JP2017214329A - Pharmaceutical composition and food product - Google Patents
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- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 19
- 235000013305 food Nutrition 0.000 title claims abstract description 13
- 239000000284 extract Substances 0.000 claims abstract description 62
- 208000015114 central nervous system disease Diseases 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims description 46
- 229940079593 drug Drugs 0.000 claims description 37
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- 238000000605 extraction Methods 0.000 description 25
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- 241000196324 Embryophyta Species 0.000 description 3
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 3
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
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- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
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- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 240000003538 Chamaemelum nobile Species 0.000 description 1
- 235000007866 Chamaemelum nobile Nutrition 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010027915 Glutamate Receptors Proteins 0.000 description 1
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 235000007232 Matricaria chamomilla Nutrition 0.000 description 1
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- 208000018737 Parkinson disease Diseases 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
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- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000000371 effect on dementia Effects 0.000 description 1
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- 229930195712 glutamate Natural products 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
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- 239000011777 magnesium Substances 0.000 description 1
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- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Mycology (AREA)
- Botany (AREA)
- Psychiatry (AREA)
- Nutrition Science (AREA)
- Hospice & Palliative Care (AREA)
- Food Science & Technology (AREA)
- Psychology (AREA)
- Polymers & Plastics (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
Description
本発明は、医薬組成物および食品に関し、特に中枢神経疾患の予防及び治療に用いられるカミツレを含有する医薬組成物及び食品に関するものである。 The present invention relates to a pharmaceutical composition and a food, and more particularly to a pharmaceutical composition and a food containing chamomile used for the prevention and treatment of central nervous disease.
超高齢化社会に伴う認知症患者数の増加は、深刻な社会問題となっている。今後さらに高年齢者の数が増加することが予測されており、認知症を予防または治療する医薬品（医薬組成物）、食品に対する要求は増加すると考えられる。
記憶障害改善作用と作用機構を明らかにした植物由来成分として、例えば、柑橘類果皮由来のノビレチンが知られている（特許文献１）。
The increase in the number of patients with dementia associated with a super-aging society has become a serious social problem. It is predicted that the number of elderly people will increase further in the future, and the demand for pharmaceuticals (pharmaceutical compositions) and foods for preventing or treating dementia is expected to increase.
For example, nobiletin derived from citrus peel is known as a plant-derived component whose memory impairment improving action and action mechanism have been clarified (Patent Document 1).
ノビレチンは、アルツハイマー病モデルを含む様々な記憶障害モデル動物において、その記憶障害を改善する。その作用機序として、ｐｒoｔｅｉｎ ｋｉｎａｓe Ａ（ＰＫＡ）／（ＥＲＫ）／ｃＡＭＰ（ＣＲＥ）-ｂｉｎｄｉｎｇ ｐｒｔｅｉｎ（ＣＲＥＢ）細胞内シグナル経路を活性化し、ＣＲＥ依存性転写を亢進して記憶学習関連遺伝子の発現を亢進することが知られている（例えば、特許文献２）。 Nobiletin improves its memory impairment in various memory impairment model animals including the Alzheimer's disease model. As its mechanism of action, protein kinase A (PKA) / (ERK) / cAMP (CRE) -binding protein (CREB) activates intracellular signaling pathway, enhances CRE-dependent transcription and promotes expression of memory learning-related genes It is known to increase (eg, Patent Document 2).
一方、高血圧の改善作用や血中総コレステロール、中性脂肪（トリグリセリド）及び血糖値の上昇抑制作用を有する雲南百薬が着目されている（例えば、特許文献３）。
雲南百薬は、ツルムラサキ科のつる性多年草で、中国雲南省から日本に持ち込まれたとされており、日本の一部の地域では食されている。また、雲南百薬は、蔓状に伸びる茎には地上であっても多数のむかごができるが、葉もむかごも根塊も利用でき、独特のぬるぬる成分が特徴であり、マグネシウムや、銅、カルシウム及び亜鉛の含有量が多い植物として知られているが、その研究、検討はされていない。
On the other hand, Yunnan hundred drugs that have an action to improve hypertension, blood total cholesterol, neutral fat (triglyceride), and blood glucose level increase are attracting attention (for example, Patent Document 3).
Yunnan Hyakuyaku is a vine perennial plant belonging to the family Crassaceae, and is said to have been brought into Japan from Yunnan Province, China, and is eaten in some parts of Japan. In addition, Yunnan Hyakuyaku has a lot of baskets on its vine-like stems, even on the ground, but it can also use leaves, baskets and root nodules, and is characterized by its unique slimy ingredients, magnesium, copper, calcium Although it is known as a plant with a high zinc content, its research and examination have not been conducted.
現在までに、認知症、特にその半数を占めるアルツハイマー病に対する予防や進行抑制効果を持つ決定的な臨床薬は存在していない。そのため、アルツハイマー病に対する予防や進行抑制効果を持つ医薬組成物や食品が求められている。 To date, there is no definitive clinical drug that has a preventive or suppressive effect on dementia, particularly Alzheimer's disease, which accounts for half of it. Therefore, there is a need for pharmaceutical compositions and foods that have an effect of preventing Alzheimer's disease and suppressing progression.
