JP5514360B1 - Tissue-type plasminogen activator activator and method for producing the same - Google Patents
Tissue-type plasminogen activator activator and method for producing the same Download PDFInfo
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Abstract
【課題】
t-PAそのものを身体に注射等によって接種する方法に比べて、より安価、簡便、かつ気軽な方法でt-PAを利用することができる組織プラスミノーゲン活性化因子活性化剤及びその製造方法を提供する。
【解決手段】
イグサからの芳香性を有する抽出物を有効成分として含有することを特徴とする組織型プラスミノーゲン活性化因子活性化剤及びその製造方法である。芳香性を有する抽出物は、抽出溶媒にイグサを浸漬して、抽出溶媒を-10〜30℃に維持して抽出することが好ましく、イグサは、破砕処理を施すことなく抽出溶媒としての水エタノール混合液に浸漬することが好ましい。
【選択図】図１【Task】
Tissue plasminogen activator activator that can use t-PA in a cheaper, simpler, and easier way compared to the method of inoculating the body with injection of t-PA itself, and a method for producing the same I will provide a.
[Solution]
A tissue-type plasminogen activator activator comprising a fragrant extract from rush as an active ingredient and a method for producing the same. The aromatic extract is preferably extracted by immersing rush in an extraction solvent and maintaining the extraction solvent at -10 to 30 ° C., and rush is water ethanol as an extraction solvent without crushing treatment. It is preferable to immerse in a mixed solution.
[Selection] Figure 1
Description
本発明は、イグサからの芳香性を有する抽出物を含有する組織型プラスミノーゲン活性化因子活性化剤に関する。 The present invention relates to a tissue-type plasminogen activator activator containing a fragrant extract from rush.
組織型プラスミノーゲン活性化因子（以下、t-PA(tissue Plasminogen Activator)と称する）は、５２７個のアミノ酸からなる分子量約７万の糖タンパクであり、主として血管内皮細胞で生産され循環血液中に分泌される。t-PAは、アミノ末端からフィンガー領域、EGF領域、２個のクリングル領域及び活性領域から構成され、血液線溶系において重要な働きを有する。すなわち、t-PAは、セリンプロテアーゼ活性を有しており、プラスミノーゲンを限定分解し、プラスミンを生成する。そして、プラスミンは凝固血液中のフィブリンを分解する線溶反応を担う。 Tissue-type plasminogen activator (hereinafter referred to as t-PA (tissue Plasminogen Activator)) is a glycoprotein consisting of 527 amino acids and having a molecular weight of about 70,000, and is produced mainly in vascular endothelial cells and in circulating blood. Secreted into the body. t-PA is composed of a finger region, an EGF region, two kringle regions and an active region from the amino terminus, and has an important function in the blood fibrinolytic system. That is, t-PA has serine protease activity, and plasminogen is limitedly decomposed to produce plasmin. Plasmin is responsible for a fibrinolytic reaction that degrades fibrin in the coagulated blood.
t-PAは、虚血性脳血管障害、急性肺塞栓症及び急性冠症候群に対する最も強力な血栓溶解薬として高い有効性が証明されている。例えば、非特許文献１では、遺伝子組み換え組織型プラスミノーゲン活性因子（rt-PA: recombinant tissue-type plasminogen activator）を発症３時間以内の急性期脳梗塞に投与すると、社会生活が自立した患者を優位に増加させることが明らかにされている。この治療法は、rt-PA静注療法と呼ばれている。 t-PA has proven to be highly effective as the most powerful thrombolytic agent for ischemic cerebrovascular disorders, acute pulmonary embolism and acute coronary syndrome. For example, in Non-Patent Document 1, when a recombinant tissue-type plasminogen activator (rt-PA) is administered to acute cerebral infarction within 3 hours of onset, patients who have become independent in social life It has been clarified that the increase is dominant. This treatment is called rt-PA intravenous therapy.
上述のように、t-PAは血栓溶解剤として優れた効果を発揮する。t-PAを得るには、チャイニーズハムスター卵巣の樹立細胞株にt-PAをコードする遺伝子を導入してrt-PAを発現、精製する方法が知られている。このようにして製造されるrt-PAは高価な点で問題であった。 As described above, t-PA exhibits an excellent effect as a thrombolytic agent. In order to obtain t-PA, a method for expressing and purifying rt-PA by introducing a gene encoding t-PA into an established cell line of Chinese hamster ovary is known. The rt-PA produced in this way has been a problem in terms of cost.
一方で、特許文献１ないし４には、種々の植物由来の抽出物が血栓の予防作用等の生体に有用な作用を示すことが記載されているが、特許文献１を除いて、イグサに関する言及はない。特許文献１においては、段落0076にイグサエキスが開示されているものの、イグサエキスの服用によりパネル６名の内の半数が腰痛あるいは肩こりの改善があったことが記載されているに過ぎない。 On the other hand, Patent Documents 1 to 4 describe that various plant-derived extracts exhibit useful effects on a living body such as a thrombus-preventing action. There is no. In Patent Document 1, although rush extract is disclosed in paragraph 0076, it is only described that half of 6 panelists have improved back pain or stiff shoulders by taking rush extract.
