JP6118519B2 - New polysaccharide - Google Patents
New polysaccharide Download PDFInfo
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- JP6118519B2 JP6118519B2 JP2012168776A JP2012168776A JP6118519B2 JP 6118519 B2 JP6118519 B2 JP 6118519B2 JP 2012168776 A JP2012168776 A JP 2012168776A JP 2012168776 A JP2012168776 A JP 2012168776A JP 6118519 B2 JP6118519 B2 JP 6118519B2
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- Prior art keywords
- polysaccharide
- hair
- culture
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Description
本発明は、新規多糖体及びその製造方法、並びに当該新規多糖体を含有する皮膚外用剤、育毛剤、美白剤等に関する。 The present invention relates to a novel polysaccharide, a method for producing the same, and a skin external preparation, a hair-restoring agent, a whitening agent and the like containing the novel polysaccharide.
多糖類は、医薬品、食品、工業品等幅広い分野で利用されている。医薬品や食品分野等でよく知られている酸性多糖類として、コンドロイチン硫酸やヒアルロン酸等のグリコサミノグリカン、キサンタンガム等；中性多糖類としてβ１，３グルカン、シクロデキストリン等が挙げられる。サメ軟骨やトリ鶏冠等に含まれているコンドロイチン硫酸や微生物生産が可能となったヒアルロン酸は、膝関節痛改善作用等を有することが知られている。またβ１，３グルカンは免疫賦活作用等を有することが知られている。
また、キサンタンガムは、Xanthomonas campestris細菌が生産する多糖であり、その糖の構成はＤ−グルコース、Ｄ−マンノース、Ｄ−グルクロン酸を構成糖にもつ分岐多糖であり、現在では工業的に大量生産可能であるため、増粘剤等として世界中で利用されている（例えば特許文献１）。
また、クロレラ属単細胞緑藻が細胞外に産生するガラクトース８１．４〜９１．９％含有の多糖体が、マウス肉腫細胞株に対して抗腫瘍作用を有することが記載されている（例えば特許文献２）。
さらに、種々の用途に利用可能な新規多糖体が、産業界において望まれているのが実状である。
Polysaccharides are used in a wide range of fields such as pharmaceuticals, foods, and industrial products. Examples of acidic polysaccharides well known in the pharmaceutical and food fields include glycosaminoglycans such as chondroitin sulfate and hyaluronic acid, xanthan gum, and the like, and β1,3 glucan and cyclodextrin as neutral polysaccharides. It is known that chondroitin sulfate contained in shark cartilage, chicken fowl, etc. and hyaluronic acid capable of producing microorganisms have an effect of improving knee joint pain and the like. Β1,3 glucan is known to have an immunostimulatory action and the like.
Xanthan gum is a polysaccharide produced by Xanthomonas campestris bacteria, and its sugar composition is a branched polysaccharide having D-glucose, D-mannose, and D-glucuronic acid as constituent sugars, and can now be industrially mass-produced. Therefore, it is used around the world as a thickener and the like (for example, Patent Document 1).
Moreover, it is described that the polysaccharide containing 81.4 to 91.9% galactose produced extracellularly by Chlorella unicellular green algae has an antitumor effect on mouse sarcoma cell lines (for example, Patent Document 2). ).
Furthermore, in reality, new polysaccharides that can be used in various applications are desired in the industry.
そこで、本発明は、斯かる実状に鑑み、種々の用途に利用可能な新規多糖体を提供しようとするものである。 Therefore, in view of such a situation, the present invention intends to provide a novel polysaccharide that can be used for various applications.
本発明者らは、種々の用途に利用可能な新規多糖体を鋭意検討した結果、ガラクトース
及びマンノースを主な構成糖とする新規多糖体を見出した。さらに、本発明の新規多糖体は、優れた育毛作用があること及び美白作用があることも見出した。本発明の新規多糖体は、皮膚外用剤、化粧料、食品、医薬品等にも利用可能であることを見出した。しかも、本発明の新規多糖体は、パラクロレラ属（Parachlorella）単細胞緑藻類がその細胞外で生産可能な多糖体であるので、工業的に大量生産可能であり、幅広い分野での利用が期待できる物質である。
As a result of intensive studies on novel polysaccharides that can be used for various applications, the present inventors have found novel polysaccharides containing galactose and mannose as main constituent sugars. Furthermore, it has also been found that the novel polysaccharide of the present invention has an excellent hair growth effect and a whitening effect. It has been found that the novel polysaccharide of the present invention can also be used for external preparations for skin, cosmetics, foods, pharmaceuticals and the like. Moreover, since the novel polysaccharide of the present invention is a polysaccharide that can be produced extracellularly by Parachlorella unicellular green algae, it can be industrially mass-produced and can be expected to be used in a wide range of fields. It is.
