JP2012206958A - Vascular endothelial function improving agent - Google Patents
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本発明は血管内皮機能改善剤に関し、より詳細には、米糠を圧搾して得られた粗米糠油を、アルカリ剤を用いた処理を行なわず、少なくとも水蒸気蒸留による精製処理を行なった精製米糠油からなる血管内皮機能改善剤に関する。 The present invention relates to a vascular endothelial function improving agent, and more specifically, purified rice bran oil obtained by subjecting crude rice bran oil obtained by squeezing rice bran to at least a purification process by steam distillation without performing a treatment using an alkaline agent. The vascular endothelial function improving agent which consists of.
血管内皮は、血液成分などの血管透過性、血管の収縮や弛緩反応、凝固線溶系に関与することによる血液凝固性の調節、血小板機能の制御などさまざま重要な機能を有している。これらの機能は、血管内皮細胞がさまざまな生理活性因子を産生、分泌することと深く関係している。特に、生理活性因子の一つである一酸化窒素は、前記の血管の収縮や弛緩反応、凝固線溶系に関与することによる血液凝固性の調節などと極めて深い関係にあり、血管組織の恒常性を維持する重要な因子である。一酸化窒素を血管内皮細胞が産生、分泌する際には、血管内皮型一酸化窒素産生酵素（以下、［ｅ-ＮＯＳ］と略することがある。）が重要な役割を果たしており、この酵素活性が抑制されると高血圧症、脂質異常症、上昇高脂血症、動脈硬化、血栓症、糖尿病、糖尿病合併症などの発生に影響を与えることがわかっている。加えて、ｅ-ＮＯＳの活性は、高血圧症、脂質異常症、上昇高脂血症、動脈硬化、血栓症、糖尿病、糖尿病合併症などによって血管内皮細胞が障害を受けると低下し、更に疾病の進行を促進させることも知られている。これらは慢性的な疾患であり、その予防や治療には長期間を要する必要がある。従って、長期間にわたって安全かつ効果的に、ｅ-ＮＯＳの活性低下を防止し、または活性を向上できる技術の開発が望まれている。 The vascular endothelium has various important functions such as vascular permeability of blood components, vasoconstriction and relaxation reactions, regulation of blood coagulation by participating in the coagulation and fibrinolytic system, and control of platelet function. These functions are closely related to the production and secretion of various physiologically active factors by vascular endothelial cells. In particular, nitric oxide, which is one of the physiologically active factors, has a very deep relationship with the above-mentioned blood vessel contraction and relaxation reactions, regulation of blood coagulation by participating in the coagulation and fibrinolysis system, etc., and vascular tissue homeostasis Is an important factor to maintain. When vascular endothelial cells produce and secrete nitric oxide, a vascular endothelial nitric oxide producing enzyme (hereinafter sometimes abbreviated as [e-NOS]) plays an important role. Inhibition of activity has been shown to affect the occurrence of hypertension, dyslipidemia, elevated hyperlipidemia, arteriosclerosis, thrombosis, diabetes, diabetic complications, and the like. In addition, the activity of e-NOS decreases when vascular endothelial cells are damaged by hypertension, dyslipidemia, elevated hyperlipidemia, arteriosclerosis, thrombosis, diabetes, diabetic complications, etc. It is also known to promote progression. These are chronic diseases, and the prevention and treatment thereof require a long time. Therefore, it is desired to develop a technique that can prevent or improve the activity of e-NOS safely and effectively over a long period of time.
