in FIG. 9, in the learning (memory acquisition) trial, Fr. 2 showed an effect of strongly improving the reduction of freezing behavior by treatment with MK801 to a state close to that of the control.
As shown in FIG. 10, it was confirmed that the memory failure by MK801 was significantly improved even in the confirmation (memory retention and recall) trial.

[Comparative Example 1] (Nobiletin improves learning and memory impairment (in vivo test))
In Test Example 4 above, the effect of intraperitoneal administration of nobiletin (50 mg / kg) was examined instead of sinensetin. The results are shown in FIG. 11 and FIG.

As shown in FIG. 11, in learning trials, the decrease in freezing behavior due to treatment with MK801 was not improved by intraperitoneal administration of nobiletin. As shown in FIG. 12, in the confirmation trial, the effect of nobiletin administration was recognized.
That is, it was confirmed that nobiletin (intraperitoneal administration) only has an effect of improving memory retention and recall disorder.

<Formulation example> (Production of the composition of the present invention)
The composition of the present invention was prepared as follows.
[Formulation Example 1] Fine granules A
N Chinpi 3.0 g, To-Go 3.0 g, Chotou Satoshi 3.0 g, River Kyu 3.0 g, Shirakaba 4.0 g, Kashiwa 4.0 g, Saiko 2.0 g, Licorice 1 Herbal medicines are blended so that the total amount becomes 200 to 800 kg at a ratio of 0.5 g and half-summer 5.0 g (total 28.5 g), and 60 to 8000 L of water using an extraction can. Extracted at -100 ° C for 30-180 minutes. The obtained extract is filtered at 1000 to 5000 rpm using a centrifuge and separated into solid and liquid, and then concentrated to about 10 to 40% under a reduced pressure of 8 kPa or less using a coil rotary type concentrator. Concentrated. The concentrate is spray-dried using a spray dryer at a rotation speed of 10,000 to 20,000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract (yokukansanka hanshin extract) It was.
A total amount of 50 g of an additive comprising 2.9 g of magnesium stearate, corn starch, lactose, pullulan and magnesium aluminate metasilicate was added to 6.1 g of the obtained Yokukansan Kasatsu Hanatsu extract. ~ 400 kg blended, mixed at 4 rpm for 20 minutes using a rotating container mixer, shaped using a dry granulator at a roll pressure of 490-2500 Pa and sized, sieve No. 30 ~ Particle classification (cassette screen) was performed between No. 50 to obtain fine granules A.
In 9.0 g of the obtained fine granule A, 6.1 g of Yokukansan Kachu Hanatsu extract containing 3.0 g of N Chinpi and other herbal medicines is contained. Take 9.0 g of Agent A in 2 to 3 divided doses per day for an adult.

[Formulation Example 2] Fine granule B
Tachibana bark 3.0 g, eggplant 4.0 g, Kobaku 3.0 g, cinnamon bark 3.0 g, soba 1.5 g, licorice 1.0 g, Daihuang 1.0 g, ginger 1.0 g, mixture of herbal medicines in a ratio of 1.0 g of wood incense, 1.0 g of koji koji, and 3.0 g of koji (total 22.5 g), so that the total amount is 200 to 800 kg, Extraction was performed at 8000 L using an extraction can at 60 to 100 ° C. for 30 to 180 minutes. After solid-liquid separation by filtration at a rotational speed of 1000 to 5000 rpm using a centrifuge, the solution was concentrated to a concentration of about 10 to 40% under a reduced pressure of 8 kPa or less using a coil rotary type concentrator. The concentrate was spray dried using a spray dryer at a rotation speed of 10,000 to 20,000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract (Kumisoto extract).
The total amount is 50 to 400 kg at a ratio of 2.3 g of additive consisting of magnesium stearate, corn starch, lactose, pullulan and magnesium aluminate metasilicate to 3.7 g of the obtained Kumi miso extract. , Mixed at 4 rpm for 20 minutes using a container rotary mixer, molded using a dry granulator at a roll pressure of 490-2500 Pa, and sized, between sieves 30-50 And particle classification (cassette screen) to obtain fine granule B.
In 6.0 g of the obtained fine granule B, 3.7 g of Kumi miso extract containing 3.0 g of Tachibana peel and other herbal medicines is contained. As a guide, fine granule B 6 Take 0.0 g divided into 2-3 times per day for adults.