上述のように、t-PAを遺伝子工学を利用して生産する方法は、t-PAの調達コストが嵩むという問題がある。また、t-PAは処方せん医薬品に分類されることから、処方せんなしには使用することができないため、血栓の形成を予防するために日常的に使用できるようなものではなかった。 As described above, the method of producing t-PA using genetic engineering has a problem that the procurement cost of t-PA increases. Also, since t-PA is classified as a prescription drug and cannot be used without a prescription, it was not something that could be routinely used to prevent thrombus formation.
本発明は、t-PAそのものを身体に注射等によって接種する方法に比べて、より安価、簡便、かつ気軽な方法でt-PAを利用することができる組織プラスミノーゲン活性化因子活性化剤及びその製造方法を提供することを目的とする。 The present invention is a tissue plasminogen activator activator that can use t-PA in a cheaper, simpler, and easier way compared to a method in which t-PA itself is inoculated by injection or the like. And it aims at providing the manufacturing method.
本発明者らは、イグサ由来の芳香性を有する抽出物にt-PAの分泌を促進する効果があることを見出し、本発明を完成するに至った。すなわち、本発明は、イグサからの芳香性を有する抽出物を有効成分として含有することを特徴とする組織型プラスミノーゲン活性化因子分泌促進剤（以下、t-PA分泌促進剤と称する）及びその製造方法である。前記t-PA分泌促進剤及びその製造方法では、芳香性を有する抽出物は、抽出溶媒にイグサを浸漬して、抽出溶媒を-10〜15℃に維持して抽出される。 The present inventors have found that an extract with fragrance derived from rush has an effect of promoting the secretion of t-PA , and have completed the present invention. That is, the present invention includes a tissue-type plasminogen activator secretion promoter (hereinafter referred to as a t-PA secretion promoter ) characterized by containing an extract having fragrance from rush as an active ingredient, and It is the manufacturing method. In the t-PA secretion promoter and a manufacturing method thereof, extract with Fang aroma property, extraction solvent by immersing the rush is extracted extraction solvent was maintained at -10 to 15 ° C..
上述の通り、芳香性を有する抽出物は、抽出溶媒にイグサを浸漬して、抽出溶媒を-10〜15℃に維持して抽出する。前記温度の範囲に維持して抽出することにより、イグサの芳香成分が揮発して失われることを防止することができる。 As mentioned above, the extract having aromaticity, extracted by immersing the rush solvent, the extraction solvent extracts was maintained at -10 to 15 ° C.. By performing extraction while maintaining the temperature range, it is possible to prevent the fragrance component of rush from being volatilized and lost.
t-PA分泌促進剤及びその製造方法においては、芳香性を有する成分を抽出するに際して、イグサは、ホモゲナイズ等の破砕処理を施すことなく抽出溶媒としての水エタノール混合液に浸漬することが好ましい。ホモゲナイズ等の破砕処理を行うことなく抽出溶液に浸漬することで、低分子量の芳香成分を有効成分として抽出溶媒に溶出させることができる。換言すると、破砕処理を行わないことによって分子量の大きい夾雑物が抽出溶媒に多量に溶出することを防ぐことができるので、その後の精製処理を省略することができる。 In the t-PA secretion promoter and the method for producing the same, the rush is preferably immersed in a water ethanol mixed solution as an extraction solvent without performing a crushing treatment such as homogenization when extracting the aromatic component. By immersing in an extraction solution without performing homogenization or other crushing treatment, a low molecular weight aromatic component can be eluted as an active ingredient into the extraction solvent. In other words, by not performing the crushing process, it is possible to prevent a large amount of contaminants having a high molecular weight from eluting into the extraction solvent, so that the subsequent purification process can be omitted.
芳香性を有する抽出物は、抽出溶媒に浸漬したイグサの色素が流出し、流出による抽出溶媒の色彩の変化がなくなるまで浸漬したものを抽出原液とし、組織プラスノーゲン活性化因子分泌促進剤は、30〜70容量％となるように前記原液を含有することが好ましい。抽出物の含有率を前記範囲内とすることでt-PAの分泌が最も高まる。 The extract with fragrance is the extract stock solution that has been immersed until the color of the extraction solvent is lost due to the outflow of the rush pigment immersed in the extraction solvent, and the tissue plasminogen activator secretion promoter is It is preferable to contain the said stock solution so that it may become 30-70 volume%. By making the extract content within the above range, secretion of t-PA is most enhanced.