この要望に対して、従来より種々の技術が提案されている。例えば、血管内皮成長因子や同因子のレセプターアゴニストを使用する方法（特許文献１）、ペプチドを使用する方法（特許文献２）、フェネチルニコチンアミド誘導体を使用する方法（特許文献３）、プテリン誘導体を使用する方法（特許文献４）、ポリシロキサン誘導体を使用する方法（特許文献５）、ＨＭＧ-ＣｏＡリダクターゼ阻害剤とγ-オリザノールやグルタチオンを併用する方法（特許文献６，７）などが挙げられる。しかし、これらは何れも合成された化合物を活用する技術であり、それらの技術を実施する場合には医師や薬剤師などの専門家の指示や指導を受ける必要があったり、長期間使用する場合に副作用が発生する恐れがあったり、予防的に長期間使用することが出来なかったりするなどの課題点があり、より安全性が高く、手軽に長期間使用できる効果的な技術の開発が望まれていた。 In response to this demand, various techniques have been proposed. For example, a method using a vascular endothelial growth factor or a receptor agonist of the same factor (Patent Document 1), a method using a peptide (Patent Document 2), a method using a phenethylnicotinamide derivative (Patent Document 3), a pterin derivative Examples include a method of using (Patent Document 4), a method of using a polysiloxane derivative (Patent Document 5), and a method of using a HMG-CoA reductase inhibitor in combination with γ-oryzanol or glutathione (Patent Documents 6 and 7). However, these are technologies that utilize synthesized compounds, and it is necessary to receive instructions and guidance from specialists such as doctors and pharmacists when implementing these technologies, or when they are used for a long time. There are problems such as the possibility of side effects and the inability to use prophylactically for a long period of time, and the development of effective technology that is safer and can be used for a long period of time is desired. It was.
本発明は、長期間摂取しても高い安全性に懸念のない、血管内皮細胞由来のｅ-ＮＯＳを活性化することで一酸化窒素の産生量を亢進させる、血管内皮機能改善剤を提供することを目的とする。 The present invention provides a vascular endothelial function-improving agent that enhances the production of nitric oxide by activating vascular endothelial cell-derived e-NOS without concern for high safety even when ingested for a long period of time. For the purpose.
本発明者らは、驚くべき事に、米糠を圧搾して得られた粗米糠油を、アルカリ剤を用いた処理を行なわず、少なくとも水蒸気蒸留による精製処理を行なった精製米糠油に優れたｅ-ＮＯＳ活性化効果が存在することを見出し、本発明を完成させるに至った。 Surprisingly, the inventors of the present invention are excellent in refined rice bran oil obtained by subjecting crude rice bran oil obtained by squeezing rice bran to at least steam distillation without performing treatment using an alkaline agent. The present inventors have found that a NOS activation effect exists and completed the present invention.
すなわち、本発明は例えば以下の項１〜３に係る血管内皮機能改善剤を包含する。
項１．圧搾して得られる米糠油を、少なくとも水蒸気蒸留処理を行なうことにより得られる圧搾精製米糠油からなる血管内皮機能改善剤。
項２．前記圧搾精製米糠油が、トリグリセリド８０〜９０質量％、γ-オリザノール０．５〜２．５質量％、β-シトステロール０．５〜１．５質量％、トコフェロール類（α-、β-、γ-、δ-トコフェロールの総量）０．０３〜０．０７質量％、ロウ分０.１質量％以下、遊離脂肪酸１質量％以下、水分０．１質量％以下の組成を有することを特徴とする項１に記載の血管内皮機能改善剤。
項３．アルカリ剤を用いた処理工程が含まれない製造方法を用いて得られる項１または２に記載の血管内皮機能改善剤。
That is, this invention includes the vascular endothelial function improving agent which concerns on the following items 1-3.
Item 1. An agent for improving vascular endothelial function, comprising press-purified rice bran oil obtained by subjecting rice bran oil obtained by pressing to steam distillation.
Item 2. The pressed refined rice bran oil contains 80 to 90% by mass of triglyceride, 0.5 to 2.5% by mass of γ-oryzanol, 0.5 to 1.5% by mass of β-sitosterol, tocopherols (α-, β-, γ -, The total amount of δ-tocopherol) 0.03-0.07 mass%, wax content 0.1 mass% or less, free fatty acid 1 mass% or less, moisture 0.1 mass% or less Item 2. The vascular endothelial function improving agent according to Item 1.
Item 3. Item 3. The vascular endothelial function improving agent according to Item 1 or 2, obtained using a production method that does not include a treatment step using an alkaline agent.
