JP5925728B2 - TGF-β secretion derivative of vascular endothelial cell, method for producing the same, and anti-aging agent - Google Patents
TGF-β secretion derivative of vascular endothelial cell, method for producing the same, and anti-aging agent Download PDFInfo
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- JP5925728B2 JP5925728B2 JP2013100754A JP2013100754A JP5925728B2 JP 5925728 B2 JP5925728 B2 JP 5925728B2 JP 2013100754 A JP2013100754 A JP 2013100754A JP 2013100754 A JP2013100754 A JP 2013100754A JP 5925728 B2 JP5925728 B2 JP 5925728B2
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Description
本発明は、血管内皮細胞に対してＴＧＦ−β（Ｔｒａｎｓｆｏｒｍｉｎｇ Ｇｒｏｔｈ Ｆａｃｔｏｒ−β）の分泌を誘導する血管内皮細胞のＴＧＦ−β分泌誘導物、及び血管内皮細胞のＴＧＦ−β分泌誘導物を製造する方法、並びに血管内皮細胞のＴＧＦ−β分泌誘導物を含有し、老化を抑制する抗老化剤に関する。 The present invention produces TGF-β (Transforming Groth Factor- β) TGF-β secretion-inducing substance of vascular endothelial cells to induce the secretion of, and TGF-beta secretion-inducing substance of vascular endothelial cells to vascular endothelial cells The present invention relates to a method and an anti-aging agent that contains a TGF-β secretion derivative of vascular endothelial cells and suppresses aging.
淡水性の単細胞微細藻類であるクロレラの抽出物であるクロレラエキスは、蛋白質及びアミノ酸を豊富に含有しているため調味料又は製造用剤といった食品添加物として用いられている。加えて、クロレラエキスは、ビタミンＢ１・ビタミンＢ２・ビタミンＢ６・ビタミンＢ１２・ビタミンＣ・ビタミンＥ・ビタミンＫ・ナイアシン・葉酸等のビタミン、並びに鉄分・カルシウム・マグネシウム・カリウム等のミネラル、更に食物繊維も多く含有しているため、健康食品の原料としても用いられている。 Chlorella extract, which is an extract of chlorella, which is a freshwater single-cell microalgae, is abundant in proteins and amino acids, and is therefore used as a food additive such as seasonings or manufacturing agents. In addition, chlorella extract contains vitamins such as vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin E, vitamin K, niacin, folic acid, minerals such as iron, calcium, magnesium, potassium, and dietary fiber. Is also used as a raw material for health foods.
このようなクロレラエキスについては従来より種々研究されており、新たな効能が存在することが報告されている。 Various studies have been conducted on such chlorella extract and it has been reported that a new effect exists.
例えば、後述する特許文献１及び特許文献３には、クロレラエキスが、トロンボスポンジン（ＴＳＰ）−１を誘導するとともに、血管内皮細胞のアポトーシスを招来させ、それによって血管新生を阻害する作用を有することが開示されている。ここで、ＴＳＰは、基底層のケラチノサイトを含む数種の皮膚細胞にて発現しており、真皮−表皮間の基底膜領域に堆積し、表皮の血管新生を阻害する作用を有することが知られている。一方、皮膚への紫外線照射によって生じるしわの形成には、血管内皮細胞の新生が関与しているものと言われている。そこで、特許文献１及び特許文献３には、ＴＳＰ−１を誘導するとともに血管内皮細胞のアポトーシスを招来させることによって、しわの形成を抑制し得ることが示唆されている。そして、クロレラエキスは、前述したようにＴＳＰ−１を誘導するとともに血管内皮細胞のアポトーシスを招来させるため、しわの形成を抑制し得るものと推定されている。 For example, in Patent Document 1 and Patent Document 3 to be described later, chlorella extract has an action of inducing thrombospondin (TSP) -1 and inducing apoptosis of vascular endothelial cells, thereby inhibiting angiogenesis. It is disclosed. Here, TSP is expressed in several types of skin cells including keratinocytes in the basal layer, and is known to accumulate in the basement membrane region between the dermis and the epidermis and to have an action of inhibiting epidermal angiogenesis. ing. On the other hand, it is said that the formation of wrinkles caused by ultraviolet irradiation of the skin is related to the formation of vascular endothelial cells. Thus, Patent Document 1 and Patent Document 3 suggest that wrinkle formation can be suppressed by inducing TSP-1 and inducing apoptosis of vascular endothelial cells. And it is estimated that a chlorella extract can suppress the formation of wrinkles because it induces TSP-1 and induces apoptosis of vascular endothelial cells as described above.
また、後述する特許文献２には、クロレラエキスが血管内皮増殖因子（ＶＥＧＦ：Ｖａｓｕｃｕｌａｒ ｅｎｄｏｔｈｅｌｉａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ）の発現を阻害する作用を有することが開示されている。ＶＥＧＦは、血管内皮細胞の増殖誘導と生存維持、血管透過性の亢進、血圧の調製、血小板による遊走、マクロファージに対する走化性等、多彩な機能を有しており、血管内皮細胞におけるＶＥＧＦの発現又は活性を阻害する物質は、血管新生阻害剤として皮膚老化の予防又は改善の作用を奏し得ることが特許文献２には示唆されている。そして、クロレラエキスは、前述したようにＶＥＧＦの発現を阻害する作用を有するため、皮膚老化を予防し又は改善し得るものと推定されている。 Further, Patent Document 2 described later discloses that chlorella extract has an action of inhibiting the expression of vascular endothelial growth factor (VEGF: Vascular Endothelial Growth Factor). VEGF has various functions such as induction of vascular endothelial cell proliferation and maintenance of survival, enhancement of vascular permeability, preparation of blood pressure, migration by platelets, chemotaxis to macrophages, and the expression of VEGF in vascular endothelial cells. Alternatively, Patent Document 2 suggests that a substance that inhibits activity can exert an effect of preventing or improving skin aging as an angiogenesis inhibitor. And since the chlorella extract has the effect | action which inhibits the expression of VEGF as mentioned above, it is estimated that skin aging can be prevented or improved.
前述したようにクロレラエキスに、ＴＳＰ−１を誘導するとともに血管内皮細胞のアポトーシスを招来させる作用、又は、ＶＥＧＦの発現を阻害する作用が存在するものの、いずれの場合にあっても、それらの作用によって皮膚の老化を有効に防止し得るか否かについては何らの実証もなされていない。 As described above, chlorella extract has an effect of inducing TSP-1 and inducing apoptosis of vascular endothelial cells, or an action of inhibiting the expression of VEGF. There has been no demonstration as to whether or not skin aging can be effectively prevented.
本発明は斯かる事情に鑑みてなされたものであって、皮膚の老化を有効に防止することができる血管内皮細胞のＴＧＦ−β分泌誘導物、及びその製造方法、並びに抗老化剤を提供する。 The present invention has been made in view of such circumstances, and provides a vascular endothelial cell TGF-β secretion derivative capable of effectively preventing skin aging, a method for producing the same, and an anti-aging agent. .
本願の発明者らが鋭意検討した結果、クロレラの熱水抽出液中に血管内皮細胞からのＴＧＦ−βの分泌を誘導するＴＧＦ−β分泌誘導物が存在し、これが血管のマトリクス構造の維持を増進させることによって、皮膚の老化を抑制することができるという知見を得て本発明を完成するに至った。 As a result of intensive studies by the inventors of the present application, there is a TGF-β secretion inducer that induces secretion of TGF-β from vascular endothelial cells in the hot water extract of chlorella, and this maintains the matrix structure of blood vessels. The present invention has been completed by obtaining the knowledge that skin aging can be suppressed by the enhancement.
すなわち、（１）本発明に係る血管内皮細胞のＴＧＦ−β分泌誘導物は、クロレラの熱水抽出物であり、血管内皮細胞に対してＴＧＦ−βの分泌を誘導させることを特徴とする That, (1) TGF-β secretion-inducing substance of vascular endothelial cells according to the present invention is a hot water extract of Kurore La, characterized in that inducing the secretion of TGF-beta with respect to vascular endothelial cells
本発明の血管内皮細胞のＴＧＦ−β分泌誘導物にあっては、クロレラから熱水抽出してなる熱水抽出物である。この熱水抽出は例えば次のようにして実施することができる。１００ｋｇのクロレラ粉末に対して、０．５ｍ３以上２０ｍ３以下の適宜容量の抽出用液、好ましくは１．０ｍ３以上５．０ｍ３以下の適宜容量の抽出用液を投入して撹拌懸濁し、クロレラ懸濁液を得る。このクロレラ懸濁液を６０℃以上１３０℃以下の適宜温度に昇温させ、その温度にて６０分程度から１０分程度までの適宜時間保持する、好ましくは９５℃以上１２０℃以下の適宜温度にて２５分程度から１０分程度までの適宜時間保持することによってクロレラの有用成分を抽出する。 The TGF-β secretion derivative of vascular endothelial cells of the present invention is a hot water extract obtained by hot water extraction from chlorella. This hot water extraction can be carried out, for example, as follows. To 100 kg of chlorella powder, an appropriate volume of extraction liquid of 0.5 m 3 or more and 20 m 3 or less, preferably 1.0 m 3 or more and 5.0 m 3 or less of an appropriate volume of extraction liquid is added and stirred and suspended. A chlorella suspension is obtained. The chlorella suspension is heated to an appropriate temperature of 60 ° C. or more and 130 ° C. or less, and held at that temperature for an appropriate time from about 60 minutes to about 10 minutes, preferably at an appropriate temperature of 95 ° C. or more and 120 ° C. or less. The useful component of chlorella is extracted by holding for an appropriate time from about 25 minutes to about 10 minutes.
そして、得られた熱水抽出液を室温程度まで冷却した後、それを連続遠心分離機又は濾過機に供給し、クロレラ残渣を含む沈殿物を除去した清液を回収する。得られた清液について更に、濃縮、凍結乾燥、噴霧乾燥等を実施してもよい。 And after cooling the obtained hot-water extract to room temperature grade, it supplies to a continuous centrifuge or a filter, and the clear liquid which removed the deposit containing a chlorella residue is collect | recovered. The obtained clear solution may be further subjected to concentration, freeze drying, spray drying and the like.
このようにして得られた抽出物について種々検討したところ、血管内皮細胞に対してＴＧＦ−βの分泌を誘導させていた。ＴＧＦ−βの分泌が誘導された血管内皮細胞にあっては、管腔構造が長期間に亘って維持され、これによって抗老化作用が奏される。 As a result of various examinations on the extract thus obtained, secretion of TGF-β was induced in vascular endothelial cells. In vascular endothelial cells in which TGF-β secretion has been induced, the luminal structure is maintained for a long period of time, thereby producing an anti-aging effect.
更に、前記血管内皮細胞のＴＧＦ−β分泌誘導物は、クロレラの熱水抽出物であって分子量が１万超３万未満の分画物にて構成してあることを特徴とする。 Furthermore, the TGF-β secretion derivative of vascular endothelial cells is a hot water extract of chlorella and is composed of a fraction having a molecular weight of more than 10,000 and less than 30,000 .
本発明の血管内皮細胞のＴＧＦ−β分泌誘導物にあっては、クロレラの熱水抽出物であって分子量が１万超３万未満の分画物にて構成してある。かかる分子量範囲の分画物は、ＴＧＦ−βの分泌を誘導させる作用が他の分子量範囲の分画物より高い。 The TGF-β secretion derivative of vascular endothelial cells of the present invention is a hot water extract of chlorella and is composed of a fraction having a molecular weight of more than 10,000 and less than 30,000 . Such a molecular weight range fraction has a higher effect of inducing secretion of TGF-β than other molecular weight range fractions.
（２）本発明に係る血管内皮細胞のＴＧＦ−β分泌誘導物は、前記クロレラはチクゴ株であることを特徴とする。 (2) The TGF-β secretion derivative of vascular endothelial cells according to the present invention is characterized in that the chlorella is a Chikugo strain.
本発明の血管内皮細胞のＴＧＦ−β分泌誘導物にあっては、クロレラはチクゴ株（クロレラ工業株式会社）であり、かかるチクゴ株は、その細胞壁の厚さが略２０ｎｍ程度と他のクロレラの細胞壁より薄いため、熱水抽出における抽出効率が高い。 In the TGF-β secretion derivative of vascular endothelial cells of the present invention, chlorella is a Chikugo strain (Chlorella Kogyo Co., Ltd.), and the Chikugo strain has a cell wall thickness of about 20 nm and other chlorella strains. Since it is thinner than the cell wall, the extraction efficiency in hot water extraction is high.
