JP4344030B2 - Anti-inflammatory agent - Google Patents
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- JP4344030B2 JP4344030B2 JP29761598A JP29761598A JP4344030B2 JP 4344030 B2 JP4344030 B2 JP 4344030B2 JP 29761598 A JP29761598 A JP 29761598A JP 29761598 A JP29761598 A JP 29761598A JP 4344030 B2 JP4344030 B2 JP 4344030B2
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Description
【０００１】
【発明の属する技術分野】
本発明は、抗炎症剤に関する。さらに詳しくは、くろず（黒酢）又はくろずもろみ末（くろずもろみからくろずを除去した残渣の粉末）を原料とする抗炎症剤に関する。
本発明の抗炎症剤は、おだやかな抗炎症作用を呈し、長期間にわたってまた大量に摂取しても、副作用がほとんどなく、炎症を予防或いは治療することができる。
【０００２】
【従来の技術】
一般に、生体の炎症性疾患は、病原性微生物による感染症、物理的又は化学的起炎原因による傷害、代謝異常による痛風等の正常反応性炎症と、各種のアレルギー、自己免疫疾患、間葉失調症候群等が含まれる異常反応性炎症とに区別されるが、いずれの場合も、基本的な症状及び徴候は、発熱、発赤、腫張、疼痛であり、また機能傷害を伴なうことが多い。
【０００３】
抗炎症剤（抗炎症薬）は、これらの炎症過程に拮抗するか又は炎症症状を抑制する作用を有する薬剤であり、抗ヒスタミン剤（抗アレルギー剤）、ステロイド性抗炎症剤（副腎皮質ホルモン剤）、非ステロイド性抗炎症剤、消炎酵素剤、免疫抑制剤、抗リウマチ剤、解熱性鎮痛剤等が知られている。
【０００４】
これらの抗炎症剤は、よく知られているように、おしなべて使用法が限定的である上、強い副作用を有する。例えば、ステロイド性抗炎症剤は、その抗炎症作用は強力であるが、長期間にわたって又は大量に使用すると、むくみ、ムーン・フェイス、糖尿病や胃潰瘍の悪化、各種の感染症にかかりやすくなる等の副作用を併発しやすい。しかも、その至適投与量に個人差があり、患者ごとに異なるので、投与量を決定するのは難しく、さらに投与量を誤った場合、炎症症状を促進させるという逆効果が生じることがある。また、非ステロイド性抗炎症剤の場合は、ステロイド性抗炎症剤ほどではないが、食欲不振、めまい、悪心、嘔吐、腹痛、下痢、頭痛等の副作用が生じやすく、ときには、むくみ、貧血、血圧上昇、口内炎、動悸、蛋白尿、血尿等の症状が現れることもある。その他の抗炎症剤についても、同様の副作用を起こすことが多い。このようなことから、長期間にわたりまた大量に投与しても、副作用が生じない抗炎症剤の開発は強く望まれている。
【０００５】
【発明が解決しようとする課題】
上記の事情に鑑み、本発明者は、おだやかな抗炎症作用を呈し、長期間にわたって投与しても、また大量に摂取しても副作用が生じることがない、新規な抗炎症剤の開発について鋭意努力した。その結果、種々の生理活性作用を有するくろず及びくろずもろみ末に着目し、これを原料として研究し、本発明を完成するに至った。
【０００６】
本発明は上記のようにしてなされたものであって、その発明としての課題は、おだやかな抗炎症作用を呈し、長期間にわたって摂取を続けても、また大量に投与しても、副作用がほとんど生じない、新規な抗炎症剤を提供することにある。
【０００７】
【課題を解決するための手段】
上記の課題を達成するための本発明は、くろずの有機溶媒抽出物の乾燥物を有効成分とする抗炎症剤である。