本発明の血管内皮機能改善剤は、古くから日本で食経験がある食品成分である米糠油成分から構成されることから、長期に渡って摂取しても副作用を生じるなどの安全性上の懸念が全くないことから、専門家の指示がなくても安心して使用でき、また、予防的に長期間摂取することが出来る。また、毎年、多量に産出される米糠を原料とするため、安価に入手することが可能である。この血管内皮機能改善剤を継続して摂取することにより、高血圧症、脂質異常症、上昇高脂血症、動脈硬化症、血栓症、糖尿病、糖尿病合併症などの発症予防やこれらの症状を緩和させる効果を期待することが出来る。 The vascular endothelial function improving agent of the present invention is composed of a rice bran oil component, which is a food component that has been eaten in Japan for a long time, and therefore safety concerns such as causing side effects even when ingested over a long period of time. Therefore, it can be used with peace of mind without any professional instruction, and can be taken for a long time for preventive purposes. Moreover, since rice bran produced in large quantities every year is used as a raw material, it can be obtained at low cost. Continuous intake of this vascular endothelial function improver prevents or reduces the onset of hypertension, dyslipidemia, elevated hyperlipidemia, arteriosclerosis, thrombosis, diabetes, diabetic complications, etc. You can expect the effect to make.
以下、本発明について、さらに詳細に説明する。 Hereinafter, the present invention will be described in more detail.
本発明の血管内皮機能改善剤は、米糠を圧搾して得られる米糠油の精製処理において、アルカリ剤を用いる精製処理を行なわず、少なくとも水蒸気蒸留を用いた精製処理を行うことを特徴とするものである。以下に詳しく説明する。 The agent for improving vascular endothelial function of the present invention is characterized in that, in the purification process of rice bran oil obtained by pressing rice bran, a purification process using at least steam distillation is performed without performing a purification process using an alkaline agent. It is. This will be described in detail below.
圧搾搾油に供する米糠は、赤糠が好ましい。米糠は多量の油脂を含有するため、リパーゼによる油脂分解による品質の劣化が生じやすい。そのため、生成した米糠は、できるだけ早急に、加熱水蒸気法、超高温過熱法などの方法を用いて約１００〜１３０℃まで米糠の温度を上昇させることによりリパーゼを失活させ、米糠の品質劣化を防止する処理を行なう。米糠の搾油は、通常、脱脂効率の良いヘキサンなどの有機溶媒を用いた抽出法が用いられるが、この有機溶媒を用いた抽出方法で得られた米糠油の場合、所期の効果が得られないため、米糠に圧力を負荷させることにより油分を削減する圧搾法による脱脂処理を行う。この圧搾処理には、特に限定するものではないが、スクリュー式搾油機（例：Ｓ100-400（（株）サン精機社製）、一軸エキスペラー（例：搾油機Ｖ-05（（株）スエヒロＥＰＭ社製）、低温連続圧搾機（例：ミラクルチャンバー（（株）テクノシグマ社製）などの装置を用いることができる。（以下、圧搾法を用いて得られた米糠油を「圧搾米糠油」ということがある。）抽出法により得られる米糠油は、使用する有機溶媒に溶解性を有する非極性物質から構成されるが、圧搾米糠油は、米糠中に存在する圧搾処理条件下において液状となる物質から構成されるため、両者の組成は同一ではない。
The rice bran used for pressing oil is preferably red rice bran. Rice bran contains a large amount of fats and oils, so quality is liable to deteriorate due to the decomposition of fats and oils by lipase. Therefore, the produced rice bran deactivates the lipase as soon as possible by raising the temperature of the rice bran to about 100 to 130 ° C. using a method such as a heated steam method or an ultra-high temperature superheating method, thereby reducing the quality of the rice bran. Prevent the process. Extraction of rice bran oil is usually performed using an extraction method using an organic solvent such as hexane with good degreasing efficiency. In the case of rice bran oil obtained by this extraction method using an organic solvent, the desired effect is obtained. Therefore, degreasing is performed by a pressing method that reduces oil content by applying pressure to rice bran. Although not specifically limited to this pressing process, a screw type oil press (example: S100-400 (manufactured by Sun Seiki Co., Ltd.)), a single screw expeller (example: oil press V-05 (Suehiro EPM Co., Ltd.) ), Low temperature continuous pressing machine (example: Miracle Chamber (manufactured by Techno Sigma Co., Ltd.), etc.) (hereinafter referred to as “pressed rice bran oil”) The rice bran oil obtained by the extraction method is composed of a nonpolar substance that is soluble in the organic solvent used, but the pressed rice bran oil is liquid under the pressing treatment conditions present in the rice bran. The composition of the two is not the same.