ところで、近年、ストレスの増加や食生活の変化など様々な要因によって、薄毛や抜け毛で悩む男女は増加傾向にあり、育毛剤への期待が高まっている。
例えば、特開２０１０−１５０２０３号公報には、ダイジン、ゲスチニン及びフラボステロンＳＥから選ばれる大豆イソフラボン配糖体と、６−ベンジルアミノピリンとを含有する育毛剤組成物が、抜け毛を低減すること及び硬毛化することが開示されている。
特開２０１２−０９７００８号公報には、パイナップル可食部を９０体積％エタノールで８０℃２時間抽出して得られた糖不含有のパイナップル抽出物が、毛乳頭細胞増殖促進作用があることが開示されている。
特開２０１０−１００５３６号公報には、アメリカンアンジェリカ（Angelica atropurpurea）の抽出物に含まれる所定の構造を有するアンギュラー型フロクマリン骨格を有する化合物が、ブタ毛包の毛伸長が認められたことが開示されている。
このように、幅広い化合物による様々な作用機序からの有効な育毛剤の検討が行われているのが実状である。しかしながら、本発明の多糖体は高分子系でありながら優れた育毛作用を有することは全くの意外であり、そして優れた育毛剤を提供することも可能である。
By the way, in recent years, due to various factors such as increased stress and changes in dietary habits, men and women who suffer from thinning hair and hair loss have been on the rise, and expectations for hair restorers are increasing.
For example, Japanese Patent Application Laid-Open No. 2010-150203 discloses that a hair restoration composition containing soybean isoflavone glycoside selected from daidzin, gestinin and flavosterone SE and 6-benzylaminopyrine reduces hair loss and bristle hair. Is disclosed.
JP 2012-097008 A discloses that a sugar-free pineapple extract obtained by extracting a pineapple edible portion with 90% by volume ethanol at 80 ° C. for 2 hours has an effect of promoting proliferation of hair papilla cells. Has been.
Japanese Patent Application Laid-Open No. 2010-1000053 discloses that a compound having an angular furocoumarin skeleton having a predetermined structure contained in an extract of Angelica atropurpurea was found to have a hair elongation of a pig hair follicle. ing.
Thus, the actual condition is that studies on effective hair-growth agents from various action mechanisms by a wide range of compounds are being conducted. However, it is completely unexpected that the polysaccharide of the present invention has a high hair-growth action even though it is a polymer system, and it is also possible to provide an excellent hair-growth agent.
よって、本発明は、上記課題を解決するために、多糖の基本構造の糖残基が少なくともガラクトースとマンノースで構成されており、当該ガラクトースにフラノース型が存在する多糖体を提供するものである。 Therefore, in order to solve the above problems, the present invention provides a polysaccharide in which the sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type.
また、本発明は、多糖の基本構造の糖残基が少なくともガラクトースとマンノースで構成されており、当該ガラクトースにフラノース型が存在する多糖体及び／又はパラクロレラ由来の細胞外多糖体を含有する、毛母細胞増殖賦活剤、ＦＧＦ−７産生促進剤、ＶＥＧＦ産生促進剤、育毛剤、メラニン産生抑制剤、美白剤又は皮膚外用剤を提供するものである。 In the present invention, the sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and contains a polysaccharide having a furanose type in the galactose and / or an extracellular polysaccharide derived from parachlorella. The present invention provides hair matrix cell growth activators, FGF-7 production promoters, VEGF production promoters, hair restorers, melanin production inhibitors, whitening agents, and skin external preparations.
また、本発明は、パラクロレラ属単細胞藻類又は改変体を炭素源含有培養培地で好気的な条件下で従属培養し、生産される多糖体を回収する多糖体の製造方法を提供するものである。
また、本発明は、Parachlorella kessleri-PNC1と命名され、FERM BP-11493として寄託された藻体を提供するものである。
The present invention also provides a method for producing a polysaccharide by subculturing a Parachlorella unicellular algae or a modified form in a carbon source-containing culture medium under aerobic conditions and recovering the produced polysaccharide. is there.
The present invention also provides an algal body named Parachlorella kessleri-PNC1 and deposited as FERM BP-11493.
本発明によれば、種々の用途に利用可能な新規な多糖体を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, the novel polysaccharide which can be utilized for various uses can be provided.
１．本開示の多糖体
２．本開示の多糖体の製造方法
３．本開示の多糖体の用途
1. 1. Polysaccharides of the present disclosure 2. Production method of polysaccharide of the present disclosure Uses of polysaccharides of the present disclosure
１．本開示の多糖体
本開示の多糖体は、多糖の基本構造の糖残基が少なくともガラクトース（以下「Ｇａｌ」ともいう）とマンノース（以下「Ｍａｎ」ともいう）で構成される。当該ガラクトースには、少なくともフラノース型が存在するのが好適である。
前記マンノースとガラクトースとの糖残基の比率が、マンノースを１としたときに、好ましくはＭａｎ １：Ｇａｌ ２〜４、より好ましくはＭａｎ １：Ｇａｌ ２．５〜３．５であるのが好適である。
1. Polysaccharide of the Present Disclosure In the polysaccharide of the present disclosure, the sugar residue of the basic structure of the polysaccharide is composed of at least galactose (hereinafter also referred to as “Gal”) and mannose (hereinafter also referred to as “Man”). The galactose preferably has at least a furanose type.
The ratio of the sugar residues of the mannose and galactose is preferably Man 1: Gal 2-4, more preferably Man 1: Gal 2.5-3.5, where mannose is 1. It is.