[Formulation Example 3] Fine granule C
N Chinpi 2.4 g, Tsutomu Tsuji 2.4 g, Hanatsu 2.4 g, Mumon Winter 2.4 g, Tsuji 2.4 g, Carrot 1.6 g, Windproof 1.6 g, Chrysanthemum 1 .6 g, licorice 0.8 g, ginger 0.8 g and gypsum 4.0 g (total 22.4 g) in a ratio of 200 to 800 kg in total amount, Extraction was performed at 8000 L using an extraction can at 60 to 100 ° C. for 30 to 180 minutes. After solid-liquid separation by filtration at a rotational speed of 1000 to 5000 rpm using a centrifuge, the solution was concentrated to a concentration of about 10 to 40% under a reduced pressure of 8 kPa or less using a coil rotary type concentrator. The concentrate was spray dried using a spray dryer at a rotation speed of 10,000 to 20,000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract (Chitosan extract).
A container containing 1.54 g of additive consisting of hydrous silicon dioxide, magnesium stearate and corn starch in a total amount of 50 to 400 kg to 4.48 g of the obtained Chotosan extract. Mixing for 4 minutes at 4 rpm using a rotary mixer, molding using a dry granulator at a roll pressure of 490-2500 Pa and sizing, particle classification between sieves 30-50 (cassette screen And fine granule C was obtained.
In 6.0 g of the obtained fine granule C, 4.48 g of Chotosan extract containing 2.4 g of N chimpi and other herbal medicines is contained, and as a guide, the fine granule C 6. Take 0 g in three divided doses per day for adults.

[Formulation Example 4] Fine granules D
50 kg of N Chinpi or Tachibana bark was extracted with 500-2000 L of water using an extraction can at 60-100 ° C. for 30-180 minutes. After solid-liquid separation by filtration at 1000 to 2500 rpm using a centrifuge, the solution was concentrated to a concentration of about 10 to 40% under a reduced pressure of 8 kPa or less using a coil rotary type concentrator. The concentrate was spray dried using a spray dryer at a rotational speed of 10,000 to 20,000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract.
In a ratio of 1.8 g of additive consisting of fine silicon dioxide and sucrose fatty acid ester to 7.2 g of the obtained extract, the total amount is blended in the range of 50 to 400 kg, and the container rotating type mixer Is mixed at a rotational speed of 4 rpm for 20 minutes, molded using a dry granulator at a roll pressure of 490-2500 Pa and sized, and classified between sieves 30-42 (cassette screen). Fine granules D were obtained.
The resulting fine granule D 9.0 g contains 7.2 g of an extract (corresponding to N chimpi or tachibana skin 20 g). As a guide, fine granule D 9.0 g is used for adults. Take 2 to 3 times a day.

[Formulation Example 5] Tea preparation N Chinpi or Tachibana peel was cut into 850 to 4750 μm squares, and 3 to 7 g of the cut N Chinpi or Tachibana peel was placed in a paper or cloth bag to make a tea preparation.
The tea preparation obtained is infused and taken 3 times a day for adults (1 bag / time).

[Formulation Example 6] Herbal Powder-Fine Granule E
N chimpi or Tachibana bark is roughly crushed using a pulverizer at a rotational speed of 2000 to 3500 rpm and an opening of 0.5 to 3.0 mm, and further using a milling machine at a rotational speed of 5000 to 8000 rpm and an opening of 150 μm. The powder was crushed into a crude drug powder. The ratio of the 9.2 g of the additive consisting of hydrous silicon dioxide, magnesium stearate and corn starch to 10 g of the obtained crude drug powder is blended in the total amount of 50 to 400 kg, and the container rotating type mixer Is mixed at a rotational speed of 4 rpm for 20 minutes, molded using a dry granulator at a roll pressure of 490-2500 Pa and sized, and classified between sieves 30-42 (cassette screen). Fine granules E were obtained.
The resulting fine granule E 19.2 g contains 10 g of N chimpi or tachibana peel, and as a guideline, 19.2 to 38.4 g of fine granule E 2 to 3 times a day for adults Take in divided doses.

[Preparation Example 7] Decoction 10-30 g of N chimpi or tachibana peel was decocted with 200-600 mL of 20 times the amount of water at 100 ° C. for 1 hour to half the water, and filtered to obtain a decoction.
100 to 300 mL of the obtained decoction contains 10 to 30 g of N chimpi or tachibana peel, and 100 to 300 mL of the decoction is taken in three divided doses per day for adults.

[Formulation Example 8] Capsule N Chinpi or Tachibana skin 50 kg was extracted with water 500 to 2000 L using an extraction can at 60 to 100 ° C. for 30 to 180 minutes. After solid-liquid separation by filtration at 1000 to 2500 rpm using a centrifuge, the solution was concentrated to a concentration of about 10 to 40% under a reduced pressure of 8 kPa or less using a coil rotary type concentrator. The concentrate was spray dried using a spray dryer at a rotational speed of 10,000 to 20,000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract.
3. Additive consisting of magnesium aluminate metasilicate, synthetic aluminum silicate / hydroxypropyl starch / crystalline cellulose, corn starch, light anhydrous silicic acid, magnesium stearate and carmellose calcium to 10 g of the resulting extract. A total amount of 50 to 400 kg is blended at a ratio of 75 g, mixed for 20 minutes at a rotation speed of 4 rpm using a container rotating mixer, and a roll pressure of 490 to 2500 Pa using a dry granulator. And then sized, and classified between particles No. 30-42 (cassette screen) to obtain fine particles. 400 mg of this fine granule was filled into a commercially available capsule usually used in this field to obtain a capsule.
The obtained 13 capsules (470 to 480 mg / piece) contain 3.6 g of extract (corresponding to 10 g of N Chinpi or Tachibana peel), and 13 to 26 capsules as a guide. Taking 2 to 3 times a day for adults.