得られた圧搾米糠油は、フィルタープレスなどを用いた濾過により夾雑物を除去した後に、以下の処理を順次行なう。なお、前記処理を施した圧搾米糠油を、粗圧搾米糠油という。 The obtained pressed rice bran oil is subjected to the following treatment in order after removing impurities by filtration using a filter press or the like. In addition, the pressed rice bran oil which performed the said process is called rough pressed rice bran oil.
先ず、粗圧搾米糠油に水蒸気または水を添加する。前記処理は、粗圧搾米糠油を加温、撹拌しながら、添加することにより行う。添加処理が完了した後も撹拌を継続し、その後、生成した不溶化物質を遠心分離機等で除去する。この処理工程において、水蒸気や水の代わりにクエン酸等の有機酸を溶解した水溶液を使用することもできる。水蒸気や水を添加する際の米糠油の温度は、４０〜１００℃が好ましい。
First, steam or water is added to the coarsely pressed rice bran oil. The said process is performed by adding coarsely pressed rice bran oil while heating and stirring. Stirring is continued even after the addition treatment is completed, and then the generated insolubilized material is removed with a centrifuge or the like. In this treatment step, an aqueous solution in which an organic acid such as citric acid is dissolved can be used instead of water vapor or water. As for the temperature of the rice bran oil at the time of adding water vapor | steam and water, 40-100 degreeC is preferable.
次いで、脱色処理、脱ロウ・ウィンター処理を行なう。これらの処理の順序は問わない。脱色処理は、前記の処理を行なった粗圧搾米糠油に酸性白土や活性白色土を添加し、撹拌することにより着色成分や着色原因になる成分を活性白色土に吸着させ、その後フィルタープレスなどを用いて白土を除去することにより行う。白土を除去の効率を高める目的で、白土に加えてケイソウ土などの濾過助剤を添加しても良い。処理時の粗圧搾米糠油の温度は７０〜１４０℃であり、撹拌時間（処理時間）は１０〜３０分が好ましい。また、脱ロウ・ウィンター処理は、粗圧搾米糠油を０〜１０℃まで冷却し、１０〜５０時間放置した後、公知の方法を用いて濾過すること行う。
Next, decolorization processing and dewaxing / winter processing are performed. The order of these processes does not matter. The decoloring treatment is performed by adding acidic white clay or active white clay to the coarsely-pressed rice bran oil that has been subjected to the above-mentioned treatment, and adsorbing the colored components or components causing the coloring to the active white soil by stirring, and then performing a filter press or the like. Use to remove the white clay. In order to increase the efficiency of removing white clay, a filter aid such as diatomaceous earth may be added in addition to white clay. The temperature of the coarsely pressed rice bran oil during the treatment is 70 to 140 ° C., and the stirring time (treatment time) is preferably 10 to 30 minutes. In addition, the dewaxing / winter treatment is performed by cooling the coarsely pressed rice bran oil to 0 to 10 ° C. and allowing it to stand for 10 to 50 hours, followed by filtration using a known method.
次いで、水蒸気蒸留を用いた処理を行なう。本発明における水蒸気蒸留は、５ｍｍＨｇ以下の陰圧下条件において、蒸留温度は２３０〜２４５℃、処理時間３０分〜２時間、処理する粗圧搾米糠油に対して３質量％前後の水蒸気量を用いた処理を行うことが好ましい。
Next, treatment using steam distillation is performed. The steam distillation in the present invention was performed under a negative pressure of 5 mmHg or less, a distillation temperature of 230 to 245 ° C., a processing time of 30 minutes to 2 hours, and a steam amount of about 3% by mass with respect to the crude pressed rice bran oil to be processed. It is preferable to carry out the treatment.