前記ガラクトースの含有量は、多糖体の中性単糖組成の全量を１００質量％としたとき（以下、「全中性糖中」とする）に、好ましくは５０〜７９質量％であり、より好ましくは６０〜７９質量％である。
本開示の多糖体の中性単糖組成は、マンノース及びガラクトースの他に、アラビノース（以下、「Ａｒａ」ともいう）、ラムノース（以下、「Ｒｈａ」ともいう）及びキシロール（以下、「Ｘｙｌ」ともいう）が含まれてもよい。
これらの含有量は、多糖体の中性単糖組成の全量を１００質量％としたとき（以下、「全中性糖中」とする）に、好ましくは０〜１６質量％、より好ましくは５〜１６質量％である。さらに、アラビノース、ラムノース及びキシロースの含有量は、全中性糖中、それぞれ、アラビノース ３〜７質量％、ラムノース ２〜６質量％、キシロース ０〜３質量％であるのが好適である。
このときマンノース及びガラクトースのこれら含有量は、全中性糖中、好ましくは８４〜１００質量％である。このマンノースの含有量は、全中性糖中、好ましくは１９〜２５質量％であり、またガラクトースの含有量は、全中性糖中、好ましくは６５〜７５質量％である。
The content of the galactose is preferably 50 to 79% by mass when the total amount of the neutral monosaccharide composition of the polysaccharide is 100% by mass (hereinafter referred to as “in all neutral sugars”). Preferably it is 60-79 mass%.
In addition to mannose and galactose, the neutral monosaccharide composition of the polysaccharide of the present disclosure includes arabinose (hereinafter also referred to as “Ara”), rhamnose (hereinafter also referred to as “Rha”) and xylol (hereinafter referred to as “Xyl”). May be included).
These contents are preferably 0 to 16% by mass, more preferably 5% when the total amount of the neutral monosaccharide composition of the polysaccharide is 100% by mass (hereinafter referred to as “in total neutral sugar”). ~ 16% by weight. Further, the contents of arabinose, rhamnose, and xylose are preferably 3-7% by mass of arabinose, 2-6% by mass of rhamnose, and 0-3% by mass of xylose, respectively, in the total neutral sugar.
At this time, the content of mannose and galactose is preferably 84 to 100% by mass in the total neutral sugar. The content of mannose is preferably 19 to 25% by mass in the total neutral sugar, and the content of galactose is preferably 65 to 75% by mass in the total neutral sugar.
本開示の多糖体の分子量は、ＧＰＣ測定において、好ましくは３〜９×１０４、より好ましくは５〜８×１０４である。
また、本開示の多糖体は水溶性のものが、様々な分野の用途で利用しやすいので、好適である。
The molecular weight of the polysaccharide of the present disclosure is preferably 3 to 9 × 10 4 , more preferably 5 to 8 × 10 4 in GPC measurement.
Moreover, since the polysaccharide of this indication is water-soluble, since it is easy to utilize for the use of various fields, it is suitable.
２．本開示の多糖体の製造方法
本開示の多糖体の製造方法は、本開示の多糖体を産生することが可能であれば、その手段は特に限定されず、例えば、細菌や藻類等の微生物及びこの改変体を用いることが挙げられる。当該改変体とは、本開示の多糖体を産生することが可能な機能をする微生物の遺伝子組み換え体、突然変異体等であり、必要に応じて生産性や耐久性等を向上させている微生物である。
2. The polysaccharide production method of the present disclosure is not particularly limited as long as the polysaccharide production method of the present disclosure can produce the polysaccharide of the present disclosure. For example, microorganisms such as bacteria and algae and It is mentioned to use this modification. The modified body is a genetically modified or mutant microorganism having a function capable of producing the polysaccharide of the present disclosure, and a microorganism whose productivity and durability are improved as necessary It is.
好適な本開示の多糖体の製造方法として、本開示の多糖体は、パラクロレラ属（Parachlorella）単細胞緑藻類又は改変体を独立的培養法又は従属的培養法等の任意の培養方法により培養することで製造することが可能である。
当該緑藻類を用いることで、本開示の多糖体は、藻類の細胞外に生産させて回収することが可能である。細胞外に産出させることで、遠心分離等の物理的手段等により藻体と多糖体とを分離させることが容易となる。これによって、培養培地交換等による連続培養による多糖体の生産も容易である。よって、本開示のパラクロレラ属単細胞緑藻類又は改変体を用いることは、多糖体の生産性を非常に向上させることができ、工業的生産に適しており、本開示の多糖体の安定的供給を可能とする。安定的供給性が可能となるため、本開示の多糖体は幅広い分野での利用も可能となる。
As a preferred method for producing the polysaccharide of the present disclosure, the polysaccharide of the present disclosure can be obtained by culturing Parachlorella unicellular green algae or a modified form by any culture method such as an independent culture method or a subordinate culture method. It is possible to manufacture with.