[Formulation Example 9] Coated tablet N Chinpi or Tachibana peel 50 kg was extracted with water 500 to 2000 L using an extraction can at 60 to 100 ° C. for 30 to 180 minutes. After solid-liquid separation by filtration at 1000 to 2500 rpm using a centrifuge, the solution was concentrated to a concentration of about 10 to 40% under a reduced pressure of 8 kPa or less using a coil rotary type concentrator. The concentrate was spray dried using a spray dryer at a rotational speed of 10,000 to 20,000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract.
The ratio of 9.1 g of an additive consisting of maltose, lactose, magnesium aluminate metasilicate and magnesium stearate to 10 g of the obtained extract was blended in a total amount of 50 to 400 kg, and the container was rotated. Using a mold mixer, mix for 20 minutes at a rotation speed of 4 rpm, and using a high-speed rotary tableting machine, beat at a rotation speed of 20 to 55 rpm, a primary compression of 0.6 mm or more, and a secondary compression of 0.4 mm or more. Tablets were obtained and 350 mg uncoated tablets were obtained. 1.1 g of a coating agent composed of hydroxypropylmethylcellulose, talc, titanium oxide and caramel is dissolved or suspended in a water / ethanol mixture (3: 7-10: 0), and the exhaust temperature is 40-55 ° C. using a coating machine. The uncoated tablets obtained at a flow rate of 200 to 400 mL / min and a rotation speed of 4 to 6 rpm are coated, and glazing is performed using a small amount of carnauba wax and white beeswax at a rotation speed of 1 rpm and an exhaust temperature of 20 to 30 ° C. One tablet was a 370 mg coated tablet.
The obtained 20 coated tablets contain 3.6 g of an extract (corresponding to 10 g of N chimpi or tachibana peel). As a guideline, 20 to 40 coated tablets are used in an amount of 2 to 3 per day for an adult. Take in divided doses.

[Formulation Example 10] Granules N Chinpi or Tachibana peel 50 kg was extracted with water 500 to 2000 L using an extraction can at 60 to 100 ° C. for 30 to 180 minutes. After solid-liquid separation by filtration at 1000 to 2500 rpm using a centrifuge, the solution was concentrated to a concentration of about 10 to 40% under a reduced pressure of 8 kPa or less using a coil rotary type concentrator. The concentrate was spray dried using a spray dryer at a rotational speed of 10,000 to 20,000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract.
In a ratio of 40 g of additive consisting of calcium carmellose, hydrous silicon dioxide, magnesium aluminate metasilicate and magnesium stearate to 10 g of the obtained extract, the total amount is blended in the range of 50 to 400 kg, Mixing for 20 minutes at a rotation speed of 4 rpm using a container rotating mixer, shaping using a dry granulator at a roll pressure of 490-2500 Pa and sizing, and particle classification between sieves 12-42 (Cassette screen) to obtain granules.
The obtained granule 18 g contains 3.6 g of an extract (corresponding to 10 g of N chimpi or tachibana peel). As a guideline, 18 to 36 g of granule is added 2 to 3 per day for an adult. Take in divided doses.

[Formulation Example 11] Jelly agent N Chinpi or Tachibana peel 50 kg was extracted with water 500 to 2000 L using an extraction can at 60 to 100 ° C. for 30 to 180 minutes. After solid-liquid separation by filtration at 1000 to 2500 rpm using a centrifuge, the solution was concentrated to a concentration of about 10 to 40% under a reduced pressure of 8 kPa or less using a coil rotary type concentrator. The concentrate was spray dried using a spray dryer at a rotational speed of 10,000 to 20,000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract.
In a ratio of 10 g to 30 g of additive obtained from carrageenan, locust bean gum, reduced maltose liquid sugar, xylitol, apple fragrance, lecithin and citric acid to 10 g of the obtained extract. The mixture was mixed and mixed at a rotation speed of 50 to 150 rpm using a mixing stirrer, and an equal amount of hot water of 80 ° C. or higher was added with stirring, and cooled to 15 ° C. or lower to obtain a jelly agent.
18 g of the obtained jelly agent contains 3.6 g of an extract (corresponding to 10 g of N Chinpi or Tachibana peel). As a guideline, 18 to 36 g of jelly agent is added to 2 to 3 per day for an adult. Take in divided doses.

  The composition for improving and / or treating a central neurodegenerative disease of the present invention is useful as a pharmaceutical or a food for improving a central neurodegenerative disease such as Alzheimer's disease and Parkinson's disease, and a promising fundamental therapeutic agent for the disease. It can also be used as a component. The composition for improving and / or treating a central neurodegenerative disease of the present invention is particularly useful for acquiring memory, maintaining memory and enhancing recall ability.

Claims (3)

Formula I-4:

A compound S4 represented by the formula:
A composition for improving and / or treating memory acquisition disorder or memory disorder, which does not contain nobiletin. An oral dosage form composition of claim 1. The composition according to claim 2 , wherein the oral dosage form is in the form of fine granules, tea, decoction, capsule, tablet, granule, jelly, powder, liquid, syrup or extract.

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