（３）一方、本発明に係る血管内皮細胞ＴＧＦ−β分泌誘導物の製造方法は、クロレラから熱水抽出して得られた抽出液を、分画分子量が３万の限外濾過膜を装着させた限外濾過器に供給して限外濾過を実施し、回収した濾液を分画分子量が１万の限外濾過膜を装着させた限外濾過器に供給して限外濾過を実施し、残渣を回収することを特徴とする。 (3) On the other hand, in the method for producing a vascular endothelial cell TGF-β secretion derivative according to the present invention, an extract obtained by hot water extraction from chlorella is equipped with an ultrafiltration membrane having a molecular weight cut off of 30,000. The ultrafiltrate is supplied to the ultrafilter and the collected filtrate is supplied to the ultrafilter equipped with an ultrafiltration membrane with a molecular weight cut off of 10,000 to perform ultrafiltration. The residue is recovered.
本発明の血管内皮細胞ＴＧＦ−β分泌誘導物の製造方法にあっては、クロレラから熱水抽出して抽出液を得る。例えば、前述した如く、１００ｋｇのクロレラ粉末に対して、０．５ｍ３以上２０ｍ３以下の適宜容量の抽出用液、好ましくは１．０ｍ３以上５．０ｍ３以下の適宜容量の抽出用液を投入して撹拌懸濁し、クロレラ懸濁液を得る。このクロレラ懸濁液を６０℃以上１３０℃以下の適宜温度に昇温させ、その温度にて６０分程度から１０分程度までの適宜時間保持する、好ましくは９５℃以上１２０℃以下の適宜温度にて２５分程度から１０分程度までの適宜時間保持することによってクロレラの有用成分を抽出する。 In the method for producing a vascular endothelial cell TGF-β secretion derivative of the present invention, hot water extraction is performed from chlorella to obtain an extract. For example, as previously mentioned, chlorella powder 100 kg, 0.5 m 3 or more 20 m 3 or less appropriate volume of solution for extraction, preferably 1.0 m 3 or more 5.0 m 3 or less of the appropriate volume solution for extraction of The suspension is stirred and suspended to obtain a chlorella suspension. The chlorella suspension is heated to an appropriate temperature of 60 ° C. or more and 130 ° C. or less, and held at that temperature for an appropriate time from about 60 minutes to about 10 minutes, preferably at an appropriate temperature of 95 ° C. or more and 120 ° C. or less. The useful component of chlorella is extracted by holding for an appropriate time from about 25 minutes to about 10 minutes.
この抽出液を、分画分子量が３万の限外濾過膜を装着させた限外濾過器に供給して限外濾過を実施して、濾液を回収する。これによって、分子量が３万以下の分画物を回収する。次に、回収した濾液を分画分子量が１万の限外濾過膜を装着させた限外濾過器に供給して限外濾過を実施し、残渣を回収することによって、分子量が１万超〜３万以下の分画物を回収する。 This extract is supplied to an ultrafilter equipped with an ultrafiltration membrane having a molecular weight cut off of 30,000, and ultrafiltration is performed to collect the filtrate. Thereby, a fraction having a molecular weight of 30,000 or less is recovered. Next, the recovered filtrate is supplied to an ultrafilter equipped with an ultrafiltration membrane having a molecular weight cut off of 10,000, and subjected to ultrafiltration, and the residue is recovered, so that the molecular weight exceeds 10,000 Collect less than 30,000 fractions.
前述した如く、分子量が１万超〜３万以下の分画物は、ＴＧＦ−βの分泌を誘導させる作用が他の分子量範囲の分画物より高い。一方、このように所要の分画分子量の限外濾過膜を組み合わせることによって、所要の分画物を効率的に回収することができる。 As described above, a fraction having a molecular weight of more than 10,000 to 30,000 or less has a higher effect of inducing secretion of TGF-β than a fraction having other molecular weight ranges. On the other hand, a required fraction can be efficiently recovered by combining an ultrafiltration membrane having a required fractional molecular weight in this way.
（４）本発明に係る血管内皮細胞ＴＧＦ−β分泌誘導物の製造方法は、前記クロレラとしてチクゴ株を用いることを特徴とする。 (4) The method for producing a vascular endothelial cell TGF-β secretion derivative according to the present invention is characterized by using a Chikugo strain as the chlorella.
本発明の血管内皮細胞ＴＧＦ−β分泌誘導物の製造方法にあっては、クロレラとしてチクゴ株を用いる。前述した如く、チクゴ株は、その細胞壁の厚さが略２０ｎｍ程度と他のクロレラの細胞壁より薄いため、熱水抽出における抽出効率が高い。 In the method for producing a vascular endothelial cell TGF-β secretion derivative of the present invention, Chikugo strain is used as chlorella. As described above, the Chikugo strain has a high extraction efficiency in hot water extraction because its cell wall thickness is approximately 20 nm, which is thinner than other chlorella cell walls.
（５）本発明に係る抗老化剤は、（２）に記載の血管内皮細胞のＴＧＦ−β分泌誘導物を有効成分として含有し、血管内皮細胞の管腔構造を維持させることを特徴とする。 (5) The anti-aging agent according to the present invention contains the TGF-β secretion derivative of vascular endothelial cells according to (2) as an active ingredient, and maintains the luminal structure of vascular endothelial cells. .
本発明の抗老化剤にあっては、（２）に記載した血管内皮細胞のＴＧＦ−β分泌誘導物を有効成分として含有する。かかる誘導物によってＴＧＦ−βの分泌が誘導されると、皮膚の老化が抑制される抗老化作用が奏される。 The anti-aging agent of the present invention contains the TGF-β secretion derivative of vascular endothelial cells described in (2) as an active ingredient. When secretion of TGF-β is induced by such an inducer, an anti-aging action is achieved in which skin aging is suppressed.
更に、本発明に係る抗老化剤は、血管内皮細胞の管腔構造を維持させることを特徴とする。 Furthermore, the anti-aging agent according to the present invention is characterized by maintaining the luminal structure of vascular endothelial cells.
本発明の抗老化剤にあっては、ＴＧＦ−βの分泌を誘導することによって、血管内皮細胞の細胞死及び細胞増殖が抑制され、これによって血管内皮細胞の管腔構造が長期間に亘って維持される。血管内皮細胞の管腔構造が維持されると、紫外線の照射によって皮膚弾力が低下し、またシワ又は弛みが発生することが抑制される。 In the anti-aging agent of the present invention, cell death and cell proliferation of vascular endothelial cells are suppressed by inducing the secretion of TGF-β, whereby the luminal structure of vascular endothelial cells is maintained over a long period of time. Maintained. When the luminal structure of vascular endothelial cells is maintained, the skin elasticity is reduced by the irradiation of ultraviolet rays, and the occurrence of wrinkles or slack is suppressed.
（６）本発明に係る抗老化剤は、外用剤であることを特徴とする。 ( 6 ) The anti-aging agent according to the present invention is an external preparation.
本発明の抗老化剤にあっては、外用剤であるため、皮膚に直接作用させることができ、紫外線に対する皮膚の老化を効果的に抑制することができる。 In the anti-aging agent of this invention, since it is an external preparation, it can be made to act on skin directly and the skin aging with respect to an ultraviolet-ray can be suppressed effectively.
以下、本発明を詳述する。なお、本実施の形態で説明する事柄は、本発明の趣旨を説明する一例であり、本発明はその趣旨を逸脱しない範囲での変形や改造を含むことはいうまでもない。 The present invention is described in detail below. Note that the matter described in the present embodiment is an example for explaining the gist of the present invention, and it goes without saying that the present invention includes modifications and alterations without departing from the gist.
（血管内皮細胞のＴＧＦ−β分泌誘導物）
血管内皮細胞のＴＧＦ−β分泌誘導物（以後、本誘導物ともいう。）は例えば次のように調製することができる。
(TGF-β secretion derivative of vascular endothelial cells)
A derivative of TGF-β secretion of vascular endothelial cells (hereinafter also referred to as this derivative ) can be prepared, for example, as follows.
すなわち、１００ｋｇのクロレラ粉末（例えば、クロレラ工業株式会社製）に対して、０．５ｍ３以上２０ｍ３以下の適宜容量の抽出用液、好ましくは１．０ｍ３以上５．０ｍ３以下の適宜容量の抽出用液を投入して撹拌懸濁し、クロレラ懸濁液を得る。このクロレラ懸濁液を６０℃以上１３０℃以下の適宜温度に昇温させ、その温度にて６０分程度から１０分程度までの適宜時間保持する、好ましくは９５℃以上１２０℃以下の適宜温度にて２５分程度から１０分程度までの適宜時間保持することによってクロレラの有用成分を抽出する、所謂熱水抽出を実施する。 That is, for 100 kg of chlorella powder (for example, manufactured by Chlorella Kogyo Co., Ltd.), an extraction liquid with an appropriate volume of 0.5 m 3 or more and 20 m 3 or less, preferably an appropriate volume of 1.0 m 3 or more and 5.0 m 3 or less. The extraction liquid was added and suspended by stirring to obtain a chlorella suspension. The chlorella suspension is heated to an appropriate temperature of 60 ° C. or more and 130 ° C. or less, and held at that temperature for an appropriate time from about 60 minutes to about 10 minutes, preferably at an appropriate temperature of 95 ° C. or more and 120 ° C. or less. Thus, the so-called hot water extraction is performed in which useful components of chlorella are extracted by holding for an appropriate time from about 25 minutes to about 10 minutes.
なお、抽出用液としては井水、水道水若しくは純水、又は適宜の緩衝液であってよいが、これらに適宜量のアルコールを添加したものであってもよい。この場合、アルコールの添加量は水溶性物質の抽出に実施的に影響を及ぼさない程度とする。 The extraction liquid may be well water, tap water or pure water, or an appropriate buffer solution, but may be one obtained by adding an appropriate amount of alcohol thereto. In this case, the amount of alcohol added is set so as not to affect the extraction of the water-soluble substance practically.
熱水抽出が終了すると、得られた熱水抽出液を室温程度まで冷却した後、それを連続遠心分離機又は濾過機に供給し、クロレラ残渣を含む沈殿物を除去した清液を回収する。そして、得られた清液を減圧濃縮器に供給して３５℃〜４５℃程度の適宜温度に加温しつつ、濃縮液の乾質量濃度が１００ｍｇ±２０ｍｇ／ｍｌ程度になるまで減圧濃縮して本誘導物を得る。なお、減圧濃縮によって沈殿物が生じた場合は、遠心分離又は濾過によって当該沈殿物を除去する。 When the hot water extraction is completed, the obtained hot water extract is cooled to about room temperature, and then supplied to a continuous centrifuge or a filter to collect a clear liquid from which a precipitate containing chlorella residue has been removed. Then, the obtained clear solution is supplied to a vacuum concentrator and heated to an appropriate temperature of about 35 ° C. to 45 ° C., and concentrated under reduced pressure until the dry mass concentration of the concentrate becomes about 100 mg ± 20 mg / ml. This derivative is obtained. In addition, when the deposit arises by concentration under reduced pressure, the said deposit is removed by centrifugation or filtration.
前述した濃縮液の乾質量濃度は次のようにして求める。すなわち、恒量した恒量管内に１．０ｍｌの試料を投入し、恒量管及び試料を１０５℃で１８時間、常圧乾燥することによって水分を除去した後、恒量管及び試料をデシケータ中に格納して室温まで冷却させる。冷却後、恒量管及び試料を精秤し、精秤された値から恒量管の恒量を減じることによって、試料の乾質量を求めて、当該試料の乾質量濃度を得る。 The dry mass concentration of the concentrated liquid described above is determined as follows. That is, 1.0 ml of a sample is put into a constant weight tube, the moisture is removed by drying the constant weight tube and the sample at 105 ° C. for 18 hours, and then the constant weight tube and the sample are stored in a desiccator. Allow to cool to room temperature. After cooling, the constant weight tube and the sample are precisely weighed, and the constant weight of the constant weight tube is subtracted from the precisely weighed value to obtain the dry mass of the sample, thereby obtaining the dry mass concentration of the sample.