この場合の有機溶媒としては、酢酸エチルを用いることが望ましい。
【０００８】
また本発明は、くろずもろみ末の有機溶媒抽出物の乾燥物を有効成分とする抗炎症剤である。
この場合の有機溶媒としては、メタノールを用いることが望ましい。
【０００９】
【発明の実施の形態】
本発明の抗炎症剤は、その原料としてくろず又はくろずもろみ末を使用する。本発明で原料とするくろずは、伝統的に、鹿児島の福山地方を中心として生産されており、最近では健康飲料ないし健康食品の原料としても注目されている。くろずは、その一般的な製法によれば、野天に並べた醸造用のカメに米麹と蒸米と水を仕込んで、太陽エネルギーの力で、糖化、アルコール発酵、酢酸発酵を同一のカメの中で進行させ、長い熟成期間を経ると、独特の風味と各種の有用成分を有するくろずもろみとなり、これを圧搾・ろ過してくろずとして生産される。
【００１０】
本発明において、くろずもろみとは、上記のようにくろず原料を発酵・熟成させた発酵物、すなわちくろずを混和している状態の発酵物のことを言う。また、くろずもろみ末とは、くろずもろみから液体であるくろずを除去した残渣を乾燥させ粉末状にしたものを言う。くろずもろみ末は、従来知られている種々の方法で製造することができ、例えば、特開昭61-58577号公報等に記載されている方法で製造したものを使用することができる。
【００１１】
くろずやくろずもろみ末には、食欲増進作用、消化吸収促進作用、殺菌力・防腐作用、漂白作用等の通常の酢が有する一般的効能の他、肩こり、五十肩、便秘、疲労、高血圧、動脈硬化、糖尿病、肥満、慢性肝炎、神経痛、リウマチ、眼底出血等に効果があると言われている。最近は、血糖、血中脂質、血清コレステロール含量を低下させる作用があることも発表されている。また、くろずの成分には、タンパク質、遊離アミノ酸、オリゴペプタイド等が含まれていることが解明されている。
【００１２】
本発明では、くろずを原料とする場合は、まずくろずを有機溶媒で抽出する。具体的には、くろずに塩基性物質、例えば水酸化ナトリウムや炭酸カリウムを添加して中和し、酢酸エチル、塩化メチレン、ベンゼン等の有機溶媒を加えて数回抽出を行なう。抽出効率の点で、酢酸エチルを使用するのが好ましい。酢酸エチルを使用する場合を例にして説明すると、次いでこの抽出液を、水層（酢酸エチル非可溶物層）と酢酸エチル層（酢酸エチル可溶物層）に分離し、酢酸エチル層を、例えば減圧下で濃縮し、乾固させる。
【００１３】
また、くろずもろみ末を原料とする場合は、すでにくろずもろみ末として製したものを使用するか又は前記のくろずもろみを圧搾しその搾り粕を乾燥粉末化したものを使用し、まず有機溶媒で抽出する。具体的には、くろずもろみ末にメタノールや酢酸エチル等の有機溶媒をを添加し、好ましくは音波破砕（sonication）を行ない、一晩程度攪拌・抽出する。抽出効率の点で、メタノールの使用が好ましい。次いでこの混液をろ過し、ろ液を、例えば減圧下で濃縮し乾固させる。
【００１４】
本発明では、このようにして得られたくろずの有機溶媒抽出物及び／又はくろずもろみ末の有機溶媒抽出物を有効成分として、抗炎症剤とする。くろずの有機溶媒抽出物とくろずもろみ末の有機溶媒抽出物は、厳密に区別する必要はなく、両者を混用してもさしつかえない。これらは、くろずの有機溶媒抽出物及び／又はくろずもろみ末の有機溶媒抽出物を単独で、又は他の抗炎症作用を有する原料を添加し、さらに製剤上慣用の賦形剤、滑沢剤、その他の助剤を加えて、錠剤、顆粒剤、カプセル剤等としてもよく、また散剤又はドリンク剤としてもよい。
【００１５】
本発明の抗炎症剤の投与は、通常経口によって行なわれ、投与量は、年齢、性別、症状等によって異なるが、成人男子の場合、 1日10mg〜100mg （くろず乾燥物換算）を 1回ないし数回に分けて投与するとよい。
【００１６】