そこで、本発明は、中枢神経疾患の予防及び進行を抑制する効果をもつ医薬組成物及び食品を提供することを目的とする。 Then, an object of this invention is to provide the pharmaceutical composition and foodstuff which have the effect which suppresses prevention and progression of central nervous disease.
本発明の医薬組成物は、中枢神経疾患の予防及び治療に用いられる医薬組成物であって、雲南百薬の乾燥物または抽出液を含有することを特徴とする。 The pharmaceutical composition of the present invention is a pharmaceutical composition used for the prevention and treatment of central nervous system diseases, and is characterized by containing a dried product or extract of Yunnan hundred drugs.
本発明の医薬組成物は、雲南百薬の抽出液が、熱水、アルコール類の群から選択される１種の抽出液であることを特徴とする。 The pharmaceutical composition of the present invention is characterized in that the Yunnan hundred medicine extract is one kind of extract selected from the group of hot water and alcohols.
また、本発明の食品は、中枢神経疾患の予防及び治療に用いられる食品であって、雲南百薬の乾燥物または抽出液を含有することを特徴とする。 In addition, the food of the present invention is a food used for the prevention and treatment of central nervous system diseases, and contains a dried product or extract of Yunnan hundred medicines.
本発明の医薬組成物は、雲南百薬の抽出液を含有することで、ＰＣ１２細胞のルシフェラーゼ活性を向上させ、ＣＲＥ依存性転写活性を促進し、記憶学習関連遺伝子の発現を亢進するという効果を生じる。そのため、中枢神経疾患の予防及び進行を抑制することができ、中枢神経疾患の予防及び治療に用いることができる。 The pharmaceutical composition of the present invention contains an extract of Yunnan Hundred Drugs, thereby improving the luciferase activity of PC12 cells, promoting CRE-dependent transcriptional activity, and enhancing the expression of memory learning related genes. . Therefore, the prevention and progression of central nervous system diseases can be suppressed, and it can be used for the prevention and treatment of central nervous system diseases.
また、本発明の医薬組成物において、雲南百薬を熱水、アルコール類を使用して抽出液を得ることができる。適切な抽出溶媒を選択することで、ルシフェラーゼ活性を向上させることができる。
In addition, in the pharmaceutical composition of the present invention, an extract can be obtained using Yunnan hundred medicines using hot water and alcohols. By selecting an appropriate extraction solvent, luciferase activity can be improved.
さらの本発明では、雲南百薬の乾燥物または抽出液を含有し、中枢神経疾患の予防及び治療に用いられる食品をも提供できる。 Furthermore, the present invention can also provide a food containing a dried product or extract of Yunnan Hundred Drugs and used for the prevention and treatment of central nervous system diseases.
本実施形態に係る医薬組成物は、雲南百薬の乾燥物または抽出物を含有し、中枢神経疾患の予防及び治療に用いられるものである。この医薬組成物は、特に、アルツハイマー病やパーキンソン病の予防及び治療に用いることができる。 The pharmaceutical composition which concerns on this embodiment contains the dried product or extract of Yunnan hundred medicine, and is used for prevention and treatment of a central nervous disease. This pharmaceutical composition can be used particularly for the prevention and treatment of Alzheimer's disease and Parkinson's disease.
［試料調整］
雲南百薬は、静岡県富士市産のアカサカズラ（ツルムラサキ科植物）を使用し、メタノールで抽出した。抽出した雲南百薬粉末は、濃度が１００μｇ／ｍＬ（試料１）、５００μｇ／ｍＬ（試料２）、１０００μｇ／ｍＬ（試料３）となるようにジメチルスルホキシド（Ｄｉｍｅｔｈｙｌ ｓｕｌｆｏｘｉｄｅ）（以下、ＤＭＳＯと記す。）に溶解させた。
雲南百薬は、中国語学名：藜蔓（落葵薯）、ラテン名：Ａｎｒｅｄｅｒａ ｃоｒｄｉｆоｌｉａ (Ｔｅｎоｒｅ) Ｓｔｅｅｎｉｓ、別名：藤三七（ｔｅｎｇ ｓａｎ ｑｉ）、ラテン名：Ｂоｕｓｓｉｎｇａｕｌｔｉａ ｇｒａｃｉｌｉｓ Ｍｉｅｒｓｖａｒ．ｐｓｅｕｄоｂａｓｅｌｌоｉｄｅｓ Ｂａｉｌｅｙ）である。
ノビレチンは、柑橘系植物から抽出したものを使用した（純度９９％）。ノビレチンは、濃度が２μｇ／ｍＬ（５μＭ）、６μｇ／ｍＬ（１５μＭ）、１２μｇ／ｍＬ（３０μＭ）となるようにＤＭＳＯに溶解させた。
[Sample preparation]
Yunnan Hyakuyaku was extracted with methanol using red crested vines (Flysaceae) from Fuji City, Shizuoka Prefecture. The extracted Yunnan hundred drug powder has a concentration of 100 μg / mL (Sample 1), 500 μg / mL (Sample 2), and 1000 μg / mL (Sample 3) so that dimethyl sulfoxide (hereinafter referred to as DMSO) is obtained. Dissolved in.
Yunnan Hyakuyaku is a Chinese scientific name: 藜 vine (Otaku), Latin name: Anredera certifolia (Tenоre) Steenis, also known as: teng san qi, Latin name: Biususingultia gracilis Miersvar. pseudobasileides Bailey).