なお、本実施の形態では原料として前述した如くクロレラ粉末を用いたが、本発明はこれに限らず、液体培養した生クロレラを水洗・濃縮したものを原料として用いることもできる。一方、熱水抽出における抽出効率を高くすべく、細胞壁の厚さがより薄いクロレラを原料として用いるのがよいが、チクゴ株クロレラ（クロレラ工業株式会社製）にあってはその細胞壁の厚さが略２０ｎｍ程度と他のクロレラの細胞壁より薄いため好適である。 In this embodiment, chlorella powder is used as a raw material as described above. However, the present invention is not limited to this, and raw chlorella that has been liquid-cultured and washed and concentrated can also be used as a raw material. On the other hand, in order to increase the extraction efficiency in hot water extraction, it is better to use chlorella with a thinner cell wall as a raw material. However, in Chikugo strain Chlorella (manufactured by Chlorella Kogyo Co., Ltd.), the cell wall thickness is It is preferable because it is about 20 nm thinner than other chlorella cell walls.
また、本実施の形態では減圧濃縮によって抽出液を濃縮したが、本発明はこれに限らず、限外濾過膜といった膜濃縮、吸着又は分子篩等によるカラム濃縮、目的物の沈殿・再溶解等の技術を用いて抽出液の濃縮を行ってもよい。 Further, in the present embodiment, the extract is concentrated by vacuum concentration, but the present invention is not limited to this, such as membrane concentration such as ultrafiltration membrane, column concentration by adsorption or molecular sieve, precipitation / re-dissolution of the target product, etc. The extract may be concentrated using a technique.
本誘導物はこのようにクロレラの熱水抽出液を濃縮して構成することができるが、濃縮液を更に精製してもよい。精製は次のように行うことができる。 The derivative can be constituted by concentrating the hot water extract of chlorella as described above, but the concentrate may be further purified. Purification can be performed as follows.
すなわち、前述した如く減圧濃縮して得られた濃縮液を、分子量が３万を超える物質は透過させず、分子量が３万以下の物質は限外へ排除する限外濾過膜を配設した限外濾過器に供給し、限外へ排除された濾液を回収する。回収した濾液を、分子量が１万を超える物質は透過させず、分子量が１万以下の物質は限外へ排除する限外濾過膜を配設した限外濾過器に供給し、限外へ排除されずに限外濾過器内に残った精製液である残液を本誘導物として回収する。 That is, as described above, the concentrated solution obtained by concentration under reduced pressure does not permeate substances having a molecular weight of more than 30,000, and limits the provision of an ultrafiltration membrane that excludes substances having a molecular weight of 30,000 or less. The filtrate that is supplied to the outer filter and removed to the limit is recovered. The collected filtrate is supplied to an ultrafilter equipped with an ultrafiltration membrane that does not allow permeation of substances with a molecular weight of more than 10,000 and excludes substances with a molecular weight of 10,000 or less. The remaining liquid which is the purified liquid remaining in the ultrafilter without being collected is recovered as the derivative .
なお、限外濾過するに当たっては、前述した如く濃縮液を供することもできるが、熱水抽出液の清液を限外濾過に供してもよい。この場合、前述したように限外濾過器内に残った残液を必要に応じて減圧濃縮するとよい。 In the ultrafiltration, a concentrated solution can be provided as described above, but a fresh hot water extract may be used for ultrafiltration. In this case, as described above, the residual liquid remaining in the ultrafilter may be concentrated under reduced pressure as necessary.
一方、濃縮液を限外濾過に供する場合、当該濃縮液に適宜量の水を加えることもできる。これによって、限外濾過を効率的に実施することができる。この場合、前同様、限外濾過器内に残った残液を必要に応じて減圧濃縮するとよい。 On the other hand, when the concentrate is subjected to ultrafiltration, an appropriate amount of water can be added to the concentrate. Thereby, ultrafiltration can be implemented efficiently. In this case, as before, the residual liquid remaining in the ultrafilter may be concentrated under reduced pressure as necessary.
ところで、このようにして得られた濃縮液又は精製液については、更に無菌濾過を実施してもよいし、糖類といった安定剤、及び／又はｐＨ調整剤を添加して本誘導物としてもよい。更に、濃縮液又は精製液を凍結乾燥又は噴霧乾燥等することによって粉末状の本誘導物としてもよい。 By the way, the concentrated solution or the purified solution thus obtained may be further subjected to aseptic filtration, or may be added to the present derivative by adding a stabilizer such as a saccharide and / or a pH adjusting agent. Further, the concentrated derivative or purified liquid may be freeze-dried or spray-dried to obtain the powdered derivative .
なお、本実施の形態では、精製を限外濾過によって行ったが、本発明はこれに限らず、分画沈殿、カラムクロマトグラフィー等を単独で、又は組み合わせて行ってもよいことはいうまでもない。 In this embodiment, purification is performed by ultrafiltration. However, the present invention is not limited to this, and it is needless to say that fractional precipitation, column chromatography, etc. may be performed alone or in combination. Absent.
このようにして得られた本誘導物にあっては、血管内皮細胞に対してＴＧＦ−βの分泌を誘導させる。本発明者らが鋭意検討した結果、ＴＧＦ−βが分泌されると、血管内皮細胞の増殖が抑制されるとともに、細胞死も抑制され、これによって血管の管腔構造をより長い期間に亘って維持させ得ることが判明した。 In the derivative thus obtained, TGF-β secretion is induced in vascular endothelial cells. As a result of intensive studies by the present inventors, when TGF-β is secreted, the proliferation of vascular endothelial cells is suppressed and cell death is also suppressed, whereby the luminal structure of blood vessels is extended over a longer period. It has been found that it can be maintained.
ところで、血管内皮細胞が増殖する際、及びその細胞死が生じる際には、血管内の体液の一部が血管外へ移動し易く、これによって皮膚にむくみが生じる。更に、血管内の体液が血管外へ移動することによって、血管の破錠をも招来する場合がある。血管が破錠すると当該部分への血流が消滅して、コラーゲンを産生する繊維芽細胞が死滅するため、皮膚にしわが発生する。前述したように本誘導物にあっては、血管内皮細胞の増殖及び細胞死を抑制して、血管の管腔構造を長期間維持させ得るため、血管内の体液が血管外へ移動することが可及的に抑制され、従って、皮膚のしわ及び弛みの発生を防止することができる。 By the way, when vascular endothelial cells proliferate and cell death occurs, a part of the body fluid in the blood vessel easily moves out of the blood vessel, which causes swelling in the skin. In addition, the body fluid in the blood vessel may move out of the blood vessel, leading to blood vessel rupture. When the blood vessel breaks down, the blood flow to the part disappears, and the fibroblasts that produce collagen die, causing wrinkles in the skin. As described above, in this derivative, the proliferation and cell death of vascular endothelial cells can be suppressed and the luminal structure of the blood vessel can be maintained for a long period of time. It is suppressed as much as possible, and therefore it is possible to prevent the occurrence of wrinkles and loosening of the skin.
（抗老化剤）
このように本誘導物は、しわ及び弛みの発生を防止する抗老化剤として用いることができる。かかる抗老化剤の剤形としては、液剤、軟膏剤、又はクリーム剤等、種々適用することができる。
(Anti-aging agent)
Thus, this derivative can be used as an anti-aging agent that prevents the occurrence of wrinkles and slack. As the dosage form of such an anti-aging agent, various solutions such as liquids, ointments or creams can be applied.
液剤の場合、前述した如く減圧濃縮したもの、又は凍結乾燥物を適宜の溶媒に溶解させたものを適用することができる。更に、化粧料又は外用薬等に使用される多糖類といった適宜の増粘剤を混合して、その粘性を高くしてもよい。これによって、抗老化剤を塗布した皮膚から、当該抗老化剤が落下することを防止し得、抗老化剤のムダを回避することができる。 In the case of a liquid agent, one obtained by concentration under reduced pressure as described above or one obtained by dissolving a lyophilized product in an appropriate solvent can be applied. Furthermore, the viscosity may be increased by mixing an appropriate thickener such as a polysaccharide used in cosmetics or external preparations. As a result, the anti-aging agent can be prevented from falling from the skin coated with the anti-aging agent, and waste of the anti-aging agent can be avoided.
また、軟膏は、かかる液剤とワセリン又は流動パラフィン等とを混練して調製することができる。同様にクリーム剤は、前記液剤とバニシングクリーム、コールドクリーム又はポリエチレングリコール等とを混練して調製することができる。 An ointment can be prepared by kneading such a liquid agent with petrolatum or liquid paraffin. Similarly, the cream can be prepared by kneading the liquid with vanishing cream, cold cream or polyethylene glycol.
更に、抗老化剤は、ビタミンＣ又はビタミンＥ等の抗酸化剤、にんじんエキス又はミノキシジル等の血行促進剤、メトケイヒ酸オクチル又はオキシベンゾン等の紫外線防止剤等々、皮膚の老化を防止又は予防する作用を奏する他の薬剤を含んでもよい。 Furthermore, anti-aging agents have the effect of preventing or preventing skin aging, such as antioxidants such as vitamin C or vitamin E, blood circulation promoters such as carrot extract or minoxidil, and UV-protecting agents such as octyl methanoate or oxybenzone. It may contain other drugs that play.
このような抗老化剤にあっては、血管内皮細胞にＴＧＦ−βの分泌を誘導させて、血管内皮細胞の増殖及び細胞死を抑制させ、血管の管腔構造を長期間維持させることによって、例えば血管内の体液が血管外へ移動することを可及的に抑制させ、これによって皮膚のしわ及び弛みの発生、弾力の低下を防止する。 In such an anti-aging agent, by inducing secretion of TGF-β in vascular endothelial cells, suppressing proliferation and cell death of vascular endothelial cells, and maintaining the luminal structure of blood vessels for a long period of time, For example, it is possible to suppress as much as possible the movement of bodily fluid in the blood vessel to the outside of the blood vessel, thereby preventing the generation of wrinkles and slack in the skin and the decrease in elasticity.
（実施例１）
本誘導物とアンジオポイエチン−１（以後、Ａｎｇ−１という。）とを用いて、血管内皮細胞の管腔構造の維持に与える影響を比較した結果について説明する。ここで、ヒト臍帯静脈血管内皮細胞を基底膜マトリックス上で培養すると、管腔構造を呈する血管形成が誘導されることが知られている。そして、この培養系では、長時間培養することにより、血管の管腔構造は破綻していくが、内皮細胞に発現しているレセプター型チロシンキナーゼＴｉｅ２をその特異的結合因子であるＡｎｇ−１で刺激すると、管腔構造が長期間に亘って維持される。これは、内皮細胞の細胞死が抑制されることと、内皮細胞同士の接着が誘導されることによるものと考えられている。そこで、本実施例では、本誘導物にもこのような作用があるか否かについて検討した。
Example 1
The results of comparison of the effects on the maintenance of the luminal structure of vascular endothelial cells using this derivative and angiopoietin-1 (hereinafter referred to as Ang-1) will be described. Here, it is known that when human umbilical vein vascular endothelial cells are cultured on a basement membrane matrix, blood vessel formation exhibiting a luminal structure is induced. In this culture system, the luminal structure of blood vessels is destroyed by culturing for a long time, but the receptor tyrosine kinase Tie2 expressed in the endothelial cells is expressed by its specific binding factor Ang-1. Upon stimulation, the luminal structure is maintained for an extended period of time. This is thought to be due to suppression of cell death of endothelial cells and induction of adhesion between endothelial cells. Therefore, in this example, it was examined whether or not this derivative also has such an effect.
２４穴皿（Ｆａｌｃｏｎ社製）の各穴内に、成長因子を含有する基底膜マトリックス（ＢＤマトリゲル（登録商標）：Ｂｉｏｓｃａｉｅｎｃｅ社製）を１００μｌずつ注入し、３７℃で３０分間培養して基底膜マトリックスを固定した。これら各基底膜マトリックス上にそれぞれ、１％のウシ胎児血清を含有するＲＰＭI１６４０培養液（ＳＩＧＭＡ社製）で混和したヒト臍帯静脈血管内皮細胞（ＨＵＶＥＣ：Ｋｕｒａｂｏ社製）を略１×１０５細胞／穴になるように加えた。 100 μl of a basement membrane matrix (BD Matrigel (registered trademark): Bioscience) containing a growth factor was injected into each well of a 24-well dish (Falcon) and cultured at 37 ° C. for 30 minutes. Fixed. On each of these basement membrane matrices, human umbilical vein endothelial cells (HUVEC: Kurabo) mixed with RPMI 1640 culture medium (manufactured by SIGMA) containing 1% fetal bovine serum were approximately 1 × 10 5 cells / I added it to be a hole.