次に、本発明の薬理作用について説明する。
本発明に係る抗炎症剤の有効成分は、くろず又はくろずもろみもしくはくろずもろみ末として従来から飲食に供されている成分であって、急性毒性は存在しないことはもちろん、副作用も生じない。
【００１７】
本発明者は、くろず及び／又はくろずもろみ末の抗炎症活性の指標として、細胞質ホスフォリパーゼA2（以下、cPLA2 で表す）阻害活性とO2 - の産生阻害活性を測定することにした。cPLA2 は、炎症やアレルギー反応のメディエーターであるプロシタグランジンやロイコトリエン等の前駆体であるアラキドン酸をリン脂質から特異的に遊離させる酵素である。また、cPLA2 は、別の炎症メディエターである血小板活性化因子産生律速酵素でもある。一方、白血球（リンパ球、マクロファージ、 好中球、好酸球等）は、生体感染防御の大きな役割を担っており、好中球等はO2 - の産生によって細菌による感染から生体を守っている。しかし、その一方でO2 - は炎症における組織障害、老化、又は発癌の原因であり、種々の疾患にも関与していると言われている。したがってO2 - 産生阻害物質は、抗炎症剤をはじめ、その他さまざまな疾患の治療薬の開発に重要な役割を果たすものである。
【００１８】
以上のことから、本発明者は、くろず及びくろずもろみ末の抗炎症活性の検討を行うなうにあたり、cPLA2 阻害活性とO2 - 産生阻害活性に着目し、検討することとした。
本発明の抗炎症剤の有効成分であるくろずの有機溶媒抽出物及び／又はくろずもろみ未の有機溶媒抽出物は、以下の試験例に示すように、cPLA2 の作用を阻害する働きをする。また本発明の抗炎症剤は、O2 - の産生を阻害する働きをする。
【００１９】
【発明の実施の形態】
以下、試験例と実施例をもって、本発明をさらに説明する。
【００２０】
【実施例１】
＜くろずの有機溶媒抽出物の乾燥物の製造例＞
まず、くろず500lを秤取し、これに水酸化ナトリウムの適量を添加して中和した。次いで、酢酸エチル200lを加えて 3回抽出を行ない、水層から酢酸エチル層を分離し、これを減圧下で30℃で濃縮し、乾固物100gを得た。
【００２１】
【実施例２】
＜くろずもろみ末の有機溶媒抽出物の乾燥物の製造例＞
まず、くろずもろみ末100gを秤取し、これにメタノール500ml を加えて、音波破砕を行ない、一晩かけて攪拌・抽出した後ろ過し、ろ液を減圧下で30℃で濃縮し、乾固物 12gを得た。
【００２２】
【実施例３】
＜抗炎症カプセル剤の製造例＞
実施例１で製した乾燥物100gにデキストリン100g混じた粉末にエタノールを少量添加して混捏し、カプセル充填機器によってゼラチンカプセルのボディに各1g宛詰め、キャップをして、抗炎症カプセル剤を 195個製造した。
【００２３】
【実施例４】
＜抗炎症散剤の製造例＞
実施例２で製した乾燥物をさらに微粉末化し、各2gごとに分包して、くろずもろみ末の有機溶媒抽出物の乾燥物100%からなる散剤５包を製した。
【００２４】
【試験例１】
＜cPLA2 阻害活性試験＞
実施例１と実施例２で製した乾燥物の 100mgを、それぞれ、水100ml に溶解して試料溶液とした。
cPLA2 阻害活性は、 2位のアシル基を放射標識したグリセロリン脂質を基質に用いてcPLA2 反応により生成した遊離脂肪酸を、Doleの試薬で溶媒分画し、分解量を測定することにより算定した。対照には、cPLA2 を特異的に阻害するアラキドン酸トリフルオロメチルケトン（AACOCF3:Cayman chemical company）を用いた。
すなわち、4mM CaCl2、0.1%遊離脂肪酸フリーBSA を含む100mM Tris-HCl (pH9)の 200μl に前記試料溶液 2μl を添加し、37℃で20分間インキュベートした。この上に調製基質50μl(50nmol/50,000dpm/ 50μl)を添加し、37℃で20分間反応させた。この反応により遊離した[14C]-アラキドン酸をDoleの方法(Natori,Y.etal,J.Biochem.,93,631 (1983))で回収して放射活性を計数した。その結果を図１に示す。