Nobiletin used was extracted from a citrus plant (purity 99%). Nobiletin was dissolved in DMSO to a concentration of 2 μg / mL (5 μM), 6 μg / mL (15 μM), and 12 μg / mL (30 μM).
試料１〜試料３の遺伝子転写活性を評価するためにルシフェラーゼレポーターアッセイを行った。
ルシフェラーゼアッセイは、被検物質の有する転写活性を定量的に解析するために使用されるものである。転写が活性化するためには、遺伝子の上流プロモーターに存在する様々な制御配列が必要である。
本実施例では、ＰＫＡ／ＥＲＫ／ＣＲＥＢ細胞内シグナルによって活性化される制御配列であり、かつ記憶学習関連遺伝子の上流プロモーターに存在するＣＲＥ配列と、発光酵素であるルシフェラーゼの遺伝子を連結したプラスミドを使用した。
被験物質がＣＲＥ依存性の転写を活性化した場合、その下流にあるルシフェラーゼ遺伝子が発現し、上昇してルシフェラーゼタンパク質が細胞内で増加する。そうすると、基質を添加することで蛍光を発するため、その吸光度を測定し、転写活性を定量できる。
ＰＣ１２細胞（ラット副腎髄質褐色細胞腫由来細胞株）を９６穴培養ディッシュに播種濃度４．０×１０４個／穴で播種した。その後、プラスミドをＰＣ１２細胞内に導入し、各試料を滴下した培地で培養した。培地には、１％血清を添加したα改変ダルベッコ変法イーグル培地（ＤＭＥＭ）を使用した。
ＰＣ１２細胞を所定時間培養した後、試料をそれぞれ１００μＬ加えた培地に交換し、培養ディッシュを３７℃、５％ＣＯ２雰囲気下で、５時間インキュベートした。その後、反応を停止させ、ルシフェラーゼレポーター活性を測定した。
なお、雲南百薬の培地に対する最終濃度は、以下の通りである。
試料１：１０μｇ／ｍＬ
試料２：５０μｇ／ｍＬ
試料３：１００μｇ／ｍＬ
In order to evaluate the gene transcription activity of Sample 1 to Sample 3, a luciferase reporter assay was performed.
The luciferase assay is used for quantitatively analyzing the transcriptional activity of a test substance. In order for transcription to be activated, various regulatory sequences present in the upstream promoter of the gene are required.
In this example, a plasmid in which a CRE sequence that is a control sequence activated by a PKA / ERK / CREB intracellular signal and is present in the upstream promoter of a memory learning-related gene and a gene for luciferase, which is a luminescent enzyme, is linked. used.
When a test substance activates CRE-dependent transcription, a luciferase gene downstream thereof is expressed and increased to increase luciferase protein in the cell. Then, since fluorescence is emitted by adding a substrate, the absorbance can be measured and the transcriptional activity can be quantified.
PC12 cells (rat adrenal medullary pheochromocytoma-derived cell line) were seeded in a 96-well culture dish at a seeding concentration of 4.0 × 10 4 cells / well. Thereafter, the plasmid was introduced into PC12 cells and cultured in a medium in which each sample was dropped. As the medium, α-modified Dulbecco's modified Eagle medium (DMEM) supplemented with 1% serum was used.
After culturing PC12 cells for a predetermined time, the medium was replaced with a medium containing 100 μL of each sample, and the culture dish was incubated at 37 ° C. in a 5% CO 2 atmosphere for 5 hours. Thereafter, the reaction was stopped and luciferase reporter activity was measured.
In addition, the final concentration with respect to the culture medium of Yunnan hundred medicine is as follows.
Sample 1: 10 μg / mL
Sample 2: 50 μg / mL
Sample 3: 100 μg / mL
図１は、各試料を添加した場合のルシフェラーゼレポーターアッセイの結果を示している。
ノビレチンは、濃度が２μｇ／ｍＬがルシフェラーゼ活性を発現する濃度であることが知られている。試料１〜試料３を添加した場合、いずれもノビレチン２μｇ／ｍＬを添加した場合よりもルシフェラーゼ活性が高いことが分かった。このことから、雲南百薬のメタノール抽出液には、ＰＣ１２細胞におけるルシフェラーゼを活性させる物質が含まれていることが分かった。
また、添加した雲南百薬の濃度が高くなるにつれ、ルシフェラーゼ活性が高くなることが分かった。試料２または試料３を添加した場合では、ノビレチン１２μｇ／ｍＬを添加した場合よりも顕著に高いルシフェラーゼ活性を示した。中でも、試料３を添加した場合では、ノビレチン１２μｇ／ｍＬを添加した場合の２倍のルシフェラーゼ活性を示した。
FIG. 1 shows the results of the luciferase reporter assay when each sample was added.
It is known that nobiletin is a concentration at which a concentration of 2 μg / mL expresses luciferase activity. It was found that when samples 1 to 3 were added, all had higher luciferase activity than when nobiletin 2 μg / mL was added. From this, it was found that the methanol extract of Yunnan Hyakuyaku contained a substance that activates luciferase in PC12 cells.
It was also found that luciferase activity increased as the concentration of added Yunnan hundred drugs increased. When sample 2 or sample 3 was added, the luciferase activity was significantly higher than when nobiletin 12 μg / mL was added. In particular, when sample 3 was added, luciferase activity was doubled when nobiletin 12 μg / mL was added.