このヒト臍帯静脈血管内皮細胞に本誘導物を１００μｇ／ｍｌになるように添加して本発明例（ａ）とした。また、前記ヒト臍帯静脈血管内皮細胞にＡｎｇ−１（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社製）を５００ｎｇ／ｍｌになるように添加して比較例（ｅ）とした。なお、前記ヒト臍帯静脈血管内皮細胞に何も加えないものを対照例（ｃ）とした。これらを３７℃で培養して、１８時間経過後（１８ｈｒ）、及び４０時間経過後（４０ｈｒ）の培養細胞をそれぞれ撮像した結果を図１に示す。 The derivative was added to the human umbilical vein endothelial cells so as to be 100 μg / ml to obtain Example (a) of the present invention. In addition, Ang-1 (manufactured by R & D systems) was added to the human umbilical vein endothelial cells at a concentration of 500 ng / ml to obtain Comparative Example (e). In addition, what added nothing to the said human umbilical vein vascular endothelial cell was made into the control example (c). These were cultured at 37 ° C., and the results of imaging the cultured cells after 18 hours (18 hours) and after 40 hours (40 hours) are shown in FIG.
図１から明らかなように、何も添加しない対照例（ｃ）にあっては、培養を開始してから１８時間までに密なネットワーク状の管腔構造が形成されていたが、培養を開始してから４０時間経過すると、ネットワークが疎になっており、管腔構造が退縮していた。 As is clear from FIG. 1, in the control example (c) in which nothing was added, a dense network-like lumen structure was formed by 18 hours after the start of the culture, but the culture was started. After 40 hours, the network was sparse and the lumen structure was retracted.
これに対して、比較例（ｅ）では前同様に培養を開始してから１８時間までに密なネットワーク状の管腔構造が形成されており、培養を開始してから４０時間経過した時点であっても、略同じ状態で管腔構造が維持されていた。同様に、本発明例（ａ）にあっても、培養を開始してから１８時間までに密なネットワーク状の管腔構造が形成されており、培養を開始してから４０時間経過した時点であっても、略同じ状態で管腔構造が維持されていた。 On the other hand, in Comparative Example (e), a dense network-like luminal structure was formed by 18 hours after the start of culture as before, and when 40 hours had passed since the start of culture. Even in such a case, the lumen structure was maintained in substantially the same state. Similarly, in Example (a) of the present invention, a dense network-like luminal structure is formed up to 18 hours after the start of culture, and when 40 hours have passed since the start of culture. Even in such a case, the lumen structure was maintained in substantially the same state.
（実施例２）
次に、本誘導物が細胞の増殖に対して与える影響について検討した結果について説明する。
(Example 2)
Next, the results of studying the effect of this derivative on cell proliferation will be described.
図２は、本誘導物を添加した培地と、添加しない培地とで細胞を培養して経時的に細胞数を計数した結果を示すヒストグラムであり、図中、縦軸は細胞数を、また横軸は培養日数をそれぞれ示している。 FIG. 2 is a histogram showing the results of culturing cells in a medium to which this derivative was added and a medium to which this derivative was not added and counting the number of cells over time. In the figure, the vertical axis represents the cell number and the horizontal line. Each axis indicates the number of days of culture.
ヒト臍帯静脈血管内皮細胞をＨｕＭｅｄｉａ−ＥＧ２（Ｋｕｒａｂｏ社製）で混和し、１２穴皿の各穴内にそれぞれ、１×１０５細胞／穴になるように注入した。そして、対照例（ｃ）には何も添加せず、本発明例（ａ）には本誘導物を１００μｇ／ｍｌになるように添加し、３７℃で培養し、経時的に細胞数を計数した。 Human umbilical vein endothelial cells were mixed with HuMedia-EG2 (manufactured by Kurabo) and injected into each well of a 12-well dish at 1 × 10 5 cells / well. Nothing is added to the control example (c), and the derivative is added to the inventive example (a) so as to be 100 μg / ml, cultured at 37 ° C., and the number of cells is counted over time. did.
図２から明らかなように、対照例（ｃ）にあっては、１日から３日と培養日数が増えるに連れて略一定の割合で細胞数が増加していた。これに対して、本発明例（ａ）にあっては、培養日数が１日目以降、細胞数は殆ど一定であった。この結果より、本誘導物は血管内皮細胞の増殖を抑制していることが理解できる。 As is clear from FIG. 2, in the control example (c), the number of cells increased at a substantially constant rate as the number of culture days increased from 1 to 3. On the other hand, in the present invention example (a), the number of cells was almost constant after the first day of culture. From this result, it can be understood that this derivative suppresses the proliferation of vascular endothelial cells.
（実施例３）
また、本誘導物が細胞死に与える影響について検討した結果について説明する。
(Example 3)
Moreover, the result of having examined the influence which this inducer has on cell death is demonstrated.
ヒト臍帯静脈血管内皮細胞をＨｕＭｅｄｉａ−ＥＧ２（Ｋｕｒａｂｏ社製）で混和し、１２穴皿の各穴内にそれぞれ１．２×１０５細胞／穴になるように注入し、３７℃で３時間培養することによって細胞を穴内に接着させた。次いで、各穴の培地を、１％のウシ胎児血清を含有するＲＰＭI１６４０培養液（ＳＩＧＭＡ社製）に交換することによって、培養細胞を血清飢餓状態にし、この状態で３７℃で４０時間培養した。このとき、培養に先立って、対照例には何も添加せず、比較例にはＡｎｇ−１（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社製）を５００ｎｇ／ｍｌになるように添加し、本発明例には本誘導物を１００μｇ／ｍｌになるように添加した。４０時間培養後、各穴内の細胞を皿から剥離し、細胞数を計測した。 Human umbilical vein vascular endothelial cells are mixed with HuMedia-EG2 (manufactured by Kurabo), poured into each well of a 12-well dish at 1.2 × 10 5 cells / well, and cultured at 37 ° C. for 3 hours. The cells were allowed to adhere in the holes. Subsequently, the cultured cells were serum-starved by replacing the medium in each well with RPMI 1640 culture medium (manufactured by SIGMA) containing 1% fetal calf serum, and cultured in this state at 37 ° C. for 40 hours. At this time, nothing was added to the control example before culturing, and Ang-1 (manufactured by R & D systems) was added to the comparative example so as to be 500 ng / ml . Was added to a concentration of 100 μg / ml. After culturing for 40 hours, the cells in each hole were detached from the dish, and the number of cells was counted.
更に、計数した対照例及び本発明例の両細胞群について、リン酸緩衝生理食塩水（例えば０．０１Ｍリン酸カリウム、０．１５Ｍ塩化ナトリウム、ｐＨ７．２±０．２）で２回洗浄した後、１×１０６細胞／ｍｌになるように１倍のバインディング緩衝液（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製）にそれぞれ再浮遊させた。両細胞浮遊液を１００μｌ分取してそれを１．５ｍｌのチューブ内に各別に注入し、そこにアネキシン（Ａｎｎｅｘｉｎ）Ｖ試薬（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製）を５μｌ、及びヨウ化プロピデューム（ＰＩ：ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製）を２μｌ加えて穏やかに混和し、室温、暗所で１５分間反応させた。そして、各チューブに１倍のバインディング緩衝液を４００μｌずつ加えた後、フローサイトメーター（ＢＤ ＦＡＣＳ Ｃａｌｉｂｕｒ（登録商標）：Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ社製）にそれぞれ供し、細胞死の誘導についてフローサイトメトリー法を用いて解析した。 Further, both the cell groups of the control example and the inventive example counted were washed twice with phosphate buffered saline (for example, 0.01 M potassium phosphate, 0.15 M sodium chloride, pH 7.2 ± 0.2). Thereafter, the cells were resuspended in 1 × binding buffer (BD Bioscience) at 1 × 10 6 cells / ml. Take 100 μl of both cell suspensions and inject them separately into 1.5 ml tubes, to which 5 μl of Annexin V reagent (BD Bioscience) and propidium iodide (PI: BD Bioscience) are added. 2 μl) was added and gently mixed, followed by reaction at room temperature in the dark for 15 minutes. Then, 400 μl of 1 × binding buffer was added to each tube, and each tube was supplied to a flow cytometer (BD FACS Calibur (registered trademark): Becton Dickinson), and flow cytometry was used to induce cell death. And analyzed.
ここで、細胞が細胞死を起こすと、細胞膜及び核膜が崩壊するため、核を染色するＰＩによって当該細胞が染色される。一方、細胞死が誘導され始めた細胞は、ＰＩによって染色されることはないが、そのような細胞の表面にはホスファチジルセリンが出現し、このホスファチジルセリンにアネキシンＶが結合するため、細胞死が誘導され始めた細胞を選択的に検出することができる。 Here, when a cell undergoes cell death, the cell membrane and the nuclear membrane are disrupted, so that the cell is stained with PI that stains the nucleus. On the other hand, cells in which cell death has started to be induced are not stained with PI, but phosphatidylserine appears on the surface of such cells, and annexin V binds to this phosphatidylserine. Cells that have begun to be induced can be selectively detected.
図３は、栄養飢餓状態で４０時間培養した場合のヒト臍帯静脈血管内皮細胞の細胞数を計数した結果を示すヒストグラムであり、図中、縦軸は細胞数を示している。また、（ｃ）は前述した対照例を、（ｅ）は前述した比較例を、（ａ）は前述した本発明例をそれぞれ示している。なお、（ｏ）は、培養開始時の細胞数を示している。 FIG. 3 is a histogram showing the results of counting the number of human umbilical vein endothelial cells when cultured for 40 hours in a nutrient-starved state, and the vertical axis indicates the number of cells. (C) shows the above-mentioned control example, (e) shows the above-mentioned comparative example, and (a) shows the above-mentioned example of the present invention. In addition, (o) has shown the cell number at the time of a culture | cultivation start.
図３から明らかなように、対照例（ｃ）にあっては、４０時間の培養でヒト臍帯静脈血管内皮細胞の細胞数が培養開始時に比べて略１／２に減少していた。一方、Ａｎｇ−１は細胞死を抑制する作用を奏することが知られているが、本実施例にあっても比較例（ｅ）に示したようにヒト臍帯静脈血管内皮細胞の細胞死を抑制していた。そして、本発明例（ａ）にあっても、比較例（ｅ）と同様にヒト臍帯静脈血管内皮細胞の細胞死を抑制していた。 As is clear from FIG. 3, in the control example (c), the number of human umbilical vein endothelial cells decreased to about ½ in the culture for 40 hours compared to the start of the culture. On the other hand, Ang-1 is known to have an effect of suppressing cell death, but even in this example, as shown in Comparative Example (e), it suppresses cell death of human umbilical vein endothelial cells. Was. And also in the example (a) of the present invention, cell death of human umbilical vein endothelial cells was suppressed as in the comparative example (e).
図４は、本発明例についてフローサイトメトリー法を用いて解析した結果を示す散布図であり、図５は、対照例についてフローサイトメトリー法を用いて解析した結果を示す散布図である。両図中、横軸はＰＩに係る強度を、また縦軸はアネキシンＶに係る強度をそれぞれ示している。 FIG. 4 is a scatter diagram showing the results of analysis using the flow cytometry method for the inventive example, and FIG. 5 is a scatter diagram showing the results of analyzing the control example using the flow cytometry method. In both figures, the horizontal axis represents the intensity related to PI, and the vertical axis represents the intensity related to Annexin V.
図５から明らかなように、対照例にあっては、殆どのヒト臍帯静脈血管内皮細胞はＰＩに対して陽性であるか、又はアネキシンＶに対して陽性であり、ＰＩ及びアネキシンＶのいずれに対しても陰性の細胞数は全細胞数の１４．５％であった。 As is clear from FIG. 5, in the control example, most human umbilical vein endothelial cells are positive for PI or positive for annexin V, and either PI or annexin V In contrast, the number of negative cells was 14.5% of the total number of cells.
