JPWO2013172087A1 - Composition comprising nitric oxide production inhibitory activity - Google Patents
Composition comprising nitric oxide production inhibitory activity Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/60—Fish, e.g. seahorses; Fish eggs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- 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
- A61P25/04—Centrally acting analgesics, e.g. opioids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Abstract
【課題】細胞毒性がほとんどない一酸化窒素産生阻害活性を備える組成物を提供すること。【解決手段】ドジョウおよびドジョウと棲息態様が同じ魚類の乾燥魚肉または前記乾燥魚肉の処理物を有効成分として含むことを特徴とする。【選択図】なしDisclosed is a composition having a nitric oxide production inhibitory activity with little cytotoxicity. The dried fish meat of the same fish or the processed product of the dried fish meat as an active ingredient is characterized in that the dried fish and the dried fish have the same habit. [Selection figure] None
Description
本発明は、一酸化窒素産生阻害活性を備える組成物に係り、特に細胞毒性がほとんどない一酸化窒素産生阻害活性を備える組成物に関する。 The present invention relates to a composition having nitric oxide production inhibitory activity, and particularly to a composition having nitric oxide production inhibitory activity with little cytotoxicity.
一般に、一酸化窒素（ＮＯ）はＬ−アルギニンから一酸化窒素合成酵素（ＮＯＳ）によって産生される。皮膚において一酸化窒素は、感染、紫外線照射、抗原刺激などで産生され、感染防御、創傷治癒、炎症、疼痛増強など様々な作用を持つことが報告さられている。 In general, nitric oxide (NO) is produced from L-arginine by nitric oxide synthase (NOS). In the skin, nitric oxide is produced by infection, ultraviolet irradiation, antigen stimulation, and the like, and has been reported to have various actions such as infection protection, wound healing, inflammation, and pain enhancement.
また、アトピー性皮膚炎などの慢性掻痒性皮膚疾患の痒みには、Ｈ１受容体拮抗薬が無効であることが多く、激しい痒みを伴うアトピー性皮膚炎や乾癬などの皮膚では、一酸化窒素が増加していることが報告されている。In addition, H 1 receptor antagonists are often ineffective for pruritus of chronic pruritic skin diseases such as atopic dermatitis, and nitric oxide is often used in skin such as atopic dermatitis and psoriasis with severe itching. Has been reported to increase.
また、表皮ケラチノサイトが、痒みのメディエーターであるロイコトリエンＢ４、トロンボキサンＡ２、ノシセプチン、一酸化窒素、過酸化水素を産生、遊離し、それらのメディエーターの作用として、一酸化窒素以外のメディエーターは痒みを誘発させる因子、一酸化窒素は痒みを増強させる因子であることが報告されている。In addition, epidermal keratinocytes produce and release itch mediators leukotriene B 4 , thromboxane A 2 , nociceptin, nitric oxide and hydrogen peroxide, and mediators other than nitric oxide are itched as an effect of these mediators. It has been reported that nitric oxide, a factor that induces inflammation, is a factor that enhances itching.
このように体内に産生された一酸化窒素は、例えば皮膚において感染防御、創傷治癒、炎症、疼痛増強など様々な症状の原因となるので、その体内における産生を抑える一酸化窒素産生阻害活性を備える組成物であって安全性の高いものが望まれている。 Since nitric oxide produced in the body causes various symptoms such as infection protection, wound healing, inflammation, and pain enhancement in the skin, it has a nitric oxide production inhibitory activity that suppresses production in the body. A highly safe composition is desired.
本発明はこれらの点に鑑みてなされたものであり、細胞毒性がほとんどない一酸化窒素産生阻害活性を備える組成物を提供することを目的とする。 This invention is made | formed in view of these points, and it aims at providing the composition provided with the nitric oxide production inhibitory activity which has little cytotoxicity.
本発明者等は鋭意研究し、体内に入っても安全なドジョウおよびドジョウと棲息態様が同じ魚類の乾燥魚肉や当該乾燥魚肉からの抽出物について一酸化窒素産生阻害活性を検証して、炎症マーカーである一酸化窒素産生阻害活性の存在を発見して本発明を完成させた。 The present inventors have conducted intensive research and verified the nitric oxide production inhibitory activity on dried fish meat that is safe to enter the body and dried fish meat that has the same habituation as the fish and extracts from the dried fish meat. The present invention was completed by discovering the presence of nitric oxide production inhibitory activity.