次に、雲南百薬を純水（熱水）、アルカリ還元水（熱水）、９９％エタノール、５０％エタノールで抽出した抽出液を添加した場合のルシフェラーゼ活性を調べた。
試料１群：純水の熱水抽出液
（抽出液に対する雲南百薬濃度：１５０〜５０００μｇ／ｍＬ）
（培地に対する雲南百薬濃度：１５〜５００μｇ／ｍＬ）
試料２群：アルカリ還元水の熱水抽出液
（抽出液に対する雲南百薬濃度：１５０〜５０００μｇ／ｍＬ）
（培地に対する雲南百薬濃度：１５〜５００μｇ／ｍＬ）
試料３群：９９％エタノール抽出液
（抽出液に対する雲南百薬濃度：１５０〜５０００μｇ／ｍＬ）
（培地に対する雲南百薬濃度：１５〜５００μｇ／ｍＬ）
試料４群：５０％エタノール抽出液
（抽出液に対する雲南百薬濃度：１５０〜５０００μｇ／ｍＬ）
（培地に対する雲南百薬濃度：１５〜５００μｇ／ｍＬ）
Next, luciferase activity was examined when an extract obtained by extracting Yunnan hundred medicines with pure water (hot water), alkali-reduced water (hot water), 99% ethanol, and 50% ethanol was added.
Sample 1 group: hot water extract of pure water (concentration of Yunnan hundred drugs with respect to the extract: 150-5000 μg / mL)
(Yunnan hundred drug concentration to medium: 15-500 μg / mL)
Sample 2 group: hot water extract of alkali reduced water (Yunnan hundred drug concentration with respect to the extract: 150-5000 μg / mL)
(Yunnan hundred drug concentration to medium: 15-500 μg / mL)
Sample 3 group: 99% ethanol extract (Yunnan hundred drug concentration with respect to extract: 150-5000 μg / mL)
(Yunnan hundred drug concentration to medium: 15-500 μg / mL)
Sample 4 group: 50% ethanol extract (concentration of Yunnan hundred drugs with respect to the extract: 150 to 5000 μg / mL)
(Yunnan hundred drug concentration to medium: 15-500 μg / mL)
図２は、１５０、５００、２０００、および５０００μｇ／ｍＬの雲南百薬の抽出液を添加した場合のルシフェラーゼレポーターアッセイの結果を示している。
純水抽出液またはアルカリ還元水抽出液を添加した場合を比較すると、いずれの雲南百薬の濃度においてもアルカリ還元水抽出液を添加した場合の方が、ルシフェラーゼ活性が高いことが分かった。このことから、同じ水であってもｐＨが高い水の抽出液にＰＣ１２細胞のルシフェラーゼ活性を向上させる物質が多く含有されていることが分かった。
また、濃度の異なるエタノール抽出液（９９％、５０％）を比較した場合、いずれの雲南百薬の濃度においても、９９％エタノールで抽出した抽出液を添加した場合の方が、ルシフェラーゼ活性が高いことが分かった。この結果から、エタノールで抽出した場合、高い濃度のエタノールで抽出した抽出液にＰＣ１２細胞のルシフェラーゼ活性を向上させる物質が多く含有されていることが分かった。
また、いずれの試料を添加した場合でも、ノビレチン１２μｇ／ｍＬを添加した場合よりも、ルシフェラーゼ活性が高いことが明らかとなり、雲南百薬のメタノール抽出液は、ルシフェラーゼ活性を向上させる効果を有することが分かった。
FIG. 2 shows the results of a luciferase reporter assay with the addition of 150, 500, 2000, and 5000 μg / mL Yunnan hundred drug extracts.
Comparing the case where the pure water extract or the alkaline reduced water extract was added, it was found that the luciferase activity was higher when the alkaline reduced water extract was added at any of the Yunnan hundred drug concentrations. From this, it was found that many substances that improve the luciferase activity of PC12 cells are contained in the water extract having a high pH even in the same water.
In addition, when ethanol extracts with different concentrations (99%, 50%) are compared, luciferase activity is higher when the extract extracted with 99% ethanol is added at any Yunnan hundred drug concentration. I understood. From this result, it was found that when extracted with ethanol, the extract extracted with a high concentration of ethanol contains many substances that improve the luciferase activity of PC12 cells.
Moreover, it is clear that any sample added has higher luciferase activity than when nobiletin 12 μg / mL was added, and the methanol extract of Yunnan Hyakuyaku has an effect of improving luciferase activity. It was.
雲南百薬の容量依存性を調べた結果、どの抽出液においても、雲南百薬の濃度が高い方が、ルシフェラーゼ活性が高いことが分かった。また、ブタノール抽出液、熱水抽出液、酢酸エチル抽出液の順にルシフェラーゼ活性を向上させることが分かった。
この結果より、ルシフェラーゼ活性を向上させる雲南百薬の有効成分は、ブタノール層に多く含まれ、次いで水層、酢酸エチル層に含まれていることが分かった。いずれの層に含まれる有効成分は同じ物質であると考えられ、これらの成分は熱水層に溶解していることから水溶性の物質であると考えられる。ここで、ノビレチンは水溶性ではないことが知られている。そのため、雲南百薬に含まれている有効成分は、ノビレチンとは異なる物質であると考えられる。
As a result of examining the volume dependency of Yunnan hundred drugs, it was found that the higher the concentration of Yunnan hundred drugs, the higher the luciferase activity in any of the extracts. Moreover, it turned out that a luciferase activity is improved in order of a butanol extract, a hot-water extract, and an ethyl acetate extract.