これに対して、図４から明らかなように、本発明例にあっては、ＰＩ及びアネキシンＶのいずれに対しても陰性を示したヒト臍帯静脈血管内皮細胞数は全細胞数の４２．６％であった。この結果より、本誘導物は、ヒト臍帯静脈血管内皮細胞の細胞死を抑制する作用を奏することが分かる。 On the other hand, as is clear from FIG. 4, in the example of the present invention, the number of human umbilical vein vascular endothelial cells that showed negative for both PI and annexin V was 42.6 of the total number of cells. %Met. From this result, it can be seen that this derivative exhibits an action of suppressing cell death of human umbilical vein endothelial cells.
以上のことから、本誘導物は、ヒト臍帯静脈血管内皮細胞の増殖は誘導せず、また細胞死を抑制する作用を奏する。 From the above, this derivative does not induce the proliferation of human umbilical vein vascular endothelial cells and exerts the action of suppressing cell death.
（実施例４）
ところで、前述したＡｎｇ−１は血管内皮細胞において、受容体型チロシンキナーゼであるＴｉｅ２の下流のセリン・スレオニンキナーゼであるＡｋｔというシグナル分子の活性化を誘導することにより、細胞死の抑制を誘導していることが知られている。そこで、前述した如く血管内皮細胞の細胞死を抑制する本誘導物にあっても、Ａｋｔの活性化を行っているか否かについて検討した。
Example 4
By the way, the aforementioned Ang-1 induces the suppression of cell death in vascular endothelial cells by inducing the activation of a signal molecule called Akt, a serine / threonine kinase downstream of the receptor tyrosine kinase Tie2. It is known that Therefore, as described above, whether or not Akt is activated is examined even in the present derivative that suppresses cell death of vascular endothelial cells.
ヒト臍帯静脈血管内皮細胞をＨｕＭｅｄｉａ−ＥＧ２（Ｋｕｒａｂｏ社製）で混和し、混和液を６穴皿の各穴内に、対象領域に対する細胞の占有率が略７０％〜略８０％程度になるように播種し、３７℃で一晩培養することによって接着させた。次いで、各穴の培地を、１％のウシ胎児血清を含有するＲＰＭI１６４０培養液（ＳＩＧＭＡ社製）に交換し、３７℃で４時間培養した。その後、対照例（ｃ）には何も添加せず、比較例（ｅ）にはＡｎｇ−１（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社製）を５００ｎｇ／ｍｌになるように添加し、本発明例には本誘導物を１００μｇ／ｍｌ（ａ１）、５００μｇ／ｍｌ（ａ２）、１０００μｇ／ｍｌ（ａ３）になるように添加し、１５分間培養した。 Human umbilical vein vascular endothelial cells are mixed with HuMedia-EG2 (manufactured by Kurabo), and the mixture is placed in each hole of a 6-well dish so that the cell occupancy ratio is about 70% to about 80%. Seeds and allowed to adhere by incubating overnight at 37 ° C. Next, the medium in each well was replaced with RPMI 1640 culture medium (manufactured by SIGMA) containing 1% fetal bovine serum and cultured at 37 ° C. for 4 hours. Thereafter, nothing was added to the control example (c), and Ang-1 (manufactured by R & D systems) was added to the comparative example (e) so as to be 500 ng / ml . Was added at 100 μg / ml (a1), 500 μg / ml (a2), and 1000 μg / ml (a3), and incubated for 15 minutes.
次に、各穴内の細胞をリン酸緩衝生理食塩水（例えば０．０１Ｍリン酸カリウム、０．１５Ｍ塩化ナトリウム、ｐＨ７．２±０．２）で洗浄した後、溶解用緩衝液（和光純薬工業株式会社製）を各穴内にそれぞれ注入することによって穴内の細胞を溶解し、それぞれ試料液とした。この試料液を用いて、ＳＤＳ−ＰＡＧＥ及びウェスタンブロティングを実施することによってＡｋｔ及びＥｒｋ（Ｅｘｔｒａｃｅｌｌｕｌａｒ ｓｉｇｎａｌ−ｒｅｇｕｌａｔｅｄ ｋｉｎａｓｅ）、並びにそれらのリン酸化の有無を検出した。 Next, the cells in each hole are washed with phosphate buffered saline (for example, 0.01 M potassium phosphate, 0.15 M sodium chloride, pH 7.2 ± 0.2), and then a lysis buffer (Wako Pure Chemical Industries, Ltd.). Kogyo Co., Ltd.) was injected into each hole to lyse the cells in the hole, and each was used as a sample solution. Using this sample solution, SDS-PAGE and Western blotting were performed to detect Akt and Erk (Extracellular signal-regulated kinase) and the presence or absence of phosphorylation thereof.
なお、Ａｋｔの検出には、ａｎｔｉ−Ａｋｔ抗体（Ｃｅｌｌ ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ社製）を用い、リン酸化Ａｋｔの検出には、ａｎｔｉ−ｐＡｋｔ抗体（Ｃｅｌｌ ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ社製）を用い、Ｅｒｋの検出には、ａｎｔｉ−Ｅｒｋ抗体（Ｃｅｌｌ ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ社製）を用い、リン酸化Ｅｒｋの検出には、ａｎｔｉ−ｐＥｒｋ抗体（Ｃｅｌｌ ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ社製）を用いた。 For detection of Akt, an anti-Akt antibody (manufactured by Cell signaling Technology) was used. For detection of phosphorylated Akt, an anti-pAkt antibody (manufactured by Cell signaling Technology) was used, and for detection of Erk, An anti-Erk antibody (manufactured by Cell signaling Technology) was used, and an anti-pErk antibody (manufactured by Cell signaling Technology) was used to detect phosphorylated Erk.
図６は、Ａｋｔ及びリン酸化Ａｋｔを検出するために行ったウェスタンブロティングの画像図であり、図７は、Ｅｒｋ及びリン酸化Ｅｒｋを検出するために行ったウェスタンブロティングの画像図である。 FIG. 6 is an image diagram of Western blotting performed to detect Akt and phosphorylated Akt, and FIG. 7 is an image diagram of Western blotting performed to detect Erk and phosphorylated Erk.
図６から明らかなように、Ａｎｇ−１で処理した比較例（ｅ）ではリン酸化Ａｋｔの存在を示す薄い二本目のバンドが表れていたが、本誘導物で処理した本発明例（ａ１）〜（ａ３）はいずれも、対照例（ｃ）と同様に、リン酸化Ａｋｔの存在を示す二本目のバンドは表れていなかった。従って、本誘導物によってはＡｋｔは活性化されていないものと考えられる。これに対して、図７から明らかなように、本誘導物で処理した本発明例（ａ１）〜（ａ３）はいずれも、リン酸化Ｅｒｋの存在を示す二本目のバンドが表れていた。これらの結果より、本誘導物は、Ａｋｔの活性化が介在するＡｎｇ−１とは異なり、Ｅｒｋの活性化を介して血管内皮細胞の細胞死を抑制しているものと考えられる。 As is clear from FIG. 6, the comparative example (e) treated with Ang-1 showed a thin second band indicating the presence of phosphorylated Akt, but the invention example (a1) treated with this derivative. In all of-(a3), the second band indicating the presence of phosphorylated Akt did not appear as in the control example (c). Therefore, it is considered that Akt is not activated by this derivative . On the other hand, as is clear from FIG. 7, all of the inventive examples (a1) to (a3) treated with the derivative showed a second band indicating the presence of phosphorylated Erk. From these results, it is considered that this derivative suppresses cell death of vascular endothelial cells through activation of Erk, unlike Ang-1 mediated by activation of Akt.
（実施例５）
次に、本誘導物で血管内皮細胞を刺激した場合に、当該血管内皮細胞から分泌される血管関連因子の発現について検討した結果について説明する。なお、検討対象とした血管関連因子は、ＶＥＧＦ、Ａｎｇ−１、Ａｎｇ−２及びＴＧＦ−β１である。
(Example 5)
Next, the results of examining the expression of vascular-related factors secreted from vascular endothelial cells when vascular endothelial cells are stimulated with this derivative will be described. The blood vessel-related factors to be examined are VEGF, Ang-1, Ang-2, and TGF-β1.
ヒト臍帯静脈血管内皮細胞をＨｕＭｅｄｉａ−ＥＧ２（Ｋｕｒａｂｏ社製）で混和し、混和液を６穴皿の各穴内に、対象領域に対する細胞の占有率が略７０％〜略８０％程度になるように播種し、３７℃で一晩培養することによって接着させた。次いで、各穴の培地を、１％のウシ胎児血清を含有するＲＰＭI１６４０培養液（ＳＩＧＭＡ社製）に交換し、３７℃で２４時間培養した。このとき、対照例（ｃ）には何も添加せず、本発明例には本誘導物を１００μｇ／ｍｌ（ａ）になるように前記培養液に添加しておいた。 Human umbilical vein vascular endothelial cells are mixed with HuMedia-EG2 (manufactured by Kurabo), and the mixture is placed in each hole of a 6-well dish so that the cell occupancy ratio is about 70% to about 80%. Seeds and allowed to adhere by incubating overnight at 37 ° C. Next, the medium in each well was replaced with RPMI 1640 culture medium (manufactured by SIGMA) containing 1% fetal calf serum and cultured at 37 ° C. for 24 hours. At this time, nothing was added to the control example (c), and the derivative was added to the culture solution so as to be 100 μg / ml (a) in the inventive example.
培養が終了すると、各穴内の培養液を廃棄し、各穴内の細胞群をリン酸緩衝生理食塩水（例えば０．０１Ｍリン酸カリウム、０．１５Ｍ塩化ナトリウム、ｐＨ７．２±０．２）で洗浄した後、ＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ（Ｑｉａｇｅｎ社製）を用いて、各細胞群それぞれについて全ＲＮＡの回収を行った。次に、ＥｘＳｃｒｉｐｔ ＲＴ ｒｅａｇｅｎｔ Ｋｉｔ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ：Ｔａｋａｒａ社製）を用いて、回収した各全ＲＮＡからｃＤＮＡをそれぞれ合成し、得られたｃＤＮＡを用いて、前述した各血管関連因子に係るｍＲＮＡの発現をリアルタイムＰＣＲ法によって解析した。なお、対照として解糖系酵素であるＧＡＰＤＨ（Ｇｌｙｃｅｒａｌｄｅｈｙｄｅ−３−ｐｈｏｓｐｈａｔｅ ｄｅｈｙｄｏｒｏｇｅｎａｓｅ）に係るｍＲＮＡの発現量も同様に調べた。また、リアルタイムＰＣＲは、Ｓｔｒａｔａｇｅｎｅ Ｍｘ３０００Ｐ（Ｓｔｒａｔａｇｅｎｅ社製）を用いて実施した。 When the culture is completed, the culture solution in each well is discarded, and the cells in each well are washed with phosphate buffered saline (for example, 0.01 M potassium phosphate, 0.15 M sodium chloride, pH 7.2 ± 0.2). After washing, total RNA was collected for each cell group using RNeasy Mini Kit (Qiagen). Next, using ExScript RT reagent Kit (Perfect Real Time: manufactured by Takara), cDNA was synthesized from each recovered RNA, and using the obtained cDNA, mRNA of each of the blood vessel-related factors described above was synthesized. Expression was analyzed by real-time PCR. As a control, the expression level of mRNA related to GAPDH (Glyceraldehyde-3 phosphate dehydrogenase), which is a glycolytic enzyme, was also examined in the same manner. Real-time PCR was performed using Stratagene Mx3000P (manufactured by Stratagene).
ここで、ヒトＶＥＧＦをＰＣＲで合成する際のプライマーは以下の配列のものを用いた。
５´-aac cat gaa ctt tct gct gtc ttg-３´ （配列番号１）
５´-ttc acc act tcg tga tga ttc tg-３´ （配列番号２）
Here, primers having the following sequences were used for the synthesis of human VEGF by PCR.
5'-aac cat gaa ctt tct gct gtc ttg-3 '(SEQ ID NO: 1)
5'-ttc acc act tcg tga tga ttc tg-3 '(SEQ ID NO: 2)
また、ヒトＡｎｇ−１をＰＣＲで合成する際のプライマーは以下の配列のものを用いた。
５´-tca cat agg gtg cag caa tc-３´ （配列番号３）
５´-aca gtt gcc atc gtg ttt ctg-３´ （配列番号４）
In addition, primers having the following sequences were used for the synthesis of human Ang-1 by PCR.