【００２５】
＜ｃＰＬＡ２阻害活性の評価＞
図１に示すとおり、くろずの酢酸エチル抽出物とくろずもろみ末のメタノール抽出物には、対照のＡＡＣＯＣＦ３の阻害活性（ＩＣ５０＝０．１μｇ／ｍｌ）に比べて、おだやかな、ｃＰＬＡ２阻害活性が認められた。
くろずの酢酸エチル抽出物；ＩＣ５０＝５４μｇ／ｍｌ
くろずもろみ末のメタノール抽出物；ＩＣ５０＝２８μｇ／ｍｌ
一方、くろずの水層の阻害活性は低く、１００μｇ／ｍｌでも５０％阻害に満たなかった。
【００２６】
【試験例２】
＜O2 - 産生阻害活性試験＞
好中球様HL60細胞をPMA (phorbolmyristateacetate) で刺激したときのO2 - 産生に対する阻害効果を検討した。
O2 - 産生量は、チトクロムC (cytochrome C)のO2 - による還元型チトクロムC への変換を比色定量して測定した。
すなわち、ヒト骨髄性白血病由来HL60細胞を1.4%DMSOと10%FCSを添加したRPMI1640培地で 4日間培養して好中球に分化させ、4 〜5 ×106cells/mlの細胞懸濁液に調製した。この細胞懸濁液25μl にME 50倍希釈した試料溶液25μl を添加し、37℃で20分間インキュベートした。これに、4mg チトクロムC 及び200ngPMAをMEM 1ml 中に含む反応液を50μl 添加し、37℃で 1時間インキュベートした。この波長550nm の吸光度を測定し、対照波長570nm の吸光度を測定して生成した還元型チトクロムC の定量を行ない、O2 - 産生阻害活性を算出した。その結果を図２に示す。
【００２７】
＜O2 - 産生阻害活性の評価＞
図２に示すとおり、くろずもろみ末のメタノール抽出物には、おだやかなO2- 産生阻害活性が認められた。再現性を確認するために再度抽出、活性試験を行なった。その結果、いずれの場合も 100μg/mlで活性を示した。
【００２８】
【発明の効果】
以上詳細に説明したとおり、本発明によって得られる抗炎症剤は、cPLA2 阻害作用とO2 - 産生阻害作用を奏するので、おだやかな抗炎症剤として有効である。その上、本発明に係る抗炎症剤は、長期間にわたって摂取を続けても、また大量に投与しても、副作用がほとんど生じない。したがって、本発明の抗炎症剤は、安全であるので、抗炎症作用により、リウマチ、皮膚病、肩関節周囲炎（いわゆる肩凝り）等の疾病や疾患の予防及び治療にきわめて適しており、ヒトの健康維持に大きな効果を奏するものである。
特に、長期間投与して抗炎症作用を発揮するので、健康食品ないし機能性食品としても有用である。
【図面の簡単な説明】
【図１】本発明のくろずの酢酸エチル抽出物又はくろずもろみ末のメタノール抽出物を有効成分とする抗炎症剤の細胞質ホスフォリパーゼA2阻害活性を示す。
【図２】本発明のくろずもろみ末のメタノール抽出物を有効成分とする抗炎症剤のO2- 産生阻害活性を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an anti-inflammatory agent. More specifically, the present invention relates to an anti-inflammatory agent using as a raw material kurozu (black vinegar) or kurozu moromi powder (residue powder obtained by removing kurozu from kurozu moromi).