From this result, it was found that the active ingredient of Yunnan hundred drugs that improve luciferase activity is contained in a large amount in the butanol layer, and then in the aqueous layer and the ethyl acetate layer. The active ingredients contained in any layer are considered to be the same substance, and these ingredients are considered to be water-soluble substances because they are dissolved in the hot water layer. Here, it is known that nobiletin is not water-soluble. Therefore, the active ingredient contained in Yunnan hundred drugs is considered to be a substance different from nobiletin.
次に、ＰＤ９８０５９による阻害効果の検討を行った。
遺伝子の転写活性についての阻害剤であるＰＤ９８０５９を添加することで、遺伝子の転写活性を阻害させる。この遺伝子の転写活性剤が添加されて阻害活性が抑制されるということは、遺伝子の転写活性を維持または増加させることでの遺伝子転写活性を増強させる可能性が示唆される。脳機能において、遺伝子転写活性が活性化させる物質の投与によって、脳機能が維持されると考えられている。
本実験では、ブタノール抽出液、熱水抽出液、酢酸エチル抽出液をそれぞれ添加した培地における、ＰＤ９８０５９による遺伝子の転写活性阻害効果の検討を行った。ＰＤ９８０５９は、５０μＭ、１００μＭに調整し、遺伝子の転写活性阻害効果試験に使用した。
Next, the inhibitory effect by PD98059 was examined.
By adding PD98059, which is an inhibitor of gene transcription activity, gene transcription activity is inhibited. The suppression of the inhibitory activity by the addition of this gene transcription activator suggests the possibility of enhancing the gene transcription activity by maintaining or increasing the gene transcription activity. In brain function, it is thought that brain function is maintained by administration of a substance that activates gene transcription activity.
In this experiment, the effect of inhibiting gene transcription activity by PD98059 was examined in media supplemented with a butanol extract, a hot water extract, and an ethyl acetate extract, respectively. PD98059 was adjusted to 50 μM and 100 μM and used for the gene transcription activity inhibition effect test.
ＰＤ９８０５９による阻害実験の結果（図３）から、酢酸エチル抽出液（５０００μｇ／ｍＬ）、ブタノール抽出液（５０００μｇ／ｍＬ）、熱水抽出液（５０００μｇ／ｍＬ）をそれぞれ添加した培地に、ＰＤ９８０５９（５０μＭ）を添加すると、顕著に遺伝子の転写活性の阻害が生じることが分かった。また、ＰＤ９８０５９（１００μＭ）を添加した場合、さらに遺伝子の転写活性を阻害することが分かった。
阻害剤ＰＤ９８０５９を添加することにより、雲南百薬による遺伝子転写活性が阻害されるため、雲南百薬の抽出液の記憶遺伝子転写メカニズムは、ＣＲＥＢの関与したＣＲＥ依存性の記憶遺伝子転写活性作用によるものであると考えられる。これは、ノビレチンと同様な作用によるものである。
以上の結果から、雲南百薬の抽出液は、ＣＲＥ依存性転写を促進し、記憶学習関連遺伝子の発現を亢進するという効果を有することが分かった。
From the result of the inhibition experiment with PD98059 (FIG. 3), PD98059 (50 μM) was added to each medium supplemented with ethyl acetate extract (5000 μg / mL), butanol extract (5000 μg / mL), and hot water extract (5000 μg / mL). ) Was found to significantly inhibit the transcriptional activity of the gene. Moreover, when PD98059 (100 micromol) was added, it turned out that the transcriptional activity of a gene is inhibited further.
The addition of the inhibitor PD98059 inhibits the gene transcription activity of Yunnan hundred drugs, so the memory gene transcription mechanism of the extract of Yunnan hundred drugs is due to the CRE-dependent memory gene transcriptional activity involved in CREB. it is conceivable that. This is due to the same action as nobiletin.
From the above results, it was found that the extract of Yunnan hundred drugs has the effect of promoting CRE-dependent transcription and enhancing the expression of memory learning related genes.
次に、雲南百薬のブタノール抽出液をさらにフラクション１Ａ〜１Ｋに分画し、質量換算で１００ｍｇ／ｍＬとなるようにＤＭＳＯに溶解させ、その溶液の転写活性をルシフェラーゼアッセイにより測定した。 Next, the butanol extract of Yunnan Hyakuyaku was further fractionated into fractions 1A to 1K, dissolved in DMSO to a mass conversion of 100 mg / mL, and the transcription activity of the solution was measured by luciferase assay.
図４は、分画した各フラクションを添加した場合のルシフェラーゼレポーターアッセイの結果を示している。
フラクション１Ｇが、最も高いルシフェラーゼ活性を有することが分かった。次に、フラクション１Ｈが高く、フラクション１Ｆ、フラクション１Ｄ、フラクション１Ｉの順でルシフェラーゼ活性が高いことが分かった。
また、生薬換算の濃度に注目すると、フラクション１Ｈが、少ない生薬量で高いルシフェラーゼ活性を有することが分かる。
フラクション１Ｄには、フラクション１Ｆ、フラクション１Ｇ、フラクション１Ｈ、フラクション１Ｉとは別の物質が含有されている可能性があると考えられる。
これらのフラクションには、ルシフェラーゼ活性を向上させる成分が含まれていると考えられ、各分画同士がルシフェラーゼ活性を向上させる、増強作用を検討する必要がある。
FIG. 4 shows the results of the luciferase reporter assay when each fractionated fraction was added.