5'-tca cat agg gtg cag caa tc-3 '(SEQ ID NO: 3)
5'-aca gtt gcc atc gtg ttt ctg-3 '(SEQ ID NO: 4)
同様に、ヒトＡｎｇ−２をＰＣＲで合成する際のプライマーは以下の配列のものを用いた。
５´-ata agc agc atc agc caa cc-３´ （配列番号５）
５´-aag ttg gaa gga cca cat gc-３´ （配列番号６）
Similarly, primers having the following sequences were used for the synthesis of human Ang-2 by PCR.
5′-ata agc agc atc agc caa cc-3 ′ (SEQ ID NO: 5)
5′-aag ttg gaa gga cca cat gc-3 ′ (SEQ ID NO: 6)
また、ヒトＴＧＦ−β１をＰＣＲで合成する際のプライマーは以下の配列のものを用いた。
５´-ggg act atc cac ctg caa ga-３´ （配列番号７）
５´-cct cct tgg cgt agt agt cg-３´ （配列番号８）
Further, primers having the following sequences were used in synthesizing human TGF-β1 by PCR.
5′-ggg act atc cac ctg caa ga-3 ′ (SEQ ID NO: 7)
5'-cct cct tgg cgt agt agt cg-3 '(SEQ ID NO: 8)
一方、ヒトＧＡＰＤＨをＰＣＲで合成する際のプライマーは以下の配列のものを用いた。
５´-acc cag aag act gtg gat gg-３´ （配列番号９）
５´-ccc tgt tgc tgt agc caa at-３´ （配列番号１０）
On the other hand, primers having the following sequences were used for the synthesis of human GAPDH by PCR.
5′-acc cag aag act gtg gat gg-3 ′ (SEQ ID NO: 9)
5′-ccc tgt tgc tgt agc caa at-3 ′ (SEQ ID NO: 10)
図８は血管内皮細胞のＶＥＧＦに係るｍＲＮＡの発現量を測定した結果を示すヒストグラムであり、図９は血管内皮細胞のＡｎｇ−１に係るｍＲＮＡの発現量を測定した結果を示すヒストグラムであり、図１０は血管内皮細胞のＡｎｇ−２に係るｍＲＮＡの発現量を測定した結果を示すヒストグラムであり、図１１は血管内皮細胞のＴＧＦ−β１に係るｍＲＮＡの発現量を測定した結果を示すヒストグラムである。各図中、縦軸は各発現量をＧＡＰＤＨに係るｍＲＮＡの発現量に対する比率で示している。また、各図中、（ｃ）は対照を示しており、（ａ）は本誘導物で処理した場合を示している。 FIG. 8 is a histogram showing the result of measuring the expression level of mRNA related to VEGF in vascular endothelial cells, and FIG. 9 is a histogram showing the result of measuring the expression level of mRNA related to Ang-1 in vascular endothelial cells, FIG. 10 is a histogram showing the results of measuring the expression level of mRNA related to Ang-2 of vascular endothelial cells, and FIG. 11 is a histogram showing the results of measuring the expression level of mRNA related to TGF-β1 of vascular endothelial cells. is there. In each figure, the vertical axis represents each expression level as a ratio to the expression level of mRNA related to GAPDH. Moreover, in each figure, (c) has shown the control | contrast and (a) has shown the case where it processes with this derivative | guide_body .
図８〜図１０から明らかなように、ＶＥＧＦ、Ａｎｇ−１及びＡｎｇ−２にあっては、本誘導物で処理した場合（ａ）と、本誘導物で処理していない対照（ｃ）との間に有意差は見られなかった。これに対して、図１１から明らかなように、ＴＧＦ−β１にあっては、本誘導物で処理していない対照（ｃ）では対応するｍＲＮＡが殆ど発現していないが、本誘導物で処理した場合（ａ）、対応するｍＲＮＡの発現量が９０程度と有意に増大していた。これにより、本誘導物は、血管内皮細胞に対してＴＧＦ−β１を誘導していることが分かる。 As is clear from FIGS. 8 to 10, VEGF, Ang-1 and Ang-2 were treated with this derivative (a) and a control (c) not treated with this derivative. There was no significant difference between the two. In contrast, as is clear from FIG. 11, in TGF-β1, the corresponding mRNA was hardly expressed in the control (c) not treated with this derivative, but treated with this derivative. (A), the corresponding mRNA expression level was significantly increased to about 90. Thereby, it turns out that this inducer induces TGF-β1 to vascular endothelial cells.
（実施例６）
次に、本誘導物によるＴＧＦ−β１の誘導が血管内皮細胞以外の細胞に対しても奏されているのかについて検討した結果について説明する。
(Example 6)
Next, the results of examining whether the induction of TGF-β1 by this derivative is also exerted on cells other than vascular endothelial cells will be described.
６匹の雌のヘアレスマウス（Ｈｏｓ：ＨＲ−１、株式会社星野試験動物飼育所製）を８週齢になるまで予備飼育した後、３匹ずつ２つの群に分け、一方の群のマウスの背部には本誘導物を配合した本誘導物クリームを塗布し、他方の群のマウスにはプラセボクリームを塗布して、紫外線を照射する試験を２週間行った。なお、予備飼育では固形飼料（ＣＥ−２、日本クレア株式会社製）を自由に摂取させた。 Six female hairless mice (Hos: HR-1, manufactured by Hoshino Test Animal Breeding Co., Ltd.) were preliminarily raised until the age of 8 weeks, and then divided into 2 groups of 3 mice. back this derivates cream blended with the induction was applied to, the mice of the other group by applying a placebo cream was carried out 2 weeks a test of irradiating ultraviolet rays. In preliminary breeding, solid feed (CE-2, manufactured by CLEA Japan, Inc.) was freely ingested.
そして、各マウスの背部からそれぞれ皮膚を採取し、得られた各皮膚を後述するように酵素処理することによって当該皮膚を構成する細胞にそれぞれ分解して全皮膚細胞群とした。また、得られた各全皮膚細胞群から一部をそれぞれ分取し、分取した各全皮膚細胞群に抗ＣＤ３１抗体（ｐｈａｒｍｉｎｇｅｎ社製）及び抗ＣＤ４５抗体（ｐｈａｒｍｉｎｇｅｎ社製）を添加し、ＣＤ３１陽性でありＣＤ４５陰性である細胞をそれぞれ分画して、それらを血管内皮細胞群とした。このようにして得た各全皮膚細胞群及び各血管内皮細胞群について、前同様にして全ＲＮＡ及びｃＤＮＡを合成し、ＴＧＦ−β１に係るｍＲＮＡの発現量をリアルタイムＰＣＲ法によって測定した。 Then, each skin was collected from the back of each mouse, and each obtained skin was enzymatically treated as described later to be decomposed into cells constituting the skin to obtain a total skin cell group. Further, a part of each whole skin cell group obtained was fractionated, and anti-CD31 antibody (manufactured by Pharmingen) and anti-CD45 antibody (manufactured by Pharmingen) were added to each of the collected whole skin cell groups, and CD31 Cells that were positive and CD45 negative were fractionated, and these were used as vascular endothelial cell groups. For each skin cell group and each vascular endothelial cell group thus obtained, total RNA and cDNA were synthesized as before, and the expression level of mRNA related to TGF-β1 was measured by a real-time PCR method.
ここで、前記本誘導物クリームの組成及び配合比は、８７ｍｇ／ｍｌの本誘導物が５１質量％、プロピレングリコールが１０質量％、エタノールが１０質量％、固形パラフィンが５質量％、流動パラフィンが５質量％、エマレックス８１００（モノステアリン酸ポリエチレングリコール）（日本エマルジョン株式会社製）が５質量％、ツイーン２０（ポリオキシエチレン（２０）ソルビタンモノラウレート）（和光純薬工業株式会社製）が５質量％、ステアリン酸が２質量％、セチルアルコールが２質量％、ステアリン酸グリセロールが２質量％、メントールが２質量％、ヒアルロン酸ナトリウムが１質量％である。また、プラセボクリームは、本誘導物に代えて水を用いた以外は前記本誘導物クリームと同じ組成及び配合比になしたものを用いた。 Here, the composition and blending ratio of the derivative cream were: 51% by mass of the derivative of 87 mg / ml, 10% by mass of propylene glycol, 10% by mass of ethanol, 5% by mass of solid paraffin, and liquid paraffin. 5% by mass, 5% by mass of Emalex 8100 (polyethylene glycol monostearate) (manufactured by Nippon Emulsion Co., Ltd.), Tween 20 (polyoxyethylene (20) sorbitan monolaurate) (manufactured by Wako Pure Chemical Industries, Ltd.) 5% by mass, 2% by mass of stearic acid, 2% by mass of cetyl alcohol, 2% by mass of glycerol stearate, 2% by mass of menthol, and 1% by mass of sodium hyaluronate. Moreover, the placebo cream, except that water was used in place of the induction was used was without the same composition and mixing ratio with the present derivates cream.
また、前述した紫外線照射試験は、３回／週で合計６回の紫外線照射を実施し、１週目はそれぞれ３６ｍＪ／ｃｍ２／回の強度で、また２週目はそれぞれ４７ｍＪ／ｃｍ２／回の強度で照射した。そして、この間、各マウスの背部の皮膚に、前記本誘導物クリーム又はプラセボクリームを、５回／週、合計で１０回塗布した。なお、この間、セロハンテープによってマウスの背部の皮膚をストリッピングするテープストリッピングを２回／週、合計で４回実施し、１回当たりのストリッピング回数は４回行った。 Moreover, the ultraviolet irradiation test mentioned above implemented a total of 6 times of ultraviolet irradiation 3 times / week, the intensity | strength of 36 mJ / cm < 2 > / times in the 1st week, and 47 mJ / cm < 2 > / each in the 2nd week respectively. Irradiated with twice the intensity. During this time, the derivative cream or placebo cream was applied to the skin on the back of each mouse 10 times, 5 times / week. During this time, tape stripping for stripping the skin on the back of the mouse with cellophane tape was performed twice a week for a total of four times, and the number of stripping times per time was four times.
一方、前述した皮膚の分解は次のようにして行った。すなわち、回収した皮膚をハサミで細断し、これをディスパーゼＩＩ（エーディア株式会社製）酵素溶液中に投入して、３７℃で適宜時間振盪させた後、酵素溶液を除去した。得られた皮膚をコラゲナーゼタイプＩ（和光純薬工業株式会社製）酵素溶液中に投入して、前同様に酵素処理した後、酵素溶液を除去した。更に、得られた皮膚をコラゲナーゼタイプＩＩ（Ｗｏｒｔｈｉｎｇｔｏｎ社製）酵素溶液中に投入して、前同様に酵素処理した後、１０ｍｌの容量のシリンジ（テルモ株式会社製）を用いて、酵素処理した皮膚細胞を吸引・排出する操作を１０回繰り返して、機械的作用によっても細胞を分散させた。この溶液をメッシュサイズが７０μｍのセルストレイナー（Ｆａｉｃｏｎ社製）にて濾過することによって、皮膚残渣を濾別して、分散させた細胞を回収した。このようにして得られた細胞に１倍のＲＢＣ溶解用緩衝液（Ｂｅｃｔｏｎ Ｃｏｕｌｔｅｒ社製）を適宜量添加して赤血球を溶解させることによって、全皮膚細胞群を得た。 On the other hand, the above-described skin decomposition was performed as follows. That is, the collected skin was chopped with scissors, put into dispase II (manufactured by Aedia Co., Ltd.) enzyme solution, shaken at 37 ° C. for an appropriate time, and then removed. The obtained skin was put into collagenase type I (manufactured by Wako Pure Chemical Industries, Ltd.) enzyme solution, subjected to enzyme treatment as before, and then the enzyme solution was removed. Furthermore, after putting the obtained skin into collagenase type II (manufactured by Worthington) enzyme solution and carrying out the enzyme treatment in the same manner as before, the skin treated with the enzyme using a 10 ml syringe (manufactured by Terumo Corporation). The operation of sucking and discharging the cells was repeated 10 times, and the cells were also dispersed by mechanical action. By filtering this solution with a cell strainer (manufactured by Faicon) having a mesh size of 70 μm, the skin residue was separated by filtration and the dispersed cells were collected. An appropriate amount of 1-fold RBC lysis buffer (Becton Coulter) was added to the cells thus obtained to lyse erythrocytes, thereby obtaining a whole skin cell group.