前記処理を経て得られた、本発明の血管内皮機能改善剤に使用する精製圧搾米糠油は、トリグリセリドが８０〜９０質量％、γ-オリザノールが０．５〜２.５質量％、β-シトステロールが０．５〜１．５質量％、トコフェロール類（α-、β-、γ-、δ-トコフェロールの総量）０．０３〜０．０７質量％、ロウ分が０.１質量％以下、遊離脂肪酸が１質量％以下、水分０．１質量％以下の組成を有することが好ましい。 The purified compressed rice bran oil used for the vascular endothelial function improving agent of the present invention obtained through the above-mentioned treatment has a triglyceride of 80 to 90% by mass, γ-oryzanol of 0.5 to 2.5% by mass, β-sitosterol. 0.5 to 1.5 mass%, tocopherols (total amount of α-, β-, γ-, and δ-tocopherol) 0.03 to 0.07 mass%, wax content is 0.1 mass% or less, free It is preferable that the fatty acid has a composition of 1% by mass or less and a water content of 0.1% by mass or less.
本発明の血管内皮機能改善剤はそのまま使用でき、必要に応じて、カプセル化、粉末化、顆粒化することが出来る。また、所期の効果を損なわない限り、通常の経口組成物に配合することで使用することもできる。前記経口組成物としては、医薬品組成物、医薬部外品組成物、口腔用組成物および食品組成物が挙げられる。本発明の血管内皮機能改善剤を配合したこれらの経口組成物は、血管内皮機能を改善することで、高血圧症、脂質異常症、上昇高脂血症、動脈硬化症、血栓症、糖尿病、糖尿病合併症などの発症や症状を緩和させる効果が期待できることから、これらの疾病を発症している人や発症するリスクの高い人（例えば、遺伝的なリスクを有している人、メタボリックシンドロームや肥満に該当する人、それぞれの疾病において「境界域」に該当する「血圧が高めの人」「血中性脂肪の気になる人」「コレステロールが高めで気になる人」「食後の血糖値が気になる人」）用の経口組成物として使用できる。 The vascular endothelial function improving agent of the present invention can be used as it is, and can be encapsulated, powdered or granulated as necessary. Moreover, as long as the desired effect is not impaired, it can also be used by mix | blending with a normal oral composition. Examples of the oral composition include pharmaceutical compositions, quasi-drug compositions, oral compositions, and food compositions. These oral compositions containing the vascular endothelial function improving agent of the present invention improve the vascular endothelial function, so that hypertension, dyslipidemia, elevated hyperlipidemia, arteriosclerosis, thrombosis, diabetes, diabetes Because it can be expected to reduce the onset and symptoms of complications, people with these diseases or those who are at high risk (for example, people with genetic risk, metabolic syndrome or obesity) People who fall under the category, “Persons with high blood pressure”, “People who are worried about blood neutral fat”, “People who are worried about high cholesterol” It can be used as an oral composition for those who are interested ”).
以下、本発明を具体的に説明するが、本発明は下記の例に限定されるものではない。