Fraction 1G was found to have the highest luciferase activity. Next, it was found that fraction 1H was high, and luciferase activity was high in the order of fraction 1F, fraction 1D, and fraction 1I.
Further, when attention is paid to the concentration in terms of herbal medicine, it can be seen that fraction 1H has high luciferase activity with a small amount of herbal medicine.
It is considered that the fraction 1D may contain a substance different from the fraction 1F, the fraction 1G, the fraction 1H, and the fraction 1I.
These fractions are considered to contain components that improve luciferase activity, and it is necessary to examine the enhancing action of each fraction to improve luciferase activity.
次に、少ない生薬量で高いルシフェラーゼ活性を有するフラクション１Ｈをさらに分画した。フラクション１Ｈをフラクション２Ａ〜２Ｉに分画し、質量換算で１００μｇ／ｍＬとなるようにＤＭＳＯに溶解させ、上記と同様な方法で、ルシフェラーゼ活性を測定した。 Next, fraction 1H having high luciferase activity with a small amount of crude drug was further fractionated. Fraction 1H was fractionated into fractions 2A to 2I, dissolved in DMSO to a mass conversion of 100 μg / mL, and luciferase activity was measured by the same method as described above.
図５は、各フラクションを添加した場合のルシフェラーゼレポーターアッセイの結果を示している。
フラクション２Ｅを添加した場合が、最も高いルシフェラーゼ活性を示すことが分かった。フラクション２Ｂは、フラクション１Ｈと同等のルシフェラーゼ活性を示した。次に、フラクション２Ｆおよびフラクション２Ｂを添加した場合に、高い活性を示した。
高い活性を示したフラクション２Ｆ及びフラクション２Ｅの成分を薄層クロマトグラフィー（ＴＬＣ）で確認した。ＴＬＣ画像より、ほぼ同じ成分が含まれていることが分かった。このことから、フラクション２Ｆとフラクション２Ｅは、同じ成分がルシフェラーゼ活性を向上させていると考えられる。また、フラクション１Ｈへの含有量は少量であるが、フラクション２Ｂにも、ルシフェラーゼ活性を向上させる成分が含有されていることが分かった。
FIG. 5 shows the results of the luciferase reporter assay when each fraction was added.
It was found that the highest luciferase activity was exhibited when fraction 2E was added. Fraction 2B showed luciferase activity equivalent to that of fraction 1H. Next, when fraction 2F and fraction 2B were added, high activity was shown.
Components of Fraction 2F and Fraction 2E showing high activity were confirmed by thin layer chromatography (TLC). From the TLC image, it was found that almost the same components were included. From this, it is considered that the same components of fraction 2F and fraction 2E improve the luciferase activity. Moreover, although the content to fraction 1H is a small amount, it turned out that the component which improves a luciferase activity is contained also in the fraction 2B.
次に、亜臨界抽出によって、雲南百薬の成分を抽出した。抽出後、雲南百薬の残渣物をメタノールで抽出した。亜臨界抽出は、１２０℃〜１８０℃の温度範囲で行った。各抽出条件は以下の通りである。
抽出条件１：１２０℃、（飽和水蒸気圧：１９８６．６ ｈＰａ）
抽出条件２：１４０℃、（飽和水蒸気圧：３６１６．６ ｈＰａ）
抽出条件３：１５０℃、（飽和水蒸気圧：４７６４．０ ｈＰａ）
抽出条件４：１６０℃、（飽和水蒸気圧：６１８６．６ ｈＰａ）
抽出条件５：１８０℃、（飽和水蒸気圧：１００３７．０ ｈＰａ）
Next, components of Yunnan Hyakuyaku were extracted by subcritical extraction. After extraction, the residue of Yunnan Hyakuyaku was extracted with methanol. Subcritical extraction was performed in the temperature range of 120 ° C to 180 ° C. Each extraction condition is as follows.
Extraction condition 1: 120 ° C. (saturated water vapor pressure: 1986.6 hPa)
Extraction condition 2: 140 ° C. (saturated water vapor pressure: 3616.6 hPa)
Extraction condition 3: 150 ° C. (saturated water vapor pressure: 4764.0 hPa)
Extraction condition 4: 160 ° C. (saturated water vapor pressure: 6186.6 hPa)
Extraction condition 5: 180 ° C. (saturated water vapor pressure: 1003.0 hPa)
そして、各抽出液から得られた抽出物をメタノールで抽出し、抽出液に対する雲南百薬の濃度が５００μｇ／ｍＬ、１０００μｇ／ｍＬの試料を調整し、ルシフェラーゼ活性を測定した。 And the extract obtained from each extract was extracted with methanol, the sample whose density | concentration of Yunnan hundred medicine with respect to an extract is 500 microgram / mL and 1000 microgram / mL was adjusted, and the luciferase activity was measured.