ここで、マウスＴＧＦ−β１のｃＤＮＡを合成する際のプライマーは以下のものを用いた。
５´-gac tct cc acct gca aga cc-３´ （配列番号１１）
５´-gac tgg cga gcc tta gtt tg-３´ （配列番号１２）
Here, the following primers were used in synthesizing mouse TGF-β1 cDNA.
5'-gac tct cc acct gca aga cc-3 '(SEQ ID NO: 11)
5'-gac tgg cga gcc tta gtt tg-3 '(SEQ ID NO: 12)
また、マウスＧＡＰＤＨのｃＤＮＡを合成する際のプライマーは以下のものを用いた。
５´-aac ttt ggc att gtg gaa gg-３´ （配列番号１３）
５´-gga tgc agg gat gat gtt ct-３´ （配列番号１４）
The following primers were used for synthesizing mouse GAPDH cDNA.
5′-aac ttt ggc att gtg gaa gg-3 ′ (SEQ ID NO: 13)
5′-gga tgc agg gat gat gtt ct-3 ′ (SEQ ID NO: 14)
なお、前述したＣＤ３１陽性でありＣＤ４５陰性である血管内皮細胞の分画は、セルソーター（ＦＡＣＳＡｒｉａ（登録商標）：Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ社製）によって行った。 The above-described fractionation of vascular endothelial cells that are CD31 positive and CD45 negative was performed with a cell sorter (FACSAria (registered trademark) manufactured by Becton Dickinson).
図１２は、全皮膚細胞群についてＴＧＦ−β１に係るｍＲＮＡの発現量を測定した結果を示すヒストグラムであり、また、図１３は血管内皮細胞群についてＴＧＦ−β１に係るｍＲＮＡの発現量を測定した結果を示すヒストグラムである。なお、両図中、縦軸はＴＧＦ−β１に係るｍＲＮＡの発現量をＧＡＰＤＨに係るｍＲＮＡの発現量に対する相対発現量で示している。また、両図中、（ｃ）は、本誘導物を塗布していない場合を、また（ａ）は本誘導物を塗布した場合をそれぞれ示している。 FIG. 12 is a histogram showing the results of measuring the expression level of mRNA related to TGF-β1 for all skin cell groups, and FIG. 13 shows the expression level of mRNA related to TGF-β1 for vascular endothelial cell groups. It is a histogram which shows a result. In both figures, the vertical axis indicates the expression level of mRNA related to TGF-β1 as a relative expression level relative to the expression level of mRNA related to GAPDH. Moreover, in both figures, (c) is a case not coated with the induction was also (a) shows the case of applying the present derivates, respectively.
図１２から明らかなように、全皮膚細胞群にあっては、本誘導物クリームを塗布した場合におけるＴＧＦ−β１に係るｍＲＮＡの発現量と、プラセボクリームを塗布した場合におけるＴＧＦ−β１に係るｍＲＮＡの発現量とは略同じ値であった。これに対して、図１３から明らかなように、血管内皮細胞群にあっては、本誘導物クリームを塗布した場合（ａ）におけるＴＧＦ−β１に係るｍＲＮＡの発現量は、プラセボクリームを塗布した場合（ｃ）におけるＴＧＦ−β１に係るｍＲＮＡの発現量の略１．３倍に亢進されていた。これらの結果より、本誘導物は、血管内皮細胞に対して特異的にＴＧＦ−β１の分泌を誘導していることが分かる。 As is clear from FIG. 12, in the whole skin cell group, the expression level of mRNA related to TGF-β1 when this derivative cream is applied, and the mRNA related to TGF-β1 when placebo cream is applied The expression level was approximately the same value. On the other hand, as is clear from FIG. 13, in the vascular endothelial cell group, the expression level of mRNA related to TGF-β1 in the case where the present derivative cream was applied was applied with the placebo cream. In case (c), the expression level of mRNA related to TGF-β1 was increased to about 1.3 times. From these results, the induced material can be seen to induce the secretion of specific TGF-.beta.1 against vascular endothelial cells.
ここで、前述した実施例１から実施例６までの結果より、本誘導物にあっては、血管内皮細胞に対して特異的にＴＧＦ−β１の分泌を誘導し、それによってＥｒｋの活性化を介して、血管内皮細胞の細胞死及び血管内皮細胞の増殖を抑制し、これによって血管の管腔構造を長期間に亘って安定的に維持させているものと言える。 Here, from the results of Example 1 to Example 6 described above, the present derivative specifically induces secretion of TGF-β1 to vascular endothelial cells, thereby activating Erk. Thus, it can be said that the cell death of vascular endothelial cells and the proliferation of vascular endothelial cells are suppressed, thereby stably maintaining the luminal structure of blood vessels over a long period of time.
（実施例７）
次に、このような本誘導物の作用が皮膚に与える影響を検討した結果について説明する。
皮膚に与える影響は、皮膚弾力試験、並びに皮膚のシワ及び弛みの評価試験によって検討した。
(Example 7)
Next, the results of studying the effect of such an inducer on the skin will be described.
The effect on the skin was examined by a skin elasticity test and an evaluation test for skin wrinkles and looseness.
２０匹の雌のヘアレスマウス（Ｈｏｓ：ＨＲ−１、株式会社星野試験動物飼育所製）を８週齢になるまで予備飼育した後、無処理例に４匹、対照例に８匹、本発明例に８匹振り分け、本発明例のマウスの背部には本誘導物を配合した本誘導物クリームを塗布し、対照例のマウスにはプラセボクリームを塗布して、紫外線を照射する試験を８週間行った。なお、無処理例のマウスには紫外線の照射、本誘導物クリーム又はプラセボクリームの塗布、及び後述するテープストリッピングのいずれも実施していない。また、予備飼育では固形飼料（ＣＥ−２、日本クレア株式会社製）を自由に摂取させた。 Twenty female hairless mice (Hos: HR-1, manufactured by Hoshino Test Animal Breeding Co., Ltd.) were preliminarily raised until they reached 8 weeks of age, then 4 in the untreated case and 8 in the control case. sorting 8 rats example, this induction was cream to the back of the mouse of the present invention example formulated with the induction was applied, by applying the placebo cream in mice in the control example, 8-week test of irradiating ultraviolet rays went. In addition, the mouse | mouth of an untreated example is not implementing any of ultraviolet irradiation, application | coating of this derivative cream or placebo cream, and tape stripping mentioned later. Moreover, solid feed (CE-2, manufactured by Nippon Claire Co., Ltd.) was freely ingested in the preliminary breeding.
ここで、前記本誘導物クリーム及びプラセボクリームの組成及び配合比は、実施例６で説明した本誘導物クリーム及びプラセボクリームの組成及び配合比と同じである。 Here, the composition and compounding ratio of the present derivates cream and placebo cream, the same as the composition and compounding ratio of the derivates cream and placebo cream described in Example 6.
また、前述した紫外線照射試験は、３回／週で合計２４回の紫外線照射を実施し、１週目はそれぞれ３６ｍＪ／ｃｍ２／回の強度とし、１週毎に照射量を増加させ、合計で２５７７ｍＪ／ｃｍ２の強度で照射した。そして、この間、各マウスの背部の皮膚に、前記本誘導物クリーム又はプラセボクリームを、５回／週、合計で４０回塗布した。なお、この間、セロハンテープによってマウスの背部の皮膚をストリッピングするテープストリッピングを２回／週、合計で１６回実施し、１回当たりのストリッピング回数は４回行った。 In addition, the ultraviolet irradiation test described above was performed 24 times in total at 3 times / week, the intensity was 36 mJ / cm 2 / time for the first week, and the amount of irradiation was increased every week. And irradiated at an intensity of 2577 mJ / cm 2 . During this time, the derivative cream or placebo cream was applied to the skin on the back of each mouse for 5 times / week for a total of 40 times. During this time, tape stripping for stripping the skin of the back of the mouse with cellophane tape was performed twice / week for a total of 16 times, and the number of times of stripping per time was 4 times.
そして、各マウスについて皮膚弾力試験、並びに皮膚のシワ及び弛みの評価試験を実施した。ここで、皮膚弾力試験は、Ｔｓｕｋａｈａｒａらの方法（Ｂｒｉｔｉｓｈ Ｊｏｕｒｎａｌ ｏｆ Ｄｅｒｍａｔｏｌｏｇｙ、１５１：９８４−９９４、２００４）に準じて実施した。すなわち、マウスに麻酔を施し、当該マウスを伏せさせた状態にさせて、背中の一番高い第１箇所、及び該第１箇所から腹側へ１ｃｍ隔てた第２箇所にそれぞれ印を付けた。マウスに十分に麻酔が効いた状態で、第１箇所の皮膚をピンセットでつまみ、そのまま鉛直上方向へ、第２箇所の皮膚が背中筋肉から浮くまで引き上げる。そして、ピンセットから皮膚を解放させてから、当該皮膚が元の状態に復帰するまでに要した時間を計測した。 Each mouse was subjected to a skin elasticity test and an evaluation test for skin wrinkles and looseness. Here, the skin elasticity test was performed according to the method of Tsukahara et al. (British Journal of Dermatology, 151: 984-994, 2004). That is, the mouse was anesthetized, and the mouse was placed in a lying state, and the first place with the highest back and the second place separated 1 cm from the first place to the ventral side were marked. With the mouse sufficiently anesthetized, the skin at the first location is pinched with forceps and pulled up vertically until the skin at the second location floats from the back muscles. Then, after the skin was released from the tweezers, the time required for the skin to return to the original state was measured.
一方、シワ及び弛みの評価試験は、Ｂｉｓｓｅｔｔらの方法（Ｐｈｏｔｏｃｈｅｍ Ｐｈｏｔｏｂｉｏｌ、４６：３６７−３７８、１９８７）に従って実施した。すなわち、「健康的な構造は見られない。多くの深く粗いシワが背骨を横切って存在する（頭から尻尾の方向に垂直の線）消えることはない」又は「全ての場所に健康的な構造は見られない。強烈なたるみが首と脇腹に沿って見られる。背中に強烈なシワがある。肌色は消失している」を３と、また、「健康的な構造は見られない。いくつかの粗いシワが背骨を横切って存在する（頭から尻尾の方向に垂直の線）消えることはない」又は「全ての場所に健康的な構造は見られない。中程度のたるみが首と脇腹に沿って見られる。背中に中程度シワがある。肌色は消失している」を２と、また、「背中に沿った部分に健康的な構造は見られない。少しの浅い粗いシワが背中を横切って（交差して）存在する（頭から尻尾の方向に垂直の線）。動きに応じて見え隠れする」又は「首と肩と脇腹に沿った部分に健康的な構造は見られない。首にいくらかのたるみがある（たるみは頭から尻尾の方向に垂直の線）。背中の皮膚にいくつかのシワの節がある。首と肩部分の肌色は消失している。背中の上部の皮膚はわずかに白くなっている」を１と、更に、「背中と脇腹一面に多数の線がある（頭から尻尾の方に向かい健康的な構造の線）。動きに応じて見え隠れする」又は「背中と脇腹一面に多数の線がある（頭から尻尾の方に向かい健康的な構造の線）。肌色はパープルピンク」を０とした。 On the other hand, the evaluation test of wrinkles and slack was performed according to the method of Bissett et al. (Photochem Photobiol, 46: 367-378, 1987). That is, “healthy structures are not seen. Many deep rough wrinkles do not disappear across the spine (vertical line from head to tail)” or “healthy structures everywhere Strong sagging can be seen along the neck and flank, intense wrinkles on the back, skin color has disappeared, and “healthy structure is not seen.” No rough wrinkles exist across the spine (a line perpendicular to the head to tail) "or" No healthy structure is seen everywhere. Moderate sagging is on the neck and flank “There are moderate wrinkles on the back. The skin color has disappeared.” 2 and “There is no healthy structure in the area along the back. Some shallow rough wrinkles are on the back. Exists across (crossing) the line (vertical from head to tail). Or "healthy structure is not seen along the neck, shoulders and flank. There is some slack in the neck (sag is a line perpendicular to the head to tail). There are some wrinkles on the skin, the skin color of the neck and shoulders has disappeared, the skin on the upper back is slightly white, and 1 and many more on the back and flank There is a line (a line of healthy structure from head to tail). It appears and hides according to movement "or" There are many lines on the back and flank (healthy structure from head to tail) The skin color was set to 0 for “purple pink”.