By using the green algae, the polysaccharide of the present disclosure can be produced and recovered outside the cells of the algae. By producing it extracellularly, it becomes easy to separate algal bodies and polysaccharides by physical means such as centrifugation. Thereby, it is easy to produce polysaccharides by continuous culture such as culture medium exchange. Therefore, using the Parachlorella unicellular green algae or the modified form of the present disclosure can greatly improve the productivity of the polysaccharide, is suitable for industrial production, and provides a stable supply of the polysaccharide of the present disclosure. Make it possible. Since stable supply is possible, the polysaccharide of the present disclosure can be used in a wide range of fields.
前記パラクロレラ属単細胞緑藻類のうち、好ましくはパラクロレラ属ケッセリ（Parachlorella kessreli）であり、より好ましくはParachlorella kessleri-PNC1株(FERM BP-11493)が好適である。
独立行政法人 国立環境研究所 微生物系統保存施設（NIESコレクション）から、NIES-2152〜NIES-2162、2177、2178、2179の14株を、パナック株式会社新規事業開発第二部が入手した。この入手した株を継代培養しながら、これらの中から、細胞外多糖体の生産性が高く、かつ従属培養に適した株を探索した。
具体的には、NIES-2152株を複数に分けて、後記実施例に示す従属培養条件下にて、継代培養を繰り返した。他の株についても同様に継代培養を繰り返し行った。継代培養を繰り返しながら、これらの中から、細胞外多糖体の生産性が高く、かつ従属培養に適した株を見出し、これをPNC1株とした。
当該PNC1株に関する形態観察及びrRNA遺伝子配列に基づく分子系統樹により、パラクロレラ属ケッセリ（Parachlorella kessreli）単細胞緑藻類と判断し、Parachlorella kessleri-PNC1株と命名した。この株は、新規な藻類として、2012年7月19日、〒305-8566 茨城県つくば市東1-1-1 つくばセンター中央第6、独立行政法人 製品評価技術基盤機構 特許生物寄託センター（NITE-IPOD）に、Parachlorella kessleri-PNC1株(FERM BP-11493)として寄託した。
Of the Parachlorella unicellular green algae, Parachlorella kessreli is preferable, and Parachlorella kessleri-PNC1 strain (FERM BP-11493) is more preferable.
Panac Co., Ltd. New Business Development Department 2 obtained 14 strains of NIES-2152 to NIES-2162, 2177, 2178, and 2179 from the National Institute for Environmental Studies Microbial System Storage Facility (NIES Collection). While subculturing the obtained strain, a strain having a high extracellular polysaccharide productivity and suitable for subculture was searched for.
Specifically, the NIES-2152 strain was divided into a plurality of cells, and subculture was repeated under the subculture conditions shown in Examples below. Subculture was repeated in the same manner for other strains. While repeating subculture, a strain having high productivity of extracellular polysaccharide and suitable for subculture was found out of these, and this strain was designated as PNC1 strain.
Based on morphological observation and molecular phylogenetic tree based on the rRNA gene sequence, the PNC1 strain was judged to be a Parachlorella kessreli unicellular green algae and named Parachlorella kessleri-PNC1 strain. As a new algae, this strain was founded on July 19, 2012, 1-1-1 Higashi Tsukuba, Tsukuba City, Ibaraki 305-8566 IPOD) was deposited as Parachlorella kessleri-PNC1 strain (FERM BP-11493).
本開示のパラクロレラ属単細胞緑藻類を培養して多糖体を製造させる際に用いる培地として、一般的な単細胞緑藻類が培養可能な基礎培地に、グルコース等の炭素源を含有させた炭素源含有の培養培地が好適である。単細胞緑藻類が培養可能な基礎培地とは、例えば、〔Appl Microbiol Biotechnol. 2011 Jul;91(1):31-46. Best practices in heterotrophic high-cell-density microalgal processes: achievements, potential and possible limitations.Bumbak F, Cook S, Zachleder V, Hauser S, Kovar K〕に記載されている培地が挙げられる。
基礎培地中の無機塩としてＫＨ２ＰＯ４、ＭｇＳＯ４等の微量無機成分が挙げられ、窒素源としては硫酸アンモニウム、尿素等が挙げられる。この基礎培地として、本開示のパラクロレラ属単細胞緑藻類を培養する際に好適なものとしては表１に示す培地の組成が挙げられ、各成分の含有量は±１０％の範囲であることが好ましい。
前記炭素源として、グルコース、果糖等の単糖類；ショ糖等のオリゴ糖等が挙げられる。これらから１種又は２種以上選択して基礎培地に添加してもよい。
前記炭素源の濃度は、特に限定されないが、培養培地中、好ましくは０．１〜３０質量％、より好ましくは１〜１０質量％とするのが好適である。
As a medium used when culturing the single cell green algae of the genus Parachlorella of the present disclosure to produce a polysaccharide, a culture containing a carbon source containing a carbon source such as glucose in a basic medium capable of culturing general single cell green algae A medium is preferred. The basic medium in which unicellular green algae can be cultured is, for example, (Appl Microbiol Biotechnol. 2011 Jul; 91 (1): 31-46.Best practices in heterotrophic high-cell-density microalgal processes: achievements, potential and possible limitations. F, Cook S, Zachleder V, Hauser S, Kovar K].