本発明の第１の態様である一酸化窒素産生阻害活性を備える組成物は、ドジョウおよびドジョウと棲息態様が同じ魚類の乾燥魚肉または前記乾燥魚肉の処理物を有効成分として含むことを特徴とする。 The composition having nitric oxide production inhibitory activity according to the first aspect of the present invention is characterized by containing as a active ingredient dried fish meat having the same habit as dojo and dojo, or a processed product of the dried fish meat. .
本発明の第２の態様である一酸化窒素産生阻害活性を備える組成物は、第１の態様において、前記処理物が、前記乾燥魚肉から冷浸抽出した抽出物であることを特徴とする。 The composition having nitric oxide production inhibitory activity according to the second aspect of the present invention is characterized in that, in the first aspect, the treated product is an extract extracted by cold immersion from the dried fish meat.
本発明の第３の態様である一酸化窒素産生阻害活性を備える組成物は、第２の態様において、前記抽出物が、アルコール類をもって冷浸抽出した抽出物であることを特徴とする。 The composition having nitric oxide production inhibitory activity according to the third aspect of the present invention is characterized in that, in the second aspect, the extract is an extract extracted by cold immersion with alcohols.
本発明の一酸化窒素産生阻害活性を備える組成物は、ドジョウの乾燥魚肉を有効成分としているために、細胞毒性がなく一酸化窒素産生阻害活性を発揮することができるという優れた作用効果を発揮することができる。 Since the composition having the nitric oxide production inhibitory activity of the present invention uses dried salmon roe as an active ingredient, it exhibits an excellent effect of being capable of exhibiting nitric oxide production inhibitory activity without cytotoxicity. can do.
以下、本発明の実施形態を製造方法とともに説明する。 Hereinafter, an embodiment of the present invention will be described together with a manufacturing method.
＜基本素材＞
基本素材としてドジョウの乾燥魚肉を用いた。
まず、ドジョウをざる等に取出して窒息死させ、その後１日間に亘って天日乾燥若しくは真空乾燥を施して乾燥魚肉からなる基本素材を得た。
この基本素材を粉末としたり、丸薬状としたり、カプセル内に詰めて傾向投与可能な状態とした。
また、本発明においては基本素材としてドジョウのほかにドジョウと棲息態様が同じ魚類、例えば、ウナギ、八目ウナギ、ナマズ、雷魚等を用いることができる。<Basic material>
Dry fish from loach was used as the basic material.
First, a loach was taken out and killed by suffocation, followed by sun drying or vacuum drying for one day to obtain a basic material made of dried fish meat.
This basic material was powdered, made into a pill form, or packed in a capsule so that it could be easily administered.
In the present invention, in addition to loach, fish having the same habit as the loach can be used as the basic material, for example, eel, eight-eyed eel, catfish, thunderfish and the like.
＜基本素材からの抽出物＞
１）ドジョウの乾燥魚肉から抽出物の調製
ドジョウをヘキサン、クロロホルム、メタノールの順に冷浸抽出（各溶媒１週間冷浸抽出を３回）し、その後、熱水で温浸抽出（２時間温浸抽出を１回）した。
得られた各抽出液について減圧下濃縮あるいは凍結乾燥して、ヘキサン抽出物（Ｆｒ．Ａ）、クロロホルム抽出物（Ｆｒ．Ｂ）、メタノール抽出物（Ｆｒ．Ｃ）、水抽出物（Ｆｒ．Ｄ）を調製した。<Extracts from basic materials>
1) Preparation of extract from dried fish of loach Extract loach from hexane, chloroform and methanol in this order (3 times for 1 week for each solvent), then digestion with hot water (2 hours digestion) Extraction was performed once).
Each obtained extract was concentrated or lyophilized under reduced pressure to obtain a hexane extract (Fr. A), a chloroform extract (Fr. B), a methanol extract (Fr. C), or a water extract (Fr. D). ) Was prepared.