図６は、各亜臨界抽出した抽出液を添加した場合のルシフェラーゼ測定結果を示している。
抽出条件１〜抽出条件５で抽出した抽出液を添加した場合、ノビレチン溶液を添加したものよりもルシフェラーゼ活性を向上させることが分かった。中でも、抽出条件２および抽出条件３によって抽出した抽出液を添加すると、高いルシフェラーゼ活性を示すことが分かった。
残渣物の抽出液を添加した場合では、ルシフェラーゼ活性を示すものの、亜臨界抽出した抽出液を添加したものと比較すると、ルシフェラーゼ活性は低いことが分かった。このことから、ルシフェラーゼ活性を向上させる有効成分は、亜臨界抽出した抽出液に多く含まれていることが分かった。
以上のことから、雲南百薬を亜臨界抽出した抽出液（抽出物）には、ＰＣ１２細胞のルシフェラーゼ活性を向上させる成分（物質）が含有されていることが明らかとなった。
FIG. 6 shows the luciferase measurement results when each subcritically extracted extract was added.
It was found that when the extract extracted under extraction condition 1 to extraction condition 5 was added, the luciferase activity was improved as compared with the addition of nobiletin solution. In particular, it was found that when an extract extracted under extraction conditions 2 and 3 was added, high luciferase activity was exhibited.
When the residue extract was added, it showed luciferase activity, but it was found that the luciferase activity was low compared to the addition of the subcritically extracted extract. From this, it was found that the active ingredient that improves luciferase activity is contained in a large amount in the subcritically extracted extract.
From the above, it has been clarified that the extract (extract) obtained by subcritical extraction of Yunnan hundred drugs contains a component (substance) that improves the luciferase activity of PC12 cells.
次に、雲南百薬の亜臨界メタノール抽出液を添加したときのＰＣ１２細胞の形態を位相差顕微鏡により観察した。その位相差顕微鏡像を図７及び図８に示す。 Next, the morphology of PC12 cells when a subcritical methanol extract of Yunnan hundred drugs was added was observed with a phase contrast microscope. The phase-contrast microscope images are shown in FIGS.
位相差顕微鏡の画像から、０．１％ＤＭＳＯを添加した培地では、ほとんどのＰＣ１２細胞が、丸い形状のまま培養ディッシュに接着している。一方、ノビレチン溶液を添加した培地では、細胞が培養ディッシュに伸展して、扁平状に接着している。
雲南百薬の亜臨界抽出液（抽出条件１〜５）を添加した培地においても、ＰＣ１２細胞は培養ディッシュに接着していることが分かった。さらに、その接着している細胞の形態は、仮足を伸ばし、伸展していることが分かった。
また、抽出条件１〜５の違いによって、接着している形態の相違は見られなかった。
以上のことから、亜臨界抽出した抽出液によって、接着しているＰＣ１２細胞の形態に大きな影響は与えず、細胞は接着および増殖することが明らかとなった。
From the phase contrast microscope image, in the medium supplemented with 0.1% DMSO, most PC12 cells adhere to the culture dish in a round shape. On the other hand, in the medium to which the nobiletin solution is added, the cells extend to the culture dish and adhere in a flat shape.
It was also found that PC12 cells were adhered to the culture dish even in the medium supplemented with Yunnan hundred drug subcritical extract (extraction conditions 1-5). Furthermore, it was found that the shape of the adhered cells was extended and extended.
Moreover, the difference in the form which adhere | attached by the difference of extraction conditions 1-5 was not seen.
From the above, it has been clarified that the subcritically extracted extract does not significantly affect the shape of the PC12 cells adhering, and the cells adhere and proliferate.
次に、雲南百薬の亜臨界メタノール抽出液がＮＭＤＡ（Ｎ−ｍｅｔｈｙｌ−Ｄ−ａｓｐａｒｔａｔｅ）受容体ＮＲ１遺伝子発現へ作用する効果を調べた。
ＮＭＤＡ受容体は、興奮性神経伝達物質グルタミン酸のイオンチャンネル型受容体であり、記憶や学習に深く関わる受容体であることが知られている。中枢神経疾患の一つであるアルツハイマー型痴呆症の治療薬には、ＮＭＤＡ型グルタミン酸受容体の部分を作動させるものがある。
本実験では、雲南百薬の亜臨界メタノール抽出液を添加した培地で培養したＰＣ１２細胞のＮＭＤＡ受容体ＮＲ１遺伝子の発現をｒｅａｌ−ｔｉｍｅ ＰＣＲ法によって調べた。コントロールは、０．２％のＤＭＳＯを使用した。
Next, the effect of Yunnan hundred drug subcritical methanol extract on NMDA (N-methyl-D-aspartate) receptor NR1 gene expression was examined.
The NMDA receptor is an ion channel type receptor for the excitatory neurotransmitter glutamate, and is known to be a receptor deeply involved in memory and learning. Some therapeutic agents for Alzheimer-type dementia, which is one of central nervous system diseases, activate the NMDA-type glutamate receptor portion.
In this experiment, the expression of the NMDA receptor NR1 gene in PC12 cells cultured in a medium supplemented with a subcritical methanol extract of Yunnan Hyakuyaku was examined by a real-time PCR method. As a control, 0.2% DMSO was used.