図１４は、皮膚弾力試験を行った結果を示すヒストグラムであり、縦軸は時間を示している。また、図中、（ｏ）は無処理例を、（ｃ）は対照例を、（ａ）は本発明例をそれぞれ示している。 FIG. 14 is a histogram showing the results of the skin elasticity test, and the vertical axis shows time. In the figure, (o) shows an untreated example, (c) shows a control example, and (a) shows an example of the present invention.
図１４から明らかなように、無処理例（ｏ）のマウスは平均１．４秒程度であるのに対し、対照例（ｃ）のマウスは平均１．７秒程度であり、紫外線の照射によって皮膚弾力が低下していた。これに対して、本発明例（ａ）のマウスは平均１．５秒程度であり、紫外線の照射による皮膚弾力の低下が抑制されていた。 As is apparent from FIG. 14, the mice of the non-treated example (o) averaged about 1.4 seconds, while the mice of the control example (c) averaged about 1.7 seconds. Skin elasticity was decreasing. In contrast, the mice of the invention example (a) had an average of about 1.5 seconds, and the decrease in skin elasticity due to ultraviolet irradiation was suppressed.
図１５は、シワの評価試験を行った結果を示すヒストグラムであり、縦軸は点数を示している。また、図中、（ｏ）は無処理例を、（ｃ）は対照例を、（ａ）は本発明例をそれぞれ示している。 FIG. 15 is a histogram showing the results of the wrinkle evaluation test, and the vertical axis shows the score. In the figure, (o) shows an untreated example, (c) shows a control example, and (a) shows an example of the present invention.
図１５から明らかなように、無処理例（ｏ）のマウスは平均０．３点程度であるのに対し、対照例（ｃ）のマウスは平均１．０点程度であり、紫外線の照射によってシワが増大していた。これに対して、本発明例（ａ）のマウスは平均０．７点程度であり、紫外線の照射によるシワの増大が抑制されていた。 As is clear from FIG. 15, the mice of the non-treated example (o) averaged about 0.3 points, whereas the mice of the control example (c) averaged about 1.0 points. Wrinkles were increasing. On the other hand, the mice of the present invention example (a) had an average of about 0.7 points, and the increase in wrinkles due to ultraviolet irradiation was suppressed.
図１６は、弛みの評価試験を行った結果を示すヒストグラムであり、縦軸は点数を示している。また、図中、（ｏ）は無処理例を、（ｃ）は対照例を、（ａ）は本発明例をそれぞれ示している。 FIG. 16 is a histogram showing the results of the slack evaluation test, and the vertical axis shows the score. In the figure, (o) shows an untreated example, (c) shows a control example, and (a) shows an example of the present invention.
図１６から明らかなように、無処理例（ｏ）のマウスは平均０．３点程度であるのに対し、対照例（ｃ）のマウスは平均０．７点程度であり、紫外線の照射によって弛みが増大していた。これに対して、本発明例（ａ）のマウスは平均０．４点程度であり、紫外線の照射による弛みの増大が抑制されていた。 As is clear from FIG. 16, the mice of the untreated example (o) averaged about 0.3 points, whereas the mice of the control example (c) averaged about 0.7 points. The slack increased. On the other hand, the mice of the invention example (a) had an average of about 0.4 points, and an increase in slack due to ultraviolet irradiation was suppressed.
これらの結果より、本誘導物にあっては前述した作用を介して、皮膚の弾力を維持し、並びに皮膚のシワ及び弛みを抑制することができる。皮膚の弾力が低下し、シワ及び弛みが発生するということは皮膚の老化が進行していることであり、本誘導物はかかる皮膚の老化の進行を抑制しているのである。従って、本誘導物は、そのまま、或いは前述した如きクリーム、ローション、パック剤等になすことによって、皮膚の抗老化剤として適用することができる。 From these results, it is possible to maintain the elasticity of the skin and to suppress wrinkles and slackness of the skin through the effects described above in the present derivative . The fact that the elasticity of the skin is reduced and wrinkles and sagging occurs means that the skin is aging, and this derivative suppresses the progress of such aging of the skin. Therefore, this derivative can be applied as an anti-aging agent for skin as it is or by making it into a cream, lotion, pack or the like as described above.
（実施例８）
次に、本誘導物の物性を検討した結果について説明する。
本実施例では、本誘導物の物性として分子量について検討した。すなわち、１００ｇのクロレラ粉末（クロレラ工業株式会社製）に１Ｌの純水を加えて撹拌し、クロレラを十分に懸濁させた。この懸濁液を１００℃に昇温させ、その状態で２０分間保持することによって熱水抽出した後、室温まで急冷させた。この液体を１００００ｒｐｍで２０分間遠心分離し、上清を回収して抽出液とした。この抽出液をロータリーエバポレータを用いて濃縮した。得られた濃縮液について乾物質量濃度を測定したところ８７ｍｇ／ｍｌであった。
(Example 8)
Next, the results of studying the physical properties of this derivative will be described.
In this example, the molecular weight was examined as a physical property of the derivative . That is, 1 L of pure water was added to 100 g of chlorella powder (manufactured by Chlorella Kogyo Co., Ltd.) and stirred to sufficiently suspend the chlorella. This suspension was heated to 100 ° C. and kept in that state for 20 minutes, followed by hot water extraction, and then rapidly cooled to room temperature. This liquid was centrifuged at 10,000 rpm for 20 minutes, and the supernatant was recovered to obtain an extract. The extract was concentrated using a rotary evaporator. It was 87 mg / ml when the dry matter amount density | concentration was measured about the obtained concentrate.
この濃縮液に含まれる物質を分子量別に分画した。
本実施例では、限外濾過膜を具備するフィルターユニットが遠心チューブ内に着脱可能に嵌合させて構成してあるアミコンウルトラフィルタ−１５（メルク株式会社製）を用いた。前記濃縮液１２ｍｌを分画分子量が１０万のアミコンウルトラフィルタ−１５のフィルターユニット内に注入し、全量が遠心チューブ内へ濾過されるまで遠心分離した。次に、得られた濾液を分画分子量が５万のアミコンウルトラフィルタ−１５のフィルターユニット内に注入し、全量が遠心チューブ内へ濾過されるまで遠心分離した。一方、分画分子量が１０万のフィルターユニットの内部を純水で洗浄して、分子量が１０万を超える分画物を回収し、回収液に純水を加えて全量を１２ｍｌになした。
Substances contained in this concentrate were fractionated according to molecular weight.
In this example, Amicon Ultra Filter-15 (manufactured by Merck & Co., Inc.) was used in which a filter unit having an ultrafiltration membrane was removably fitted into a centrifugal tube. 12 ml of the concentrated solution was poured into a filter unit of Amicon Ultra Filter-15 having a molecular weight cut-off of 100,000, and centrifuged until the entire amount was filtered into a centrifuge tube. Next, the obtained filtrate was poured into a filter unit of Amicon Ultra Filter-15 having a molecular weight cut-off of 50,000, and centrifuged until the entire amount was filtered into a centrifuge tube. On the other hand, the inside of the filter unit having a fractional molecular weight of 100,000 was washed with pure water to collect fractions having a molecular weight exceeding 100,000, and pure water was added to the collected liquid to make the total volume 12 ml.
同様の操作を、分画分子量が３万、１万、及び３千のアミコンウルトラフィルタ−１５を用いて順次実施することによって、分子量が１０万超、５万〜１０万、３万〜５万、１万〜３万、３千〜１万、３千未満の分画物を含む分画液をそれぞれ得た。なお、分画分子量が３千のアミコンウルトラフィルタ−１５の遠心チューブ内へ濾過された濾液は、全量を回収し、この回収液に純水を加えて全量を１２ｍｌになした。 By carrying out the same operation sequentially using Amicon Ultra Filter-15 having a molecular weight cut off of 30,000, 10,000, and 3,000, the molecular weight is more than 100,000, 50,000 to 100,000, and 30,000 to 50,000. Fractions containing 10,000 to 30,000, 3,000 to 10,000, and less than 3,000 fractions were obtained. The filtrate filtered into the centrifugal tube of Amicon Ultra Filter-15 having a fractional molecular weight of 3,000 was recovered in its entirety, and pure water was added to this recovered solution to make the total volume 12 ml.
ここで、各分画液の乾燥質量濃度は、分子量が１０万超のものが３６．４ｍｇ／ｍｌであり、分子量が５万超〜１０万以下のものが３．３ｍｇ／ｍｌであり、分子量が３万超〜５万以下のものが２．２ｍｇ／ｍｌであり、分子量が１万超〜３万以下のものが５．８ｍｇ／ｍｌであり、分子量が３千超〜１万以下のものが６．５ｍｇ／ｍｌであり、分子量が３千未満のものが３７．５ｍｇ／ｍｌであった。なお、濃縮液及び各分画液の乾燥質量濃度は前述した方法によって測定した。 Here, the dry mass concentration of each fraction solution is 36.4 mg / ml when the molecular weight exceeds 100,000, and 3.3 mg / ml when the molecular weight is more than 50,000 to 100,000 or less. Having a molecular weight of more than 30,000 to 50,000 or less is 2.2 mg / ml, a molecular weight of more than 10,000 to 30,000 or less is 5.8 mg / ml, and a molecular weight of more than 3,000 to 10,000 or less Of 6.5 mg / ml and a molecular weight of less than 3,000 was 37.5 mg / ml. The dry mass concentration of the concentrated solution and each fraction was measured by the method described above.
このようにして得た各分画液について、実施例５で説明した方法と同様の方法によって、ＴＧＦ−β１に係るｍＲＮＡの発現量を測定した。 For each of the fractions thus obtained, the expression level of mRNA related to TGF-β1 was measured by the same method as described in Example 5.
図１７は、分子量で分画した各分画液について、ＴＧＦ−β１に係るｍＲＮＡの発現量を測定した結果を示すヒストグラムであり、図中、縦軸はＴＧＦ−β１に係るｍＲＮＡの発現量をＧＡＰＤＨに係るｍＲＮＡの発現量に対する比率で示している。また、図中、（ｃ）は何ら処理していない対照を示しており、（ａ）は前述した濃縮液で処理した場合を示しており、（ａ１）〜（ａ６）はそれぞれ分子量が１０万超の分画液、分子量が５万超〜１０万以下の分画液、分子量が３万超〜５万以下の分画液、分子量が１万超〜３万以下の分画液、分子量が３千超〜１万以下の分画液、分子量が３千未満の分画液で処理した場合を示している。 FIG. 17 is a histogram showing the results of measuring the expression level of mRNA related to TGF-β1 for each fractionated solution by molecular weight. In the figure, the vertical axis shows the expression level of mRNA related to TGF-β1. It is shown as a ratio to the expression level of mRNA related to GAPDH. Moreover, in the figure, (c) shows a control that has not been treated at all, (a) shows the case of treatment with the concentrated liquid described above, and (a1) to (a6) each have a molecular weight of 100,000. Ultra-fractionation liquid, fractionation liquid with molecular weight of more than 50,000 to 100,000 or less, fractionation liquid with molecular weight of more than 30,000 to 50,000 or less, fractionation liquid with molecular weight of more than 10,000 to 30,000 or less, molecular weight The case where it processed with the fraction liquid of more than 3,000-10,000 or less and the molecular weight is less than 3,000 is shown.
図１７から明らかなように、分子量が１万超〜３万以下の分画液（ａ４）で処理した場合、ＴＧＦ−β１に係るｍＲＮＡの発現量が最も亢進されていた。また、この亢進の程度は、前述した濃縮液（ａ）で処理した場合と比べて有意に高いものであった。 As is clear from FIG. 17, when treated with a fraction (a4) having a molecular weight of more than 10,000 to 30,000 or less, the expression level of mRNA related to TGF-β1 was most enhanced. Further, the degree of this enhancement was significantly higher than that in the case of treatment with the concentrated liquid (a) described above.
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