２）一酸化窒素産生阻害活性試験および細胞毒性試験（１）
前記抽出物Ｆｒｓ．Ａ〜Ｄ（各濃度５０μｇ／ｍＬ）について、一酸化窒素産生阻害活性試験および細胞毒性試験を行った。
一酸化窒素産生は生細胞により行われるため、試料自体（ドジョウ抽出物あるいはその分離画分）に細胞への毒性（細胞死作用）があると、一酸化窒素酸性が低下し、一酸化窒素産生阻害活性試験において産生阻害活性を示してしまう。そこで、一酸化窒素産生阻害活性試験と併せて細胞毒性試験を実施して、一酸化窒素産生阻害活性が細胞毒性によるものなのか、あるいは細胞自体には影響はなく何らかのメカニズムによるものなのかを判断した。2) Nitric oxide production inhibitory activity test and cytotoxicity test (1)
The extract Frs. A to D (each concentration 50 μg / mL) were subjected to a nitric oxide production inhibitory activity test and a cytotoxicity test.
Since nitric oxide production is performed by living cells, if the sample itself (loach extract or separated fraction thereof) is toxic to cells (cell death effect), nitric oxide acidity decreases, and nitric oxide production occurs. Inhibitory activity test shows production inhibitory activity. Therefore, a cytotoxicity test is performed in conjunction with the nitric oxide production inhibitory activity test to determine whether the nitric oxide production inhibitory activity is due to cytotoxicity, or whether it is due to some mechanism without affecting the cells themselves. did.
２−１）ＮＯ産生阻害活性試験
（Ｇｒｉｅｓｓ法による一酸化窒素合成阻害率の測定方法）
マウスマクロファージ細胞株Ｊ７７４．１細胞を９６穴マイクロプレートに５×１０４個／２００μＬ／ウェル播種し、一晩培養した。次に、培養液を交換し、１００Ｕ／ｍＬ ＩＦＮ−γ、０．５μｇ／ｍＬ ＬＰＳ、となるように添加した。被検試料（ドジョウ抽出物あるいはその分離画分）は終濃度６．２５〜２００μＭになるようにＩＦＮ−γ、ＬＰＳと同じタイミングで添加した。
２４時間培養後に培養上清１００μＬを別のμプレートに移し、Ｇｒｉｅｓｓ試薬（０．１％N-(1-naphtyl)ethylenediamine dihydorochloride、 １％sulfanilamide、２％リ
ン酸、用時調製）１００μＬを添加し室温で１０分間静置した後マイクロプレートリーダー（バイオラッドラボラトリーズ株式会社、東京、Ｍｏｄｅｌ５５０）で波長５４０ｎｍの吸光度を測定した。１００μＭのＮａＮＯ２溶液を段階的に希釈し、同様に処理して検量線を作成し、ＮＯ２ −濃度を求めた。
ＩＦＮ−γ、ＬＰＳを添加したときの一酸化窒素濃度に対する被検薬の作用を一酸化窒素合成阻害率（％）として求めた（培養上清中ＮＯ２ −濃度を一酸化窒素生成量の指標とした）。2-1) NO production inhibitory activity test
(Measurement method of inhibition rate of nitric oxide synthesis by Griess method)
Mouse macrophage cell line J774.1 cells were seeded in 96-well microplates at 5 × 10 4 cells / 200 μL / well and cultured overnight. Next, the culture solution was exchanged, and 100 U / mL IFN-γ and 0.5 μg / mL LPS were added. The test sample (loach extract or separated fraction thereof) was added at the same timing as IFN-γ and LPS so that the final concentration was 6.25 to 200 μM.
After culturing for 24 hours, 100 μL of the culture supernatant was transferred to another μ plate, and 100 μL of Griess reagent (0.1% N- (1-naphtyl) ethylenediamine dihydorochloride, 1% sulfonanilamide, 2% phosphoric acid, prepared at the time of use) was added. After standing at room temperature for 10 minutes, the absorbance at a wavelength of 540 nm was measured with a microplate reader (BioRad Laboratories, Tokyo, Model 550). Diluted stepwise with NaNO 2 solution of 100 [mu] M, a calibration curve is prepared similarly treated, NO 2 - concentrations were determined.