血管内皮細胞におけるｅ−ＮＯＳの活性化
血管内皮細胞由来のｅ−ＮＯＳは、自身がリン酸化されることで活性化し、一酸化窒素の産生を亢進することが知られていることから、血管内皮細胞を用いたリン酸化ｅ−ＮＯＳ量を測定することで血管内皮機能改善効果を評価した。
具体的には下記の手順に従って、評価した。
評価には ウシ大動脈血管内皮細胞 (以下「ＢＡＥＣ」と略する。)を使用した。ＢＡＥＣの培養は、３７℃、ＣＯ２５％の存在下で、１０％ウシ胎児血清（以下、「ＦＢＳ」と略する。）、ペニシリン１００Ｕ/ｍｌおよび ストレプトマイシン１００μｇ/ｍｌを含有するＭ１９９培地 (Ｓｉｇｍａ Ａｌｄｒｉｃｈｓ社製) にて培養し、継代を行った。継代数７のＢＡＥＣをＩ型コラーゲンで被覆した６well培養フラスコ (住友ベークライト（株)社製） に１.６ ×１０５/ wellの濃度で播種した。細胞がコンフルエントになった後、１％のＦＢＳを含有するＭ１９９培地に交換し、２４時間後、被験物質を１％のＦＢＳを含有するＭ１９９培地に目的の濃度になるよう溶解することにより評価した。被検物質としては、下記の組成を有する圧搾抽出した精製米糠油、ヘキサン抽出法で製造された米糠油およびγ―オリザノールを用いた。各々の被検物質は、ウシ血清アルブミン (ナカライテスク（株)社製） にコンジュゲートさせてから、所定の濃度になるように培養細胞に添加した。２４時間培養した後に、培養した細胞を冷ＰＢＳ (-) で洗浄し、氷上でプロテアーゼ阻害剤 (Thermo Scientific社製) を添加したＲＩＰＡ緩衝液(Thermo Scientific社製) を用いて細胞を回収した。遠心分離処理で得られた上清に、ＳＤＳサンプル緩衝液(Thermo Scientific社製) を添加し、加熱することでタンパク質変性した。その後ＳＤＳ−ＰＡＧＥを用いた電気泳動を行い、分離したタンパク質をニトロセルロース膜に転写した。タンパク質を転写したニトロセルロース膜を５％スキムミルクを含む０．０５％ツイーン−２０含有ＰＢＳにてブロッキングし、次いで、定法に従い、1次抗体としてマウス抗リン酸化ｅ―ＮＯＳ抗体 (BD Bioscience社製)、ウサギ抗ｅＮＯＳ抗体 (Cell Signaling Technology社製)、２次抗体として、ＨＲＰ標識抗マウスＩｇＧ抗体 (Invitrogen社製)、ＨＲＰ標識抗ウサギＩｇＧ抗体 (Santa Cruz社製) と反応させた。基質としてＥＣＬ ｐｌｕｓ(GE Healthcare社製) を用いてニトロセルロース膜を化学発光させ、ＬＡＳ４０００ｍｉｎｉ (GE Healthcare社製) システムを用いてリン酸化ｅ−ＮＯＳと総ｅ−ＮＯＳの発光強度を数値化し、血管内皮細胞の生理活性機能の指標になりうるリン酸化ｅ−ＮＯＳ/総ｅ−ＮＯＳ値を求めた。
測定結果を、表１に示す。
評価に供した精製圧搾米糠油について
クッキング処理を施した赤糠を、ミラクルチャンバーを用いて圧搾し、得られた米糠油を、フィルタープレスを用いた濾過により夾雑物を除去し、粗圧搾米糠油を得た。粗圧搾米糠油は、加熱、撹拌条件で水蒸気を添加した後に、不溶物質を除去した。脱色処理、脱ロウ・ウィンター処理の順番で処理した後に、水蒸気蒸留を行い、得られた圧搾精製米糠油を得た。
粗圧搾米糠油は、トリグリセリドが８３．０質量％、γ-オリザノールが１．７７質量％、β-シトステロールが０．９６質量％、トコフェロール類（α-、β-、γ-、δ-トコフェロールの総量）０．０５８質量％の組成を有し、前記処理を行うことにより得られた精製圧搾米糠油は、トリグリセリドが８５．０質量％、γ-オリザノールが０．８０質量％、β-シトステロールが０．７７質量％、トコフェロール類（α-、β-、γ-、δ-トコフェロールの総量）０．０５０質量％、ロウ分が０．０６質量％、遊離脂肪酸が０．１４質量％、水分が０．０２質量％の組成を有していた。
評価に供した精製ヘキサン抽出米糠油について
米糠からヘキサンを用いて油成分を抽出し、蒸留処理を行なうことでヘキサンを除去し粗米糠油を得る。得られた粗米糠油は、ガム質を除去した後に、低温で析出するロウ分の除去工程、水蒸留工程、アルカリ成分を用いた脱酸処理工程、活性白土を用いた脱色工程及び脱臭工程を経て得られたヘキサン抽出による精製米糠油である。
Activation of e-NOS in Vascular Endothelial Cells Since it is known that e-NOS derived from vascular endothelial cells is activated by phosphorylation itself and enhances production of nitric oxide. The effect of improving vascular endothelial function was evaluated by measuring the amount of phosphorylated e-NOS using cells.
Specifically, it evaluated in accordance with the following procedure.