Examples of inorganic salts in the basal medium include trace inorganic components such as KH 2 PO 4 and MgSO 4, and examples of nitrogen sources include ammonium sulfate and urea. Examples of the basal medium suitable for culturing the Parachlorella unicellular green algae of the present disclosure include the composition of the medium shown in Table 1, and the content of each component is preferably in the range of ± 10%. .
Examples of the carbon source include monosaccharides such as glucose and fructose; oligosaccharides such as sucrose. One or more of these may be selected and added to the basal medium.
The concentration of the carbon source is not particularly limited, but is preferably 0.1 to 30% by mass, more preferably 1 to 10% by mass in the culture medium.
本開示の多糖体を製造するための培養に際し、好気的な条件下で培養することが、上記多糖体を安定的に生産させることができるので好適である。通気培養することが、多糖体の生産性を向上させる点で、好ましい。通気手段として、例えば、撹拌、振盪、通気及びバブリング等が挙げられる。これらを単独で又は２種以上組み合わせて行うことが可能である。これにより、培養培地中に適度な気体が混合されるようになる。
培養培地中の溶存酸素（ＤＯ）は、好気的な条件になるように調整すればよく、好ましくは３ｐｐｍ以上、より好ましくは５〜１３ｐｐｍとするのが好適である。
培養温度は、特に限定されないが、５〜４０℃の常温程度であればよい。
また、培養培地中のｐＨ（２０℃）は、好ましくは４〜９、より好ましくは５〜８とするのが好適である。
培養期間は、特に限定されないが、４日〜２週間程度を１サイクルとするのが、多糖体を産出させるのが好適である。また、前培養及び本培養を行う際には、本培養の期間は、４日〜１週間程度であればよい。この際、光照射を行なってもよいが、光照射を行わなくとも、多糖体を生産することが可能である。
また、独立栄養培養条件下にて藻体数を多くする場合、太陽光；植物栽培用ランプ、ＬＥＤ等の人工光源等の光を用いることが可能であり、炭酸ガスの補給と撹拌をすることが好ましい。
In culturing for producing the polysaccharide of the present disclosure, culturing under an aerobic condition is preferable because the polysaccharide can be stably produced. Aeration culture is preferable from the viewpoint of improving the productivity of polysaccharides. Examples of the aeration means include stirring, shaking, aeration, and bubbling. These can be performed alone or in combination of two or more. Thereby, an appropriate gas comes to be mixed in the culture medium.
What is necessary is just to adjust dissolved oxygen (DO) in a culture medium so that it may be aerobic conditions, Preferably it is 3 ppm or more, More preferably, it is 5-13 ppm.
Although culture temperature is not specifically limited, What is necessary is just about the normal temperature of 5-40 degreeC.
Further, the pH (20 ° C.) in the culture medium is preferably 4-9, more preferably 5-8.
The culture period is not particularly limited, but it is preferable to produce polysaccharides by setting one cycle to 4 days to 2 weeks. Moreover, when performing preculture and main culture, the period of main culture should just be about 4 days-1 week. At this time, light irradiation may be performed, but a polysaccharide can be produced without light irradiation.
In addition, when increasing the number of algal bodies under autotrophic culture conditions, it is possible to use sunlight; light from artificial light sources such as lamps for plant cultivation and LEDs, and supply and agitate carbon dioxide. Is preferred.
また、本開示の多糖の生産方法は、バッチ式、連続式の何れでもよいが、連続式が生産性向上のため好ましい。また、開放系培養及び閉鎖系培養の何れでもよく、例えば、培養タンク内の密閉培養及び開放系の露天培養等が挙げられる。培養条件管理の点で、閉鎖系培養が好ましい。 In addition, the polysaccharide production method of the present disclosure may be either a batch type or a continuous type, but the continuous type is preferable for improving productivity. Moreover, any of open system culture and closed system culture may be sufficient, for example, closed culture in a culture tank, open system open culture, etc. are mentioned. Closed culture is preferable from the viewpoint of culture condition management.
本開示の多糖体を前記単細胞緑藻類又は改変体に産生させた後に、水溶液洗浄、超音波、遠心分離、ろ過等の物理的手段及び化学的手段にて、藻体から多糖体を分離し、本開示の多糖体が含まれる上清液を得るのが好適である。このとき、培養後の藻体表面から多糖体を分離することがその藻体を用いて連続的に多糖体を生産することも可能であるので好ましい。例えば、遠心分離の際には、５０００〜９０００ｒｐｍ、５分間程度でよい。さらに濾過助剤及びろ過フィルター等を単独で又は組み合わせて用いてろ過を行うことが不純物をより除去できるので好ましい。
得られた本開示の多糖体は、希釈液、濃縮液又は乾燥物等の状態に適宜調整してもよい。乾燥手段として凍結乾燥が好ましい。
After the polysaccharide of the present disclosure is produced in the unicellular green algae or the modified body, the polysaccharide is separated from the alga body by physical means and chemical means such as aqueous solution washing, ultrasonic wave, centrifugation, filtration, etc. It is preferred to obtain a supernatant containing the disclosed polysaccharide. At this time, it is preferable to separate the polysaccharide from the surface of the cultured alga, since it is also possible to continuously produce the polysaccharide using the alga. For example, at the time of centrifugation, it may be 5000 to 9000 rpm for about 5 minutes. Further, it is preferable to perform filtration using a filter aid and a filtration filter alone or in combination because impurities can be further removed.