The effect of the test drug on the nitric oxide concentration when IFN-γ and LPS were added was determined as the nitric oxide synthesis inhibition rate (%) (the NO 2 − concentration in the culture supernatant was an indicator of the amount of nitric oxide produced) )
２−２）細胞毒性試験
（ＭＴＴ法による細動生存率の測定方法）
マウスマクロファージ細胞株Ｊ７７４．１細胞を９６穴マイクロプレートに１×１０４個２００μＬ／ウェル播種し、一晩培養した。その後、被検試料（ドジョウ抽出物あるいはその分離画分）を終濃度６．２５〜２００μＭになるように添加し、２４時間培養後に、５ｍｇ／ｍＬのＭＴＴ液を１０μＬ／ウェル加えて、３７℃で３時間インキュベートし、ホルマザンを生成させた。上清を除去し、dimethylsulfoxide（ＤＭＳＯ）を１００μＬ／ウェル加え振盪してホルマザンを溶解させ、波長５４０ｎｍの吸光度をマイクロプレートリーダーにて測定した。ＭＴＴはテトラゾリコム塩の一種で、細胞内に取り込まれると、生細胞においてのみ活性を示すミトコンドリアの吸収鎖のスクシネート−テトラゾリウム還元酵素システムによってホルマザンに分解される。従って、ホルマザン色素量は生細胞数に比例する。
何も添加してないときの生存率を１００％として、生存率に対する被検試料の影響を調べた。2-2) Cytotoxicity test
(Measuring method of fibrillation survival rate by MTT method)
Mouse macrophage cell line J774.1 cells were seeded in 96-well microplates at 1 × 10 4 200 μL / well and cultured overnight. Thereafter, a test sample (loach extract or a fraction thereof) was added to a final concentration of 6.25 to 200 μM, and after 24 hours of incubation, 5 mg / mL of MTT solution was added at 10 μL / well, and the mixture was incubated at 37 ° C. For 3 hours to form formazan. The supernatant was removed, 100 μL / well of dimethylsulfoxide (DMSO) was added and shaken to dissolve formazan, and the absorbance at a wavelength of 540 nm was measured with a microplate reader. MTT is a kind of tetrazolicom salt, and when taken into cells, it is broken down into formazan by the succinate-tetrazolium reductase system of the mitochondrial absorption chain that is active only in living cells. Therefore, the amount of formazan dye is proportional to the number of living cells.
The survival rate when nothing was added was defined as 100%, and the influence of the test sample on the survival rate was examined.
２−３）結果
一酸化窒素産生阻害活性試験の結果を図１に示した。
細胞毒性活性は、ドジョウ抽出物のいずれの画分にも認められなかった。
これらの結果より、ドジョウの乾燥魚肉には一酸化窒素産生阻害活性を有する成分が存在することが明らかとなった。これらの画分の中で最も強い活性を認められたメタノール抽出物（Ｆｒ．Ｃ）について更に分離・精製を行うこととした。
なお、アルコールとしての機能を備えているエタノール等の他のアルコール類もメタノールと同様に抽出成分として用いることができる。2-3) Results The results of the nitric oxide production inhibitory activity test are shown in FIG.
Cytotoxic activity was not observed in any fraction of loach extract.
From these results, it was clarified that dried fish meat of loach contains a component having an activity of inhibiting nitric oxide production. The methanol extract (Fr. C), which showed the strongest activity in these fractions, was further separated and purified.
It should be noted that other alcohols such as ethanol having a function as an alcohol can be used as an extraction component in the same manner as methanol.
３）ドジョウ抽出物の分離（１）
メタノール抽出物Ｆｒ．Ｃについて、９０％メタノールに溶解し、ヘキサンで分配し、下層を減圧下濃縮後、Ｓｅｐｈａｄｅｘ ＬＨ−２０カラムクロマトグラフィー（５０ｍｍ ｉ．ｄ． ×３００ｍｍ、８０％メタノール）で４つの画分（Ｆｒｓ．Ｃ−１〜Ｃ−４）に分画した。3) Separation of loach extract (1)
Methanol extract Fr. About C, it melt | dissolves in 90% methanol, It distributes with hexane, After concentrating a lower layer under pressure reduction, it is four fractions (Frs.) By Sephadex LH-20 column chromatography (50mm id * 300mm, 80% methanol). C-1 to C-4) were fractionated.
３−１）一酸化窒素産生阻害活性試験および細胞毒性試験（２）
前記画分Ｆｒｓ．Ｃ−１〜Ｃ−４（各濃度５０μ／ｍＬ）について、一酸化窒素産生阻害試験および細胞毒性試験を行った。
一酸化窒素産生阻害活性試験の結果を図２に示した。
細胞毒性活性は、いずれの画分にも認められなかった。
これらの結果から、次に一酸化窒素産生阻害活性が最も強い画分Ｆｒ．Ｃ−１について更に分離・精製を進めることとした。3-1) Nitric oxide production inhibitory activity test and cytotoxicity test (2)
Said fraction Frs. C-1 to C-4 (each concentration 50 μ / mL) were subjected to a nitric oxide production inhibition test and a cytotoxicity test.