抽出溶媒に浸漬するイグサは、浸漬する前に50〜60℃の低温又は天日干しで乾燥させたものを使用することが好ましい。これにより、抽出液の原料となるイグサからの芳香成分が揮発することを防止することができる。 The rush immersed in the extraction solvent is preferably used after drying at a low temperature of 50-60 ° C. or sun-drying before immersion. Thereby, it can prevent that the aromatic component from the rush which becomes a raw material of an extract liquid volatilizes.
本発明のt-PA分泌促進剤は、生細胞に適用することでt-PAの分泌を高める効果を発揮する。したがって、本発明のt-PA分泌促進剤は、例えば、マッサージオイルなどの皮膚用塗布剤としてヒトの体に塗って使用することで、ヒトの細胞が発現するt-PAの分泌を高める効果が得られる。また、本発明のt-PA分泌促進剤は、アロマオイルや芳香剤などの吸引投与組成物として使用することができる。アロマオイルとして使用する場合は、t-PA分泌促進剤を耐熱容器に入れて、耐熱容器を適宜の方法によって加熱してイグサの抽出液の芳香成分を揮発させたり、t-PA分泌促進剤を適宜の容器に入れてイグサの抽出液の芳香成分を自然に揮発させたりして使用する。温水を満たした容器にt-PA分泌促進剤を添加して、揮発する芳香成分を吸引してもよい。芳香剤として使用する場合は、容器にt-PA分泌促進剤を入れて、室内に設置して使用する。アロマオイルや芳香剤等の吸引組成物として使用する場合、イグサからの抽出物に含まれる芳香成分が揮発して、呼吸によって肺の静脈から体内に取り込まれる。アロマオイルや芳香剤として使用すれば、芳香成分を嗅ぐだけで体内のt-PAの分泌を高めることができる。 The t-PA secretion promoter of the present invention exerts an effect of enhancing the secretion of t-PA when applied to living cells. Therefore, the t-PA secretion enhancer of the present invention has an effect of enhancing the secretion of t-PA expressed by human cells by being applied to the human body as a skin coating agent such as massage oil. can get. Moreover, the t-PA secretion promoter of the present invention can be used as a composition for suction administration such as aroma oil and fragrance. When using as an aroma oil, put a t-PA secretion promoter in a heat-resistant container and heat the heat-resistant container by an appropriate method to volatilize the aromatic component of the extract of rush or use a t-PA secretion promoter . Place it in a suitable container and use it by volatilizing the aromatic component of the rush extract. A t-PA secretion promoter may be added to a container filled with warm water to suck the volatile aromatic component. When using as a fragrance, put a t-PA secretion promoter in a container and install it indoors. When used as an inhalation composition such as aroma oil or fragrance, the fragrance component contained in the extract from the rush volatilizes and is taken into the body from the pulmonary vein by breathing. If used as an aroma oil or fragrance, it is possible to increase the secretion of t-PA in the body simply by smelling the fragrance component.
t-PAは血栓溶解作用だけでなく、学習能力の向上や記憶の形成、神経細胞の移動、神経の発達、神経細胞の樹状突起上のスパインの形成等に寄与することが報告されている。したがって、本発明のt-PA分泌促進剤を吸入することで学習能力の向上や記憶力の向上を図る効果が期待される。アロマオイルや芳香剤として使用すれば、簡便に抽出物に含まれる芳香成分を吸入することができるので、学習室や病室の片隅に設置して気軽に使用することができる。後述するように、イグサからの芳香性を有する抽出物を有効成分として含有するt-PA分泌促進剤は、それ自体が血小板の凝集を抑制する効果を有し、t-PA分泌促進剤それ自体が血栓形成防止剤としての効果を有する。したがって、本発明のt-PA分泌促進剤は、線溶反応を促進する効果と血小板凝集を抑制する効果との両面で血栓形成を防止することができる。使用方法も上述の通り簡便なものであり、かつ安価で提供することができるのでt-PAを予防的に積極的に使用することが可能になる。 It has been reported that t-PA contributes not only to thrombolytic activity but also to improvement of learning ability and memory formation, nerve cell migration, nerve development, and spine formation on nerve cell dendrites . Therefore, inhalation of the t-PA secretion promoter of the present invention is expected to have an effect of improving learning ability and memory ability. If it is used as an aroma oil or fragrance, the fragrance component contained in the extract can be easily inhaled, so it can be used easily by installing it in one corner of a learning room or hospital room. As will be described later, a t-PA secretion promoter containing an extract having a fragrance from rush as an active ingredient has an effect of suppressing platelet aggregation itself, and the t-PA secretion promoter itself Has an effect as a thrombus formation inhibitor. Therefore, the t-PA secretion promoter of the present invention can prevent thrombus formation in both the effect of promoting the fibrinolytic reaction and the effect of suppressing platelet aggregation. The method of use is also simple as described above, and can be provided at low cost, so t-PA can be used proactively and actively.
以下、発明を実施するための形態について説明する。 Hereinafter, modes for carrying out the invention will be described.