The obtained polysaccharide of the present disclosure may be appropriately adjusted to a diluted solution, a concentrated solution, a dried product, or the like. As the drying means, freeze-drying is preferable.
本開示の多糖体は、適宜公知の分離・精製技術、例えば液々分液、固液分液、濾過膜、活性炭、吸着樹脂、イオン交換樹脂等の方法によって不活性な不純物を除去し、更に精製してもよい。
本開示の多糖体を水溶液に溶解後、不溶性画分を除去し、水溶性画分を回収することが好適である。水に溶解の際に混合することが好ましく、この水温は、５〜４０℃であるのが好ましい。溶解後、低温（例えば１〜１０℃）に放置（例えば５〜１５時間）することが好ましい。
不溶性画分の除去は、遠心分離及び限界ろ過膜等の物理的手段にて行うことが好ましい。これにより、より水溶性の高い多糖体を得ることができるので、種々の用途に利用可能である。さらに得られた水溶性多糖体を、凍結乾燥することが好ましい。
The polysaccharides of the present disclosure can remove inert impurities by a known separation / purification technique, such as liquid-liquid separation, solid-liquid separation, filtration membrane, activated carbon, adsorption resin, ion exchange resin, and the like. It may be purified.
It is preferable to dissolve the polysaccharide of the present disclosure in an aqueous solution, then remove the insoluble fraction and collect the water-soluble fraction. It is preferable to mix when dissolving in water, and the water temperature is preferably 5 to 40 ° C. After dissolution, it is preferably left at a low temperature (for example, 1 to 10 ° C.) (for example, 5 to 15 hours).
The insoluble fraction is preferably removed by physical means such as centrifugation and ultrafiltration membrane. Thereby, since a polysaccharide with higher water solubility can be obtained, it can be used for various applications. Further, the obtained water-soluble polysaccharide is preferably lyophilized.
３．本開示の多糖体の用途
本開示の多糖体は、後記実施例に示すように、線維芽細胞増殖因子のファミリーの一つである線維芽細胞増殖因子−７（ＦＧＦ−７）の量が増加することが認められている。よって、本開示の多糖体は、ＦＧＦ−７産生促進作用、線維芽細胞増殖賦活作用、皮膚の抗老化防止作用、シワ改善作用、毛母細胞増殖作用及び育毛作用等を有する。
3. Use of Polysaccharides of the Present Disclosure The polysaccharides of the present disclosure increase the amount of fibroblast growth factor-7 (FGF-7), which is one of the family of fibroblast growth factors, as shown in the Examples below. Is allowed to do. Therefore, the polysaccharide of the present disclosure has an FGF-7 production promoting action, a fibroblast proliferation activation action, a skin anti-aging prevention action, a wrinkle improvement action, a hair matrix cell proliferation action, a hair growth action and the like.
ここで、線維芽細胞増殖因子は、血管新生作用、コラーゲンやフィブロネクチンの合成抑制作用等を有することや、ヘパリンに対して強い親和性を有していることが知られている。
また、線維芽細胞は、真皮に含まれており、線維芽細胞の増殖機能が維持されることによって、皮膚の水分量、柔軟性、弾力性等が良好な状態に保たれ、美しく健康的な皮膚（肌）の状態が維持される。また、線維芽細胞の増殖機能が低下することによって、皮膚の線維成分及び基質の産生量が減少する。これは、皮膚の表面形状及び物理的性状に変化を及ぼす要因となり、皮膚のガサつき、肌荒れ、しわ、たるみ等の原因となる。よって、線維芽細胞増殖賦活作用を有すると、皮膚の抗老化防止作用、シワ改善作用等に寄与すると考えられる。
また、ＦＧＦ−７は毛乳頭細胞から分泌される因子であり、このＦＧＦ−７が毛母細胞に作用し、毛母細胞の増殖を促進し、毛髪成長が促進すると考えられている。よって、ＦＧＦ−７産生促進作用を有すると、毛母細胞増殖作用、育毛作用等に寄与すると考えられる。
Here, it is known that the fibroblast growth factor has an angiogenesis action, an action for inhibiting the synthesis of collagen and fibronectin, and has a strong affinity for heparin.
In addition, fibroblasts are contained in the dermis, and by maintaining the proliferative function of fibroblasts, the moisture content, flexibility, elasticity, etc. of the skin are maintained in a good state, which is beautiful and healthy. The condition of the skin (skin) is maintained. Moreover, the production amount of the fiber component and the substrate of the skin decreases due to the decrease in the proliferation function of fibroblasts. This causes changes in the surface shape and physical properties of the skin, and causes skin roughness, rough skin, wrinkles, sagging, and the like. Therefore, it is considered that having a fibroblast proliferation activation action contributes to an anti-aging prevention action, wrinkle improvement action and the like of the skin.