The results of the nitric oxide production inhibitory activity test are shown in FIG.
Cytotoxic activity was not observed in any fraction.
From these results, the fraction Fr. It was decided to further separate and purify C-1.
４）ドジョウの抽出物の分離（２）
画分Ｆｒ．Ｃ−１を１０％メタノールにて懸濁させ、Ｄｉａｉｏｎ ＨＰ−２０カラムクロマトグラフィー（６０ｍｍ ｉ．ｄ．×５０ｍｍ）に付し、１０％メタノール、５０％メタノール、メタノール、酢酸エチルで順次極性を下げながら溶出させ、１０％メタノール溶出画分（Ｆｒ．Ｃ−１Ａ）、５０％メタノール溶出画分（Ｆｒ．Ｃ−１Ｂ）、メタノール溶出画分（Ｆｒ．Ｃ−１Ｃ）、酢酸エチル溶出画分（Ｆｒ．Ｃ−１Ｄ）の４つの画分に分画した。4) Separation of loach extract (2)
Fraction Fr. C-1 was suspended in 10% methanol and subjected to Diaion HP-20 column chromatography (60 mm id x 50 mm), and the polarity was decreased successively with 10% methanol, 50% methanol, methanol and ethyl acetate. 10% methanol elution fraction (Fr.C-1A), 50% methanol elution fraction (Fr.C-1B), methanol elution fraction (Fr.C-1C), ethyl acetate elution fraction ( Fr. C-1D) was fractionated into four fractions.
４−１）一酸化窒素産生阻害活性試験および細胞毒性試験（３）
画分Ｆｒｓ．Ｃ−１Ａ〜Ｃ−１Ｄ（各濃度５００μｇ／ｍＬ）について、一酸化窒素産生阻害活性試験および細胞毒性試験を行った。
一酸化窒素産生阻害活性試験の結果を図３に示した。
更に、強い一酸化窒素産生阻害活性を示した画分Ｆｒ．Ｃ−１ＣとＦｒ．Ｃ−１Ｄについて、一酸化窒素産生阻害活性と用量との関係を検討した。その結果を図４に示した。どちらの画分も相関係数が１に近い値（０．９６４３）であったことから、一酸化窒素産生阻害活性の用量依存性が明らかになった。4-1) Nitric oxide production inhibitory activity test and cytotoxicity test (3)
Fraction Frs. C-1A to C-1D (each concentration of 500 μg / mL) were subjected to nitric oxide production inhibitory activity test and cytotoxicity test.
The results of the nitric oxide production inhibitory activity test are shown in FIG.
Furthermore, the fraction Fr. which showed strong nitric oxide production inhibitory activity. C-1C and Fr. Regarding C-1D, the relationship between nitric oxide production inhibitory activity and dose was examined. The results are shown in FIG. Since the correlation coefficient of both fractions was a value close to 1 (0.9643), the dose dependency of nitric oxide production inhibitory activity was revealed.
５）ドジョウの抽出物の分離（３）
画分Ｆｒ．Ｃ−１Ｃを８０％メタノールで溶解し、Sep-P Plus Ｃ１８ environmental cartridges (WAT023635) に通した溶出画分をＦｒ．Ｃ−１Ｃ１（８９８ｍｇ）とし、メタノールで回収した画分をＦｒ．Ｃ−１Ｃ２とした。5) Separation of loach extract (3)
Fraction Fr. The elution fraction obtained by dissolving C-1C with 80% methanol and passing through Sep-P Plus C18 environmental cartridges (WAT023635) was obtained as Fr. C-1C1 (898 mg), and the fraction collected with methanol was Fr. C-1C2.
６）ドジョウの抽出物の分離（４）
画分Ｆｒ．Ｃ−１Ｃ１について、次の条件下、分取逆相ＨＰＬＣにより３つの画分（Ｆｒ．Ｃ−１Ｃ１Ａ〜Ｃ−１Ｃ１Ｃ）に分画した。
ＨＰＬＣ条件
Pump : Hitachi L-6250, Flow : 9.0mL/min
Detector : Hitachi L-4000, 210nm
Column : Senshu Pak DOCOSIL SP-100 (20mm i.d. × 250mm,Senshu)
Solvent : MeOH-H2O (4:1)6) Separation of loach extract (4)
Fraction Fr. C-1C1 was fractionated into three fractions (Fr. C-1C1A to C-1C1C) by preparative reverse phase HPLC under the following conditions.