図９は、ＮＭＤＡ受容体ＮＲ１遺伝子発現の結果を示している。
全ての亜臨界抽出液において、コントロールよりもＮＭＤＡ受容体ＮＲ１遺伝子発現が上昇することが分かった。
中でも、抽出条件１の抽出液は、高いＮＭＤＡ受容体ＮＲ１遺伝子発現を生じ、ノビレチン１００μｇ／ｍＬと同等の効果があることが分かった。
このことから、雲南百薬の亜臨界抽出液には、ＮＭＤＡ受容体を活性させる成分（物質）が含有され、その成分は低温・短時間で亜臨界抽出した抽出液に多く含まれていることが明らかとなった。
FIG. 9 shows the results of NMDA receptor NR1 gene expression.
It was found that in all subcritical extracts, NMDA receptor NR1 gene expression was higher than that in the control.
Among these, the extract under extraction condition 1 produced high NMDA receptor NR1 gene expression, and was found to have the same effect as nobiletin 100 μg / mL.
From this, the subcritical extract of Yunnan Hyakuyaku contains a component (substance) that activates the NMDA receptor, and that component is often contained in the extract sublimated at low temperature and in a short time. It became clear.
現在、雲南百薬は、健康食品の食品素材として使用されている。そのため、雲南百薬の抽出物は、中枢神経疾患の予防及び治療に用いられる食品に適用することもできる。 Currently, Yunnan Hyakuyaku is used as a food material for health foods. Therefore, the extract of Yunnan Hyakuyaku can also be applied to foods used for the prevention and treatment of central nervous disease.
以上、本実施形態について説明したが、これ以外にも、本発明の主旨を逸脱しない限り、上記実施の形態で挙げた構成を取捨選択したり、他の構成に適宜変更することが可能である。
例えば、本実施形態では、熱水、メタノール、ブタノール、酢酸エチルの抽出溶媒を使用したが、他の抽出溶媒として、プロピレングリコール、１，３−ブチレングリコール等の多価アルコールやアセトン等のケトン類や酢酸エチルエステル等のエステル類、やジエチルエーテル、ジオキサン、アセトニトリル、キシレン、ベンゼン、クロロホルム等を使用することもできる。また、これらの抽出溶媒は、水性溶媒と有機溶媒との混合液として使用してもよい。
Although the present embodiment has been described above, the configuration described in the above embodiment can be selected or changed to other configurations as appropriate without departing from the gist of the present invention. .
For example, in this embodiment, an extraction solvent of hot water, methanol, butanol, and ethyl acetate was used, but as other extraction solvents, polyhydric alcohols such as propylene glycol and 1,3-butylene glycol, and ketones such as acetone And esters such as ethyl acetate, diethyl ether, dioxane, acetonitrile, xylene, benzene, chloroform and the like can also be used. Moreover, you may use these extraction solvents as a liquid mixture of an aqueous solvent and an organic solvent.
Claims (3)
雲南百薬の乾燥物または抽出液を含有する、
ことを特徴とする医薬組成物。 A pharmaceutical composition used for the prevention and treatment of central nervous disease,
Containing dry products or extracts of Yunnan Hyakuyaku,
The pharmaceutical composition characterized by the above-mentioned.
ことを特徴とする請求項１に記載された医薬組成物。 The extract of Yunnan hundred medicines is one kind of extract selected from the group of hot water and alcohols,
The pharmaceutical composition according to claim 1, wherein:
雲南百薬の乾燥物または抽出液を含有する、
ことを特徴とする食品。 A food used for the prevention and treatment of central nervous disease,
Containing dry products or extracts of Yunnan Hyakuyaku,
A food characterized by that.
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| JP2010507651A (en) * | 2006-10-24 | 2010-03-11 | エスケー ケミカルズ カンパニー リミテッド | Oleanane triterpene saponin compounds effective in treating dementia and mild cognitive impairment and improving cognitive function |
| JP2014141438A (en) * | 2013-01-24 | 2014-08-07 | Sankyo:Kk | Formulation, food and drink containing novel madeira vine product |
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| JP2010507651A (en) * | 2006-10-24 | 2010-03-11 | エスケー ケミカルズ カンパニー リミテッド | Oleanane triterpene saponin compounds effective in treating dementia and mild cognitive impairment and improving cognitive function |
| JP2014141438A (en) * | 2013-01-24 | 2014-08-07 | Sankyo:Kk | Formulation, food and drink containing novel madeira vine product |
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| ESPADA, A. ET AL.: "Boussingoside D1, a new triterpenoid saponin from Boussingaultia baselloides", LIEBIGS ANNALEN DER CHEMIE, JPN6017028469, 1991, pages 291 - 293, XP055553349, ISSN: 0003892890, DOI: 10.1002/jlac.199119910149 * |
| ESPADA, A. ET AL.: "Boussingoside E, a new triterpenoid saponin from the tubers of Boussingaultia baselloides", JOURNAL OF NATURAL PRODUCTS, vol. 60, JPN6017028470, 1997, pages 17 - 19, XP055553356, ISSN: 0003892891 * |
| ESPADA, A. ET AL.: "Hypoglucaemic triterpenoid saponins from Boussingaultia baselloides", CANADIAN JOURNAL OF CHEMISTRY, vol. 68, no. 11, JPN6017028471, 1990, pages 2039 - 2044, XP055553373, ISSN: 0003892892, DOI: 10.1139/v90-312 * |
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