The anti-inflammatory agent of the present invention exhibits a mild anti-inflammatory action, and can prevent or treat inflammation with few side effects even when taken in a large amount over a long period of time.
[0002]
[Prior art]
In general, inflammatory diseases in the body include infections caused by pathogenic microorganisms, injuries caused by physical or chemical inflammation, normal reactive inflammation such as gout due to metabolic abnormalities, various allergies, autoimmune diseases, mesenchymal ataxia It is distinguished from abnormally responsive inflammation, including syndromes, etc., but in any case, the basic symptoms and signs are fever, redness, swelling, pain, and often accompanied by functional impairment .
[0003]
Anti-inflammatory agents (anti-inflammatory agents) are drugs that antagonize these inflammatory processes or suppress inflammatory symptoms. Antihistamines (anti-allergic agents), steroidal anti-inflammatory agents (corticosteroids), Non-steroidal anti-inflammatory agents, anti-inflammatory enzymes, immunosuppressive agents, anti-rheumatic agents, antipyretic analgesics and the like are known.
[0004]
As is well known, these anti-inflammatory agents generally have limited use and have strong side effects. For example, steroidal anti-inflammatory agents have a strong anti-inflammatory effect, but when used over a long period or in large quantities, they are susceptible to swelling, moon face, diabetes and stomach ulcer deterioration, and various infectious diseases. It is easy to have side effects. In addition, the optimum dose varies among individuals and varies from patient to patient. Therefore, it is difficult to determine the dose, and if the dose is incorrect, the adverse effect of promoting inflammatory symptoms may occur. Non-steroidal anti-inflammatory drugs are not as good as steroidal anti-inflammatory drugs, but side effects such as loss of appetite, dizziness, nausea, vomiting, abdominal pain, diarrhea, and headache are likely to occur, sometimes swelling, anemia, blood pressure Symptoms such as elevation, stomatitis, palpitation, proteinuria, hematuria may appear. Other anti-inflammatory agents often have similar side effects. For this reason, development of an anti-inflammatory agent that does not cause side effects even when administered in a large amount over a long period of time is strongly desired.
[0005]
[Problems to be solved by the invention]
In view of the above circumstances, the present inventor has earnestly developed a novel anti-inflammatory agent that exhibits a mild anti-inflammatory action and does not cause side effects even when administered over a long period of time or ingested in a large amount. Made an effort. As a result, the inventors have paid attention to the various types of bioactive activity of kurozu and kurozu moromi powder, and have studied this as a raw material, thereby completing the present invention.
[0006]
The present invention has been made as described above, and the object of the present invention is to provide a mild anti-inflammatory effect, and there are almost no side effects even if it is ingested over a long period of time or administered in a large amount. It is to provide a novel anti-inflammatory agent that does not occur.
[0007]
[Means for Solving the Problems]
The present invention for achieving the above object is an anti-inflammatory agent comprising a dried product of an organic solvent extract of kuruzu as an active ingredient.
In this case, it is desirable to use ethyl acetate as the organic solvent.
[0008]
Moreover, this invention is an anti-inflammatory agent which uses the dried material of the organic-solvent extract of kurozu moromi powder as an active ingredient.
In this case, it is desirable to use methanol as the organic solvent.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The anti-inflammatory agent of the present invention uses kurzu or kuromo moromi powder as a raw material. Kurozu, which is used as a raw material in the present invention, has been traditionally produced mainly in the Fukuyama region of Kagoshima, and has recently attracted attention as a raw material for health drinks and health foods. According to its general manufacturing method, Kurozu uses rice bran, steamed rice, and water in brewing turtles arranged in the field, and saccharification, alcoholic fermentation, and acetic acid fermentation are carried out by the power of solar energy. When it is allowed to proceed, and after a long aging period, it becomes a crumbly crumb having a unique flavor and various useful ingredients, which is produced by pressing and filtering.