HPLC conditions
Pump: Hitachi L-6250, Flow: 9.0mL / min
Detector: Hitachi L-4000, 210nm
Column: Senshu Pak DOCOSIL SP-100 (20mm id × 250mm, Senshu)
Solvent: MeOH-H2O (4: 1)
６−１）一酸化窒素産生阻害活性試験および細胞毒性試験（４）
画分Ｆｒｅ．Ｃ−１Ｃ１Ａ〜Ｃ−１Ｃ１Ｃ（各濃度２５μｇ／ｍＬ）について、一酸化窒素産生阻害活性試験および細胞毒性試験を行った。
その結果を図５に示す。
いずれの画分にも細胞毒性活性は認められず、Ｆｒ．Ｃ−１Ｃ１Ｂに６５％以上の強い一酸化窒素産生阻害活性が明らかになった。次に、画分Ｆｒ．Ｃ−１Ｃ１Ｂのについて更に分離・精製を進めることとした。6-1) Nitric oxide production inhibitory activity test and cytotoxicity test (4)
Fraction Fre. C-1C1A to C-1C1C (each concentration 25 μg / mL) were subjected to nitric oxide production inhibitory activity test and cytotoxicity test.
The result is shown in FIG.
None of the fractions showed cytotoxic activity, and Fr. C-1C1B showed a strong nitric oxide production inhibitory activity of 65% or more. Next, fraction Fr. It was decided to further separate and purify C-1C1B.
７）ドジョウ抽出物の分離（５）
画分Ｆｒ．Ｃ−１Ｃ１Ｂについて、以下の条件下、分取逆相ＨＰＬＣにより分離を行った。
ＨＰＬＣ条件
Pump : Hitachi L-6250, Flow : 2.0mL/min
Detector : Hitachi L-4000, 210nm
Column : Senshu Pak DOCOSIL SP-100 (10mm i.d. × 250mm,Senshu)
Solvent : MeOH-H2O (17:3)
分取逆相ＨＰＬＣによる分離により図６のクロマトグラムが得られ、点線で区切った保持時間で９つに分画した。ただし、ｉはメタノール回収分を含む。
その結果、９つの画分（Ｆｒｓ．Ｃ−１Ｃ１Ｂ１〜Ｃ−１Ｃ１Ｂ９）を以下に示す収量で得た。
ａ → Ｆｒ．Ｃ−１Ｃ１Ｂ１（２８．３ｍｇ）
ｂ → Ｆｒ．Ｃ−１Ｃ１Ｂ２（５．１ｍｇ）
ｃ → Ｆｒ．Ｃ−１Ｃ１Ｂ３（９．０ｍｇ）
ｄ → Ｆｒ．Ｃ−１Ｃ１Ｂ４（４．６ｍｇ）
ｅ → Ｆｒ．Ｃ−１Ｃ１Ｂ５（７．５ｍｇ）
ｆ → Ｆｒ．Ｃ−１Ｃ１Ｂ６（８．４ｍｇ）
ｇ → Ｆｒ．Ｃ−１Ｃ１Ｂ７（１３．４ｍｇ）
ｈ → Ｆｒ．Ｃ−１Ｃ１Ｂ８（２９．０ｍｇ）
ｉ → Ｆｒ．Ｃ−１Ｃ１Ｂ９（６１．１ｍｇ）7) Separation of loach extract (5)
Fraction Fr. C-1C1B was separated by preparative reverse phase HPLC under the following conditions.
HPLC conditions
Pump: Hitachi L-6250, Flow: 2.0mL / min
Detector: Hitachi L-4000, 210nm
Column: Senshu Pak DOCOSIL SP-100 (10mm id × 250mm, Senshu)
Solvent: MeOH-H2O (17: 3)
The chromatogram of FIG. 6 was obtained by separation by preparative reverse phase HPLC, and was fractionated into nine with retention times separated by dotted lines. However, i includes methanol recovery.