FGF-7 is a factor secreted from hair papilla cells, and it is considered that this FGF-7 acts on hair matrix cells to promote hair matrix cell proliferation and promote hair growth. Therefore, it is considered that having FGF-7 production promoting action contributes to hair matrix cell growth action, hair growth action and the like.
さらに、本開示の多糖体は、上述のように、ＦＧＦ−７産生促進作用及び線維芽細胞増殖賦活作用等を有することで、ＦＧＦ−７産生量減少、線維芽細胞増殖能の低減、毛母細胞増殖能の低減、線維芽細胞増殖能の低減等に起因する疾患や症状の予防、改善又は治療が可能と考えられる。
本開示のＦＧＦ−７産生量減少、線維芽細胞増殖能の低減、毛母細胞増殖能の低減等に起因する各種疾患や症状として、例えば、皮膚の老化、シワ形成、脱毛症等が挙げられる。この脱毛症としては、男性型脱毛症、女性のびまん性脱毛症、円形脱毛症等が挙げられる。
Furthermore, as described above, the polysaccharide of the present disclosure has an FGF-7 production promoting action, a fibroblast proliferation activation action, and the like, thereby reducing FGF-7 production, reducing fibroblast proliferation ability, hair matrix. It is considered possible to prevent, ameliorate, or treat diseases and symptoms caused by reduced cell proliferative ability, fibroblast proliferative ability and the like.
Examples of various diseases and symptoms resulting from a decrease in FGF-7 production, a reduction in fibroblast proliferation ability, a reduction in hair matrix proliferation ability, and the like of the present disclosure include skin aging, wrinkle formation, and alopecia. . Examples of the alopecia include male pattern alopecia, female diffuse alopecia, alopecia areata and the like.
また、本開示の多糖体は、後記実施例に示すように血管内皮増殖因子（ＶＥＧＦ）の量が増加することが認められている。よって、本開示の多糖体は、ＶＥＧＦ産生促進作用、血管内皮増殖賦活作用、血管新生促進作用、毛母細胞活性化作用、発毛促進作用等を有する。 Moreover, it is recognized that the polysaccharide of this indication increases the quantity of vascular endothelial growth factor (VEGF) as shown in the below-mentioned Example. Therefore, the polysaccharide of the present disclosure has a VEGF production promoting action, a vascular endothelial growth activating action, an angiogenesis promoting action, a hair matrix activation action, a hair growth promoting action and the like.
ここで、血管内皮増殖因子は、血管内皮細胞に働き、細胞の増殖、遊走を促進させたり、血管新生を促進させたり、また血管透過性を亢進させたりすることが知られている。このように、血管内皮増殖賦活作用を有すると、血管内皮細胞増殖促進作用、血管新生促進作用等に寄与すると考えられる。
また、本開示の多糖体は、血管内皮増殖因子（ＶＥＧＦ）を有するが、このＶＥＧＦは、毛乳頭細胞から分泌され、このＶＥＧＦが血管新生を促進し、それにより毛母細胞が活性化することで発毛が促進されると考えられている。このように、ＶＥＧＦ産生促進作用を有すると、毛母細胞活性化作用、発毛促進作用等に寄与すると考えられる。
さらに、本開示の多糖体は、上述のように、ＶＥＧＦ産生促進作用及び血管内皮増殖賦活作用を有することで、ＶＥＧＦ産生量減少、血管内皮増殖抑制、毛母細胞活性低下等に起因する各種疾患や症状の予防、改善又は治療が可能と考える。
本開示のＶＥＧＦ産生抑制、血管内皮増殖抑制、毛母細胞活性低下等に起因する各種疾患や症状として、脈管形成不全、血管系の発達異常、脱毛症等が挙げられる。
Here, vascular endothelial growth factor is known to act on vascular endothelial cells to promote cell proliferation and migration, promote angiogenesis, and enhance vascular permeability. Thus, having a vascular endothelial growth activating effect is thought to contribute to a vascular endothelial cell growth promoting action, an angiogenesis promoting action, and the like.
In addition, the polysaccharide of the present disclosure has vascular endothelial growth factor (VEGF), which is secreted from hair papilla cells, and this VEGF promotes angiogenesis, thereby activating hair matrix cells. It is thought that hair growth is promoted. Thus, having a VEGF production promoting action is considered to contribute to hair matrix activation, hair growth promoting action, and the like.
Furthermore, as described above, the polysaccharide of the present disclosure has a VEGF production promoting action and a vascular endothelial growth activating action, thereby causing various diseases caused by a decrease in VEGF production, vascular endothelial growth inhibition, hair matrix activity reduction, and the like. It is possible to prevent, improve or treat symptoms and symptoms.
Examples of various diseases and symptoms resulting from suppression of VEGF production, suppression of vascular endothelial growth, reduction of hair matrix cell activity, and the like of the present disclosure include vascular dysplasia, abnormal development of the vascular system, and alopecia.