[0010]
In the present invention, kurozu-moromi refers to a fermented product obtained by fermenting and ripening a kurukuri raw material as described above, that is, a fermented product in which kurozu is mixed. Further, the term “kurazu moromi powder” refers to a powder obtained by drying a residue obtained by removing the liquid kuruku from the kuromo moromi. Kurozu moromi powder can be produced by various conventionally known methods, for example, those produced by the method described in JP-A-61-58577 and the like can be used.
[0011]
In addition to the general effects of ordinary vinegar such as appetite enhancing action, digestion absorption promoting action, bactericidal power / antiseptic action, bleaching action, etc., Kurozu and Kurozu Moromi powder have stiff shoulders, fifty shoulders, constipation, fatigue, high blood pressure, It is said to be effective for arteriosclerosis, diabetes, obesity, chronic hepatitis, neuralgia, rheumatism, fundus bleeding, and the like. Recently, it has also been announced that it has the effect of lowering blood sugar, blood lipids, and serum cholesterol content. Further, it has been elucidated that the components of kuruzu contain proteins, free amino acids, oligopeptides and the like.
[0012]
In the present invention, when the raw material is black lead, the black lead is first extracted with an organic solvent. Specifically, a basic substance such as sodium hydroxide or potassium carbonate is added to neutralize and neutralized, and an organic solvent such as ethyl acetate, methylene chloride, or benzene is added for extraction several times. From the viewpoint of extraction efficiency, it is preferable to use ethyl acetate. The case where ethyl acetate is used will be described as an example. Next, the extract is separated into an aqueous layer (ethyl acetate insoluble material layer) and an ethyl acetate layer (ethyl acetate soluble material layer). For example, concentrate under reduced pressure and dry.
[0013]
In addition, when using kurozu moromi powder as a raw material, use the one already produced as a kurozu moromi powder or use the one obtained by squeezing the above kuromo mash and drying the squeezed rice cake, Extract with solvent. Specifically, an organic solvent such as methanol or ethyl acetate is added to the kuromo-moji powder, preferably sonicated, and stirred and extracted overnight. In view of extraction efficiency, use of methanol is preferred. The mixture is then filtered and the filtrate is concentrated to dryness, eg, under reduced pressure.
[0014]
In the present invention, the thus obtained organic solvent extract of kuruku and / or the organic solvent extract of kuromo mash is used as an active ingredient to be an anti-inflammatory agent. The organic solvent extract of Kurozu and the organic solvent extract of Kurozu Moromi do not need to be strictly distinguished, and both may be used together. These are obtained by adding an organic solvent extract of kurzu and / or an organic solvent extract of kurzu moromi powder alone or other raw materials having an anti-inflammatory action, and further using conventional excipients and lubricants in formulation. Additives and other auxiliaries may be added to form tablets, granules, capsules, etc., or powders or drinks.
[0015]
Administration of the anti-inflammatory agent of the present invention is usually carried out orally, and the dosage varies depending on age, sex, symptoms, etc. In the case of adult males, 10 mg to 100 mg (converted as dry matter) once a day Or it may be divided into several times.
[0016]
Next, the pharmacological action of the present invention will be described.
The active ingredient of the anti-inflammatory agent according to the present invention is a component that has been conventionally used for eating and drinking as kuruku or kuromo moromi or koji kuromi mash, and has no acute toxicity and of course no side effects .