本発明は、イグサからの芳香性を有する抽出物を有効成分として含有することを特徴とする組織型プラスミノーゲン活性化因子分泌促進剤及びその製造方法である。原料として使用できるイグサは、特に限定されず、例えば畳表に使用されるイグサを使用することができる。それらのうち、芳香性が高いものを選択して使用すればよい。畳表の原料として流通するイグサは、収穫後の変色を防ぐ目的で刈り取った後、泥水に浸けられている。泥水に浸漬したイグサは、泥染めイグサと呼ばれているが、本発明では泥染めしない生イグサを使用することが好ましい。 The present invention is a tissue-type plasminogen activator secretion promoter and a method for producing the same, characterized by containing a fragrant extract from rush as an active ingredient. The rush which can be used as a raw material is not specifically limited, For example, the rush used for a tatami mat can be used. Among them, a highly aromatic one may be selected and used. The rush that circulates as a raw material for the tatami surface is cut in order to prevent discoloration after harvesting and then immersed in muddy water. The rush immersed in the muddy water is called a mud-dyed rush. In the present invention, it is preferable to use a raw rush that is not mud-dyed.
芳香性を有する抽出物を得る際に使用する抽出方法としては、抽出溶媒にイグサを浸漬して、抽出溶媒を-10〜30℃の温度に維持して抽出することが好ましく、−5〜15℃の低温に維持して抽出するとより好ましい。前記温度範囲で抽出することで抽出作業中にイグサ由来の芳香成分が揮発して失われることを防ぐことができる。一方で、上記範囲を超えて低温にすると抽出効率が低下することがある。抽出溶媒には、生体毒性の比較的低い有機溶媒又は有機溶媒と水の混合液を使用することが好ましい。有機溶媒としては、エタノール、プロパノール、イソプロピルアルコール、若しくはブチルアルコールなどのアルコール類又はこれらの内の少なくとも１種以上の混合物を使用することが好ましく、中でも生体毒性の低いエタノールと水との混合液を使用することが好ましい。エタノールは生体毒性が低いため、抽出溶媒を除去せずにt-PA分泌促進剤を人体に適用することができる。抽出溶媒を除去せずに使用に供する場合は、抽出効率及び生体毒性を考慮してエタノールの濃度を10〜70容量％となるように調整することが好ましい。エタノール濃度は、10〜40容量％とすることがより好ましい。 As an extraction method used when obtaining an extract having aromaticity, it is preferable to extract by immersing rush into an extraction solvent and maintaining the extraction solvent at a temperature of −10 to 30 ° C., and −5 to 15 It is more preferable to perform extraction while maintaining a low temperature of ° C. By extracting in the temperature range, it is possible to prevent fragrance components derived from rushes from being volatilized and lost during the extraction operation. On the other hand, if the temperature is exceeded beyond the above range, the extraction efficiency may decrease. As the extraction solvent, it is preferable to use an organic solvent having a relatively low biotoxicity or a mixed solution of an organic solvent and water. As the organic solvent, it is preferable to use an alcohol such as ethanol, propanol, isopropyl alcohol, or butyl alcohol, or a mixture of at least one of these, and among them, a mixture of ethanol and water having low biotoxicity is used. It is preferable to use it. Since ethanol has low biological toxicity, a t-PA secretion promoter can be applied to the human body without removing the extraction solvent. When using it without removing the extraction solvent, it is preferable to adjust the ethanol concentration to 10 to 70% by volume in consideration of extraction efficiency and biotoxicity. The ethanol concentration is more preferably 10 to 40% by volume.
抽出溶媒に上記の乾燥イグサを浸漬する際には、ホモゲナイズ等の破砕処理をすることなく、イグサを5〜10ｍｍ長を目安に切断してから浸漬することが好ましい。浸漬中においては、スターラーなどの撹拌子で撹拌してもよい。抽出溶媒0.7リットルに対して乾燥イグサを10〜40ｇを目安として、乾燥イグサを浸漬するとよい。抽出溶媒に対する乾燥イグサの浸漬量によっても異なるが、上記の混合比で、-10〜30℃の温度で浸漬した場合、一晩浸漬すれば、抽出溶媒にイグサの芳香成分が流出して色彩の変化が起こらなくなり飽和状態に達する。この際、水エタノール混合液の色は、薄黄色に変色している。本発明では、抽出溶媒に浸漬したイグサの色素が流出し、流出による抽出溶媒の色彩の変化がなくなるまで浸漬したものを抽出原液とする。t-PA分泌促進剤は、原液をそのまま使用してもよいが、30〜70容量％となるように抽出原液を含有するように、希釈して使用することが好ましい。より好ましくは、40〜60容量％となるように希釈するとよい。後述するように、この数値範囲において特に、t-PAの分泌の飛躍的な高まりがみられる。 When immersing the dried rush in the extraction solvent, it is preferable to immerse the rush after cutting the rush to a length of 5 to 10 mm without crushing such as homogenization. During the immersion, stirring may be performed with a stirring bar such as a stirrer. The dry rush may be dipped with 10-40 g of dry rush as a guide for 0.7 liter of extraction solvent. Depending on the amount of dry rush soaked in the extraction solvent, if it is immersed at a temperature of −10 to 30 ° C. with the above mixing ratio, the fragrance component of the rush will flow out into the extraction solvent if it is immersed overnight. No change occurs and reaches saturation. At this time, the color of the water ethanol mixed solution is changed to light yellow. In the present invention, the extraction stock solution is one in which the rush pigment soaked in the extraction solvent flows out and is immersed until there is no change in the color of the extraction solvent due to the outflow. Although the stock solution may be used as it is, the t-PA secretion enhancer is preferably used by diluting it so that it contains 30 to 70% by volume of the extracted stock solution. More preferably, dilution may be performed so as to be 40 to 60% by volume. As will be described later, particularly in this numerical range, a dramatic increase in the secretion of t-PA is observed.