Bovine aortic vascular endothelial cells (hereinafter abbreviated as “BAEC”) were used for the evaluation. The culture of BAEC is M199 medium (Sigma) containing 10% fetal bovine serum (hereinafter abbreviated as “FBS”), penicillin 100 U / ml and streptomycin 100 μg / ml at 37 ° C. in the presence of 5% CO 2 . And subcultured. A 6-well culture flask (manufactured by Sumitomo Bakelite Co., Ltd.) coated with BAEC of passage number 7 with type I collagen was seeded at a concentration of 1.6 × 10 5 / well. After the cells became confluent, they were replaced with M199 medium containing 1% FBS, and 24 hours later, the test substances were evaluated by lysing the test substances in M199 medium containing 1% FBS to a target concentration. . As the test substance, purified rice bran oil extracted by pressing and having the following composition, rice bran oil produced by hexane extraction method and γ-oryzanol were used. Each test substance was conjugated to bovine serum albumin (manufactured by Nacalai Tesque Co., Ltd.), and then added to the cultured cells to a predetermined concentration. After culturing for 24 hours, the cultured cells were washed with cold PBS (−), and the cells were recovered on ice using a RIPA buffer solution (manufactured by Thermo Scientific) to which a protease inhibitor (manufactured by Thermo Scientific) was added. SDS sample buffer (manufactured by Thermo Scientific) was added to the supernatant obtained by centrifugation, and the protein was denatured by heating. Thereafter, electrophoresis using SDS-PAGE was performed, and the separated protein was transferred to a nitrocellulose membrane. The nitrocellulose membrane to which the protein was transferred was blocked with PBS containing 0.05% Tween-20 containing 5% skim milk, and then a mouse anti-phosphorylated e-NOS antibody (manufactured by BD Bioscience) as a primary antibody according to a standard method Rabbit anti-eNOS antibody (manufactured by Cell Signaling Technology) was reacted with HRP-labeled anti-mouse IgG antibody (manufactured by Invitrogen) and HRP-labeled anti-rabbit IgG antibody (manufactured by Santa Cruz) as secondary antibodies. The nitrocellulose membrane is chemiluminescent using ECL plus (manufactured by GE Healthcare) as a substrate, the luminescence intensity of phosphorylated e-NOS and total e-NOS is digitized using the LAS4000mini (manufactured by GE Healthcare) system, and blood vessels The phosphorylated e-NOS / total e-NOS value that can serve as an index of the physiologically active function of endothelial cells was determined.
The measurement results are shown in Table 1.
Red rice bran that has been subjected to cooking treatment for refined compressed rice bran oil subjected to evaluation is compressed using a miracle chamber, and the resulting rice bran oil is filtered using a filter press to remove impurities, and coarsely pressed rice bran oil Got. Crude pressed rice bran oil removed water-insoluble substances after adding water vapor under heating and stirring conditions. After processing in the order of decolorization processing and dewaxing / winter processing, steam distillation was performed, and the resulting pressed and refined rice bran oil was obtained.
Crude pressed rice bran oil is composed of 83.0% by mass of triglyceride, 1.77% by mass of γ-oryzanol, 0.96% by mass of β-sitosterol, tocopherols (of α-, β-, γ-, δ-tocopherol). The refined compressed rice bran oil having a composition of 0.058% by mass and obtained by the above treatment is 85.0% by mass of triglyceride, 0.80% by mass of γ-oryzanol, and β-sitosterol. 0.77 mass%, tocopherols (total amount of α-, β-, γ-, δ-tocopherol) 0.050 mass%, wax content 0.06 mass%, free fatty acid 0.14 mass%, moisture content It had a composition of 0.02% by weight.
About the refined hexane-extracted rice bran oil subjected to the evaluation, the oil component is extracted from the rice bran using hexane and subjected to distillation treatment to remove hexane and obtain crude rice bran oil. The obtained crude rice bran oil is subjected to a wax removal process, a water distillation process, a deoxidation process using an alkaline component, a decolorization process using an activated clay, and a deodorization process after removing the gum. This is a refined rice bran oil obtained by hexane extraction.
表１に示したとおり、精製圧搾米糠油には精製ヘキサン抽出米糠油に比べて、多くのリン酸化ｅ−ＮＯＳを産生しており、血管内皮機能をより活性化していることがわかった。 As shown in Table 1, it was found that the purified pressed rice bran oil produced more phosphorylated e-NOS than the purified hexane-extracted rice bran oil, and activated the vascular endothelial function more.
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