[0017]
The present inventors as an index of black not a and / or black not a mash at the end of anti-inflammatory activity, cytosolic phospholipase A 2 (hereinafter, represented by cPLA 2) inhibitory activity and O 2 - measuring the production inhibitory activity I made it. cPLA 2 is an enzyme that specifically releases arachidonic acid, which is a precursor of prostaglandins and leukotrienes, which are mediators of inflammation and allergic reactions, from phospholipids. CPLA 2 is also a rate-limiting enzyme that produces platelet activating factor, which is another inflammatory mediator. On the other hand, white blood cells (lymphocytes, macrophages, neutrophils, eosinophils, etc.) play a major role in protecting living infections, and neutrophils protect the living body from infection by bacteria by producing O 2 −. Yes. On the other hand, however, O 2 − is a cause of tissue damage, aging, or carcinogenesis in inflammation, and is said to be involved in various diseases. Thus O 2 - production inhibitor, including anti-inflammatory agents are those that play an important role in the development of therapeutic agents for various other diseases.
[0018]
From the above, the present inventors, when now to examine the black not a and black not a mash at the end of anti-inflammatory activity, cPLA 2 inhibitory activity and with the O 2 - Focusing on production inhibitory activity, have to be considered.
As shown in the following test examples, an organic solvent extract of kurzu and / or an unsolved organic solvent extract that is an active ingredient of the anti-inflammatory agent of the present invention acts to inhibit the action of cPLA 2. To do. The anti-inflammatory agent of the present invention functions to inhibit O 2 − production.
[0019]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be further described with test examples and examples.
[0020]
[Example 1]
<Example of production of dried product of organic solvent extract of Kurozu>
First, 500 liters of kuruzu were weighed and neutralized by adding an appropriate amount of sodium hydroxide. Subsequently, 200 l of ethyl acetate was added and extraction was performed three times. The ethyl acetate layer was separated from the aqueous layer, and this was concentrated at 30 ° C. under reduced pressure to obtain 100 g of a dried solid product.
[0021]
[Example 2]
<Example of production of dried product of organic solvent extract of Kurozu Moromi powder>
First, 100 g of Kurozu Moromi powder is weighed, 500 ml of methanol is added to this, sonicated, stirred and extracted overnight, filtered, and the filtrate is concentrated at 30 ° C. under reduced pressure and dried. 12 g of solid material was obtained.
[0022]
[Example 3]
<Example of production of anti-inflammatory capsule>
A small amount of ethanol is added to the powder of 100 g of dextrin mixed with 100 g of the dried product prepared in Example 1, and 1 g each is packed into the body of the gelatin capsule using a capsule filling machine, and the cap is capped to obtain an anti-inflammatory capsule. Individually manufactured.
[0023]
[Example 4]
<Example of production of anti-inflammatory powder>
The dried product produced in Example 2 was further pulverized and packaged every 2 g to produce 5 powdered powders made of 100% dried product of the organic solvent extract of kuromo mash.
[0024]
[Test Example 1]
<CPLA 2 inhibitory activity test>
100 mg of the dried product produced in Example 1 and Example 2 was dissolved in 100 ml of water to prepare a sample solution.
The cPLA 2 inhibitory activity was calculated by fractionating the free fatty acid produced by the cPLA 2 reaction using glycerophospholipid radiolabeled with the acyl group at position 2 with Dole's reagent and measuring the amount of degradation. . As a control, arachidonic acid trifluoromethyl ketone (AACOCF 3 : Cayman chemical company) that specifically inhibits cPLA 2 was used.
That is, 2 μl of the sample solution was added to 200 μl of 100 mM Tris-HCl (pH 9) containing 4 mM CaCl 2 and 0.1% free fatty acid-free BSA, and incubated at 37 ° C. for 20 minutes. Thereto was added 50 μl (50 nmol / 50,000 dpm / 50 μl) of the prepared substrate and allowed to react at 37 ° C. for 20 minutes. [ 14 C] -arachidonic acid released by this reaction was recovered by the method of Dole (Natori, Y. etal, J. Biochem., 93 , 631 (1983)) and the radioactivity was counted. The result is shown in FIG.
[0025]
<Evaluation of cPLA 2 inhibitory activity>
As shown in FIG. 1, the ethyl acetate extract of Kurozu and the methanol extract of Kurozu Moromi powder have a milder cPLA than the control activity of AACOCF 3 (IC 50 = 0.1 μg / ml). 2 Inhibitory activity was observed.