上述の通り、本発明では泥染めしていない生イグサを使用することが好ましい。しかし、時期によっては生イグサを調達できるとは限らない。生イグサを調達できない場合は、生イグサを低温で乾燥したものを使用するとよい。生イグサを低温乾燥することで、イグサ由来の芳香成分を乾燥の過程で損なうことなく維持することが可能になるからである。生イグサの乾燥温度は、50〜60℃の低温とすることが好ましい。乾燥方法は、とくに制限されないが例えば、温水を管内に循環させた管に乾燥した空気を送り込んで温風を作り出し、この温風を乾燥室に満たして、当該乾燥室に生イグサを吊るすことにより行うことができる。乾燥時間は12〜24時間とすることが好ましい。 As described above, in the present invention, it is preferable to use raw rush that is not mud-dyed. However, depending on the season, it is not always possible to procure raw rush. If raw rush cannot be procured, it is recommended to use raw rush dried at low temperature. This is because, by drying raw rush at low temperature, it is possible to maintain the fragrance component derived from rush without damaging the drying process. The drying temperature of the green rush is preferably 50-60 ° C. The drying method is not particularly limited. For example, by sending dry air into a pipe in which hot water is circulated in the pipe to create hot air, filling the hot air in the drying room, and suspending the green rush in the drying room. It can be carried out. The drying time is preferably 12 to 24 hours.
上述のように、本発明のt-PA分泌促進剤は、マッサージオイルなどの皮膚用塗布剤、アロマオイル又は芳香剤などの吸引投与組成物として使用することができる。その際には、抽出原液をそのまま使用してもよいが、上述のように30〜70容量％となるように抽出原液を含有するように、希釈することが好ましい。より好ましくは、40〜60容量％となるように希釈することが好ましい。希釈液としては、水にアスコルビン酸、トコフェロールなどの酸化防止剤を添加したもの等を使用することができる。 As described above, the t-PA secretion promoter of the present invention can be used as a composition for suction administration such as a skin coating agent such as massage oil, an aroma oil or a fragrance. In this case, the extraction stock solution may be used as it is, but it is preferable to dilute the extraction stock solution so as to be 30 to 70% by volume as described above. More preferably, the dilution is preferably 40 to 60% by volume. As the diluting solution, water added with an antioxidant such as ascorbic acid or tocopherol can be used.
以下、本発明の実施例を挙げてさらに具体的に説明する。 Hereinafter, examples of the present invention will be described in more detail.
〔イグサの調達〕
12月末頃に苗床に残しておいた種苗を株分けした。株分けは、１株当たり芽が２、３本含まれるようにした。株分けした苗を鉢植用の土を入れたトレイに挿して、本田の片隅においておいた。翌年の３月上旬に根が絡んで新芽が出てきたので畑に植え替えた。８月頃には苗が成長して大きくなったので、苗１株を20株程度に分けて水田（苗床）に手作業で植えた。その後、肥培管理しながら栽培し、その年の11月下旬に株を引き抜いて、株についた泥を落とした後、20センチ×60センチのカセットの中に株を解しながら詰めていった。カセットを20アール分用意したところで、カセットを本田に搬入して、本田に植え替えて、肥培管理しながら栽培した。その後翌年の６月下旬まで栽培して刈り取った。イグサの栽培は全て無農薬で行った。収穫したイグサは泥染めすることなく、そのまま乾燥機に入れて55℃で18時間乾燥させた。この乾燥は天日干しにしてもよい。
[Procurement of rush]
The seedlings left on the nursery at the end of December were separated. The stock split was made to contain a few buds per strain. The planted seedlings were inserted into a tray with potting soil and placed in a corner of Honda. In early March of the following year, roots were tangled and new shoots came out. Since seedlings grew and grew around August, 1 seedling was divided into about 20 plants and planted manually in paddy fields (nursery beds). After that, it was cultivated under fertilizer management, and the stock was pulled out in late November of that year, and after removing the mud from the stock, it was packed in a 20cm x 60cm cassette while unraveling the stock. When 20 cassettes were prepared for cassettes, the cassettes were transferred to Honda, replanted in Honda, and cultivated with fertilizer management. After that, it was cultivated and harvested until the end of June of the following year. All the rushes were grown without pesticides. The harvested rush was put in a dryer as it was without being muddy and dried at 55 ° C. for 18 hours. This drying may be sun-dried.