As a result, nine fractions (Frs. C-1C1B1 to C-1C1B9) were obtained in the yields shown below.
a → Fr. C-1C1B1 (28.3 mg)
b → Fr. C-1C1B2 (5.1 mg)
c → Fr. C-1C1B3 (9.0 mg)
d → Fr. C-1C1B4 (4.6 mg)
e → Fr. C-1C1B5 (7.5 mg)
f → Fr. C-1C1B6 (8.4 mg)
g → Fr. C-1C1B7 (13.4 mg)
h → Fr. C-1C1B8 (29.0 mg)
i → Fr. C-1C1B9 (61.1 mg)
７−１）一酸化窒素産生阻害活性試験および細胞毒性試験（５）
画分Ｆｒ．Ｃ−１Ｃ１Ｂ１〜Ｃ−１Ｃ１Ｂ９（各濃度２５μｇ／ｍＬ）について、一酸化窒素産生阻害活性試験および細胞毒性試験を行った。
その結果を図７に示す。
画分Ｆｒ．Ｃ−１Ｃ１Ｂ３、Ｆｒ．Ｃ−１Ｃ１Ｂ５に強い一酸化窒素産生阻害活性が認められた。
画分Ｆｒ．Ｃ−１Ｃ１Ｂ２、Ｆｒ．Ｃ−１Ｃ１Ｂ７〜Ｆｒ．Ｃ−１Ｃ１Ｂ９に５０％程度の一酸化窒素産生阻害活性が得られた。
また、細胞製造率がほぼ１００％であり、細胞毒性活性がないことを確認した。7-1) Nitric oxide production inhibitory activity test and cytotoxicity test (5)
Fraction Fr. C-1C1B1 to C-1C1B9 (each concentration 25 μg / mL) were subjected to a nitric oxide production inhibitory activity test and a cytotoxicity test.
The result is shown in FIG.
Fraction Fr. C-1C1B3, Fr. A strong nitric oxide production inhibitory activity was observed in C-1C1B5.
Fraction Fr. C-1C1B2, Fr. C-1C1B7-Fr. About 50% of nitric oxide production inhibitory activity was obtained for C-1C1B9.
The cell production rate was almost 100%, and it was confirmed that there was no cytotoxic activity.
８）基本素材からの抽出物による効果
前記各画分の一酸化窒素産生阻害活性試験および細胞毒性試験の結果より、基本素材からの抽出物には細胞毒性のない一酸化窒素産生阻害活性が得られることが明確となった。8) Effect of the extract from the basic material From the results of the nitric oxide production inhibitory activity test and the cytotoxicity test of each fraction, the extract from the basic material has a nitric oxide production inhibitory activity without cytotoxicity. It became clear that
＜基本素材による効果＞
花粉症を発症している青森大学薬学部の２３〜２５歳の男子学生６名に対して、１日小さじ１杯分の基本素材の粉末を経口投与して経過観察したところ、全員７日経過すると花粉症の症状が軽減した。
この花粉症軽減により一酸化窒素の産生が低減されたことが明らかになった。
従って、本発明は基本素材および基本素材からの抽出物についてもそれぞれ細胞毒性のない一酸化窒素産生阻害活性が得られることが明確となった。<Effects of basic materials>
When 6 male students aged 23 to 25 years old from Aomori University School of Pharmacy who developed hay fever were observed by oral administration of 1 teaspoon of basic material powder, all 7 days passed. The symptoms of hay fever were reduced.
It became clear that the production of nitric oxide was reduced by reducing this pollen allergy.
Therefore, it was clarified that the present invention can obtain nitric oxide production inhibitory activity without cytotoxicity for the basic material and the extract from the basic material.
本発明は、前記実施形態のものに限らずに、必要に応じて変更することができる。具体的には、ドジョウに代えて、ヒトデ、ナマコ、ウニ等の棘皮動物を原料とした乾燥肉質又は当該乾燥肉質の処理物を有効成分として含有する組成物を用いることができる。 The present invention is not limited to the embodiment described above, and can be changed as necessary. Specifically, in place of loach, a composition containing dried meat from echinoderms such as starfish, sea cucumber, sea urchin or the like or a processed product of the dried meat as an active ingredient can be used.
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| PCT/JP2013/057758 WO2013172087A1 (en) | 2012-05-17 | 2013-03-19 | Composition provided with nitric oxide production-inhibiting activity |
| JP2014515525A JPWO2013172087A1 (en) | 2012-05-17 | 2013-03-19 | Composition comprising nitric oxide production inhibitory activity |
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