Kurozu ethyl acetate extract; IC 50 = 54 μg / ml
Methanol extract of Kurozu Moromi powder; IC 50 = 28 μg / ml
On the other hand, the inhibitory activity of the aqueous layer of Kurozu was low, and even at 100 μg / ml, it was less than 50% inhibition.
[0026]
[Test Example 2]
<O 2 - production inhibitory activity test>
It was investigated the inhibitory effect on the production - O 2 when the neutrophil-like HL60 cells were stimulated with PMA (phorbolmyristateacetate).
The amount of O 2 − produced was measured by colorimetric determination of the conversion of cytochrome C into reduced cytochrome C by O 2 − .
In other words, human myeloid leukemia-derived HL60 cells were cultured in RPMI1640 medium supplemented with 1.4% DMSO and 10% FCS for 4 days to differentiate into neutrophils, resulting in a cell suspension of 4-5 × 10 6 cells / ml. Prepared. 25 μl of a sample solution diluted 50-fold ME was added to 25 μl of this cell suspension, and incubated at 37 ° C. for 20 minutes. To this, 50 μl of a reaction solution containing 4 mg cytochrome C and 200 ngPMA in 1 ml of MEM was added and incubated at 37 ° C. for 1 hour. The absorbance at a wavelength of 550nm was measured, performs quantification of reduced cytochrome C produced by measuring the absorbance of the reference wavelength 570 nm, O 2 - was calculated production inhibitory activity. The result is shown in FIG.
[0027]
<O 2 - evaluation of production inhibitory activity>
As shown in FIG. 2, a gentle O 2 − production inhibitory activity was observed in the methanol extract of Kurozu Moromi powder. In order to confirm reproducibility, extraction and activity tests were performed again. As a result, the activity was shown at 100 μg / ml in all cases.
[0028]
【The invention's effect】
As described above in detail, an anti-inflammatory agent obtained by the present invention, cPLA 2 inhibitory activity and O 2 - so achieve the production inhibitory effect and is effective as a mild anti-inflammatory agents. Moreover, the anti-inflammatory agent according to the present invention has almost no side effects even if it is ingested over a long period of time or administered in a large amount. Therefore, since the anti-inflammatory agent of the present invention is safe, it is very suitable for the prevention and treatment of diseases and disorders such as rheumatism, dermatoses, shoulder periarthritis (so-called shoulder stiffness) due to its anti-inflammatory action, and human It has a great effect on maintaining health.
In particular, since it exhibits an anti-inflammatory effect when administered for a long time, it is also useful as a health food or functional food.
[Brief description of the drawings]
FIG. 1 shows the cytosolic phospholipase A 2 inhibitory activity of an anti-inflammatory agent comprising, as an active ingredient, an ethyl acetate extract of kurozu or a methanol extract of kuromo mash.
FIG. 2 shows the O 2 − production inhibitory activity of an anti-inflammatory agent comprising as an active ingredient a methanol extract of the kurozu moromi powder of the present invention.
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP29761598A JP4344030B2 (en) | 1998-10-05 | 1998-10-05 | Anti-inflammatory agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP29761598A JP4344030B2 (en) | 1998-10-05 | 1998-10-05 | Anti-inflammatory agent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2000109432A JP2000109432A (en) | 2000-04-18 |
| JP4344030B2 true JP4344030B2 (en) | 2009-10-14 |
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| Application Number | Title | Priority Date | Filing Date |
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| JP29761598A Expired - Fee Related JP4344030B2 (en) | 1998-10-05 | 1998-10-05 | Anti-inflammatory agent |
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| JP (1) | JP4344030B2 (en) |
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| JP4681091B2 (en) * | 1999-07-15 | 2011-05-11 | 坂元醸造株式会社 | Drugs that promote the prevention and treatment of osteoporosis |
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