〔イグサ抽出液の作製〕
上記の乾燥させたイグサを5〜10mm間隔で切断したイグサ25gと25容量％に調整した水エタノール混合液700mlとを注いだビーカーに入れた後、室温においてスターラーでしばらく撹拌してから、４℃に維持して一晩浸漬した。翌朝にはイグサの色素が水エタノール混合液に流出し、液色が薄黄色になっていたので、イグサを取り除いてこれをイグサ抽出液とした。イグサ抽出液の匂いを確認したところ、イグサの芳香が抽出液に移行していた。同様の方法で作製したイグサ抽出液をもう一晩４℃で浸漬したが、液色に大きな変化は見られず、抽出液のイグサの芳香が強くなることもなかった。
[Preparation of rush extract]
After the above dried rush was cut into 5-10 mm intervals, 25 g of rush and 700 ml of a water ethanol mixture adjusted to 25% by volume were poured into a beaker, stirred at room temperature for a while with a stirrer, and then at 4 ° C. And soaked overnight. The next morning, the rush pigment flowed into the water-ethanol mixture and the liquid color was pale yellow. The rush was removed and this was used as the rush extract. When the odor of the rush extract was confirmed, the fragrance of the rush was transferred to the extract. The rush extract prepared by the same method was soaked overnight at 4 ° C., but no significant change was observed in the liquid color, and the fragrance of the rush in the extract did not increase.
〔細胞培養〕
ヒト由来の培養細胞を使用した。細胞培養液は、10容量％の牛胎児血清（FBS）を含むE-MEM培地を121℃、15分間に設定したオートクレーブに入れて加圧及び加熱処理をし、これを滅菌フィルターでろ過したものを使用した。96ウェルのプレートに細胞が3×104cells/cm2となるように播種して、37℃、5%CO2の条件下でコンフルーエントになるまで培養した。古い培養液を除去した後、培養細胞に新しい培養液56.7μlと上記ようにして調製したイグサ抽出液6.4μlとを加えてさらに24時間培養した。イグサ抽出液は、原液、1/2希釈、1/4希釈、1/8希釈、1/16希釈、1/32希釈又は1/64希釈してから添加した。希釈はいずれも容量比に基づいて超純水を添加して希釈した。比較対象のためにコントロールとして超純水を使用した。
[Cell culture]
Human-derived cultured cells were used. The cell culture solution is an E-MEM medium containing 10% by volume fetal bovine serum (FBS) put in an autoclave set at 121 ° C for 15 minutes, pressurized and heated, and filtered through a sterile filter. It was used. Cells were seeded in a 96-well plate at 3 × 10 4 cells / cm 2 and cultured at 37 ° C. under 5% CO 2 until confluent. After removing the old culture solution, 56.7 μl of a new culture solution and 6.4 μl of the rush extract prepared as described above were added to the cultured cells and further cultured for 24 hours. The rush extract was added after diluting the stock solution, 1/2 dilution, 1/4 dilution, 1/8 dilution, 1/16 dilution, 1/32 dilution or 1/64 dilution. All dilutions were performed by adding ultrapure water based on the volume ratio. For comparison purposes, ultrapure water was used as a control.
〔t-PA活性の測定〕
t-PA活性の測定には、t-PAに特異性の高い合成アミド基質であるH-D-Ile-Pro-Arg-pNA(S-2288)を使用した。基質濃度は、ジメチルスルホキシド（DMSO）で5×10-3Mに調整した。96ウェルプレートに0.1Mリン酸緩衝液(pH7.4)50μlと前記合成アミド基質10μlを混合して37℃、10分間プレインキュベーションした。さらに前記の各濃度に希釈した培養液を40μl添加して、405nmの吸光度を37℃で10分間測定した。t-PAは下記の式の反応を触媒して、pNA(p-ニトロアラニン)を遊離させる。pNAは吸光度の変化によって定量される。そして、生じたpNA量からt-PA量（unit/ml）を算出した。
[化１]
H-D-Ile-Pro-Arg-pNA→H-D-Ile-Pro-Arg + pNA
[Measurement of t-PA activity]
For measurement of t-PA activity, HD-Ile-Pro-Arg-pNA (S-2288), which is a synthetic amide substrate highly specific for t-PA, was used. The substrate concentration was adjusted to 5 × 10 −3 M with dimethyl sulfoxide (DMSO). A 96-well plate was mixed with 50 μl of 0.1 M phosphate buffer (pH 7.4) and 10 μl of the synthetic amide substrate and preincubated at 37 ° C. for 10 minutes. Further, 40 μl of the culture solution diluted to each concentration was added, and the absorbance at 405 nm was measured at 37 ° C. for 10 minutes. t-PA catalyzes the reaction of the following formula to liberate pNA (p-nitroalanine). pNA is quantified by the change in absorbance. Then, the amount of t-PA (unit / ml) was calculated from the amount of pNA produced.
[Chemical 1]
HD-Ile-Pro-Arg-pNA → HD-Ile-Pro-Arg + pNA
t-PA活性の測定結果を図１に示す。上記の各濃度のイグサ抽出液を添加した培養細胞３セットを１組として２ロット用意した。t-PA活性の測定は、この２つのロット（培養細胞１、培養細胞２）の培養細胞のそれぞれについて行った。図１の培養細胞１及び培養細胞２のグラフはそれぞれ、各希釈率における３セット分の測定結果（unit/ml）を平均した数値を示す。原液、1/2希釈（50%）、1/4希釈(25%)、1/8希釈(12.5%)、1/16希釈(6.25%)、1/32希釈(3.13%)又は1/64希釈(1.56%)にしたイグサ抽出液を上述のように培養細胞１又は培養細胞２に添加してt-PA活性を測定したところ、原液を1/2希釈した場合に最も酵素活性が高まることが確かめられた。特に培養細胞２（10919.4unit/ml）では超純水を添加したコントロール(683.3unit/ml)に比べて20倍もt-PA活性が高まること確認された。この酵素活性の高まりは、t-PAの発現量の増大及びそれに伴うt-PAの分泌量の増大に起因するものである。したがって、本発明の組織型プラスミノーゲン活性化因子活性化剤は、組織型プラスミノーゲン活性化因子の生成促進剤あるいは組織型プラスミノーゲン活性化因子の分泌促進剤として捉えることができる。 The measurement result of t-PA activity is shown in FIG. Two sets of 3 sets of cultured cells to which the rush extract of each concentration was added were prepared. The t-PA activity was measured for each of the cultured cells of these two lots (cultured cell 1, cultured cell 2). The graphs of cultured cell 1 and cultured cell 2 in FIG. 1 show the numerical values obtained by averaging the measurement results (unit / ml) for three sets at each dilution rate. Stock solution, 1/2 dilution (50%), 1/4 dilution (25%), 1/8 dilution (12.5%), 1/16 dilution (6.25%), 1/32 dilution (3.13%) or 1/64 When the diluted rush extract (1.56%) was added to cultured cell 1 or cultured cell 2 as described above and t-PA activity was measured, the enzyme activity increased most when the stock solution was diluted 1/2. Was confirmed. In particular, it was confirmed that cultured cells 2 (10919.4 unit / ml) had a 20-fold increase in t-PA activity compared to the control (683.3 unit / ml) to which ultrapure water was added. This increase in enzyme activity is attributed to the increase in the expression level of t-PA and the accompanying increase in the secretion amount of t-PA. Therefore, the tissue type plasminogen activator activator of the present invention can be regarded as a tissue type plasminogen activator production promoter or tissue type plasminogen activator secretion promoter.
〔血小板凝集の抑制能〕
次に、上述のようにして得たイグサ抽出液が血小板凝集に与える影響について調べた。ヒトの多血小板血漿（PRR: Platelet Rich Plasma）に上述のようにして得たイグサ抽出液の原液を10μl加えて、これを37℃で、5分間プレインキュベーションした。次に、凝集惹起物質としてコラーゲンを22μl(終濃度4.5μg/ml)又はADPを22μl（終濃度2.5μM）加えた。そして、37℃において5分間、血小板凝集率をアグリゴメーター（PAT-4A）で測定した。測定に際しては、貧血小板血漿の光透過率を100％とした。血小板凝集率の算出は、血小板が凝集していない状態では、血小板が比較的均一に分散していることに起因して光透過率が低いのに対して、血小板が凝集すると、光透過率が高まることを利用している。すなわち、血小板が凝集すると凝集した血小板間の間隙が大きくなり光が透過しやすくなることを利用したものである。比較対照のためにコントロールとして25容量％に調整した水エタノール混合液を使用した。結果を図２に示す。図２から明らかなように、イグサ抽出液には凝集惹起物質であるコラーゲン及びADPのいずれに対しても、血小板の凝集を抑える効果があることが明らかになった。
[Inhibition of platelet aggregation]
Next, the effect of the rush extract obtained as described above on platelet aggregation was examined. 10 μl of the stock solution of rush extract obtained as described above was added to human platelet-rich plasma (PRR) and preincubated at 37 ° C. for 5 minutes. Next, 22 μl of collagen (final concentration: 4.5 μg / ml) or 22 μl of ADP (final concentration: 2.5 μM) was added as an aggregation-inducing substance. The platelet aggregation rate was measured with an aggregometer (PAT-4A) at 37 ° C. for 5 minutes. In the measurement, the light transmittance of the poor platelet plasma was set to 100%. The platelet aggregation rate is calculated when the platelets are not aggregated, and the light transmittance is low due to the relatively uniform distribution of the platelets. Take advantage of growing. That is, when platelets aggregate, the gap between the aggregated platelets becomes large and light is easily transmitted. For comparison, a water ethanol mixture adjusted to 25% by volume was used as a control. The results are shown in FIG. As is apparent from FIG. 2, the rush extract has an effect of suppressing the aggregation of platelets with respect to both the aggregation-inducing substances collagen and ADP.
上述の1/2希釈した抽出液は、そのままマッサージオイルとして肌に塗布して使用することができた。さらに1/2希釈した抽出液は、耐熱皿に入れて当該耐熱皿を加熱することで、アロマオイルとして使用することができた。このアロマオイルは、皿に入れて自然に香気成分を揮発させて使用することもできた。また、1/2に希釈した抽出液を小瓶に詰めて芳香剤として使用することができた。希釈には、酸化防止剤を添加した滅菌水を使用した。 The 1/2 diluted extract described above could be applied to the skin as massage oil as it was. Furthermore, the extract diluted 1/2 could be used as an aroma oil by putting it in a bakeware and heating the bakeware. This aroma oil could be used by volatilizing the aroma component naturally in a dish. Moreover, the extract diluted 1/2 was packed in a small bottle and used as a fragrance. For dilution, sterilized water to which an antioxidant was added was used.
上記の方法でイグサから抽出した抽出物は、イグサをホモゲナイズ等の破砕処理をしておらず、低温の水エタノール混合液に浸漬して抽出したものであるから、水エタノール混合液に溶出した成分は低分子量の芳香成分を主成分とするものである。この芳香成分は鼻や口を経て肺に達して血中に取り込まれるため、本発明のt-PA分泌促進剤を嗅いだり吸引したりするだけで、体内でのt-PAの分泌を促し、t-PA活性を向上させて血栓の形成を予防したり、学習効果を高める効果が得られる。 The extract extracted from the rush by the above method is a component that has not been crushed, such as homogenizing, but extracted by immersing it in a low-temperature water ethanol mixed solution. Is mainly composed of a low molecular weight aromatic component. Because this fragrance component reaches the lungs through the nose and mouth and is taken into the blood, just by smelling and inhaling the t-PA secretion promoter of the present invention, it promotes the secretion of t-PA in the body, Increases t-PA activity to prevent thrombus formation and enhances learning effect.
Claims (8)
芳香性を有する抽出物は、抽出溶媒にイグサを浸漬して、抽出溶媒を-10〜15℃の低温に維持して抽出される組織型プラスミノーゲン活性化因子分泌促進剤。 A tissue-type plasminogen activator secretion promoter characterized by containing an extract having fragrance from rush as an active ingredient ,
The extract having fragrance is a tissue-type plasminogen activator secretion promoter that is extracted by immersing rush in an extraction solvent and maintaining the extraction solvent at a low temperature of −10 to 15 ° C.
組織プラスノーゲン活性化因子分泌促進剤は、30〜70容量％となるように前記抽出原液を含有するものである請求項１又は２に記載の組織型プラスミノーゲン活性化因子分泌促進剤。 The extract with fragrance is the extraction stock solution that has been immersed until the color of the extraction solvent is eliminated due to the outflow of the rush pigment immersed in the extraction solvent.
Tissue plasminogen no plasminogen activator secretion enhancers, tissue-type plasminogen activator secretion promoters described in claim 1 or 2 are those containing the extracted stock solution such that 30 to 70 volume%.
芳香性を有する抽出物は、抽出溶媒にイグサを浸漬して、抽出溶媒を-10〜15℃の低温に維持して抽出される組織型プラスミノーゲン活性化因子分泌促進剤の製造方法。 A method for producing a tissue-type plasminogen activator secretion promoter containing an aromatic extract from rush as an active ingredient,
Extract having aromaticity, extraction solvent by immersing the rush, extraction method of manufacturing a solvent -10 to 15 ° C. set Ru is extracted while maintaining the low temperature of the weave-type plasminogen activator secretion promoters.
組織プラスノーゲン活性化因子分泌促進剤は、30〜70容量％となるように前記抽出原液を希釈するものである請求項５又は６に記載の組織型プラスミノーゲン活性化因子分泌促進剤の製造方法。 The extract with fragrance is the extraction stock solution that has been immersed until the color of the extraction solvent is eliminated due to the outflow of the rush pigment immersed in the extraction solvent.
Tissue plasminogen no plasminogen activator secretion enhancers, manufacture of tissue-type plasminogen activator secretion promoters described in claim 5 or 6 is to dilute the extracted stock solution such that 30 to 70 volume% Method.
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