本開示の多糖体は、後記実施例に示すように、毛乳頭の細胞を賦活することが認められている。よって、本開示の多糖体は、上述のように、ＦＧＦ−７産生促進作用及びＶＥＧＦ産生促進作用も有していることから、非常に良好に毛母細胞増殖賦活作用、育毛作用等に寄与すると考えられる。
一般的に、育毛作用物質の探索は、アデノシンのような低分子化合物又は植物等由来の有機溶媒抽出物等から行われているが、これとは本開示の多糖体は分子量や性質も異なる高分子かつ多糖体であり、さらに新規である本開示の多糖体に優れた育毛作用があることは全くの意外である。
The polysaccharides of the present disclosure have been found to activate hair papilla cells as shown in the Examples below. Therefore, since the polysaccharide of the present disclosure also has an FGF-7 production promoting action and a VEGF production promoting action as described above, it contributes very well to hair matrix cell growth activation action, hair growth action and the like. Conceivable.
In general, the search for a hair growth active substance is performed from a low molecular weight compound such as adenosine or an organic solvent extract derived from a plant, etc., but the polysaccharides of the present disclosure are different in molecular weight and properties. It is quite surprising that the polysaccharides of the present disclosure, which are molecular and polysaccharides, and which are novel, have an excellent hair-growth effect.
よって、本開示の多糖体は、上述のような、ＦＧＦ−７産生促進作用、線維芽細胞増殖賦活作用、細胞賦活作用、毛乳頭細胞賦活作用、育毛作用、皮膚の抗老化防止作用、シワ改善作用、ＶＥＧＦ産生促進作用、血管内皮増殖賦活作用等のために使用してもよく、また、ＦＧＦ−７産生促進剤、線維芽細胞増殖賦活剤、細胞増殖賦活剤、毛乳頭細胞増殖賦活剤、育毛剤、皮膚の抗老化防止剤、シワ改善剤、ＶＥＧＦ産生促進剤、血管内皮増殖賦活剤等の上述のような使用を目的とした各種製剤に使用することができ、これら各種製剤を製造するために使用することができる。
なお、本開示における「育毛剤」とは、毛を発育させること、毛の発育を促進させること、毛の健康状態を保つこと、脱毛を予防すること、及び脱毛を低減させること等の効果を発揮することを意味し、「養毛剤」をも包含する。
Therefore, the polysaccharide of the present disclosure has the above-described FGF-7 production promoting action, fibroblast proliferation activation action, cell activation action, hair papilla cell activation action, hair growth action, skin anti-aging prevention action, and wrinkle improvement. May be used for action, VEGF production promoting action, vascular endothelial growth stimulating action, etc., and FGF-7 production promoter, fibroblast proliferation activator, cell proliferation activator, hair papillary cell proliferation activator, It can be used in various preparations intended for use as described above, such as hair restorers, anti-aging agents for skin, wrinkle improving agents, VEGF production promoters, vascular endothelial growth activators, etc., and produce these various preparations Can be used for.
In addition, the “hair growth agent” in the present disclosure has effects such as growing hair, promoting hair growth, maintaining hair health, preventing hair loss, and reducing hair loss. It means to exert, and includes “hair restorer”.
通常、皮膚においてメラニンは紫外線から生体を保護する役目も果たしているが、過剰生成や不均一な蓄積は、皮膚の黒化やシミの原因となる。一般的に、メラニンは色素細胞中で生成されるチロシナーゼの酵素の働きによって、チロシンからドーパ、ドーパからトーパキノンに変化し、次いで、５，６―ジヒドロキシインドフェノール等の中間体を経て形成される。従って、皮膚の色黒（皮膚色素沈着症）を予防又は改善するため、すなわち、美白のためには、メラニン産生過程を阻害すること、又は既に産生したメラニンを淡色化漂白することが有効と考えられている。
このようなチロキシナーゼ活性阻害作用を有する物質として、例えば、特開２００２−３７０９６２号公報には、藤茶枝葉部からのエタノール抽出物に優れたチロシナーゼ活性があることが開示されている。
また、特開２００１−０２６５３０号公報には、スフィンゴ脂質という脂質に、メラノーマＢ１６細胞のメラニン生成抑制作用があることが開示されている。
このように、従来植物抽出物やスフィンゴ脂質等の低分子系について美白作用が多く報告されているため、高分子で中性の多糖体に美白作用があるとの知見はほとんど見出されていない。
Normally, melanin also plays a role in protecting the body from ultraviolet rays in the skin, but overproduction and uneven accumulation cause skin darkening and spots. In general, melanin is changed from tyrosine to dopa, from dopa to topaquinone, and then formed through an intermediate such as 5,6-dihydroxyindophenol by the action of an enzyme of tyrosinase produced in pigment cells. Therefore, in order to prevent or improve skin darkness (skin pigmentation), that is, for whitening, it is effective to inhibit the melanin production process or to lighten and bleach the already produced melanin. It has been.
As such a substance having an inhibitory action on tyroxynase activity, for example, JP-A-2002-370962 discloses that an ethanol extract from Fuji tea branch leaves has excellent tyrosinase activity.
Japanese Patent Application Laid-Open No. 2001-026530 discloses that a lipid called sphingolipid has an inhibitory action on melanin production of melanoma B16 cells.
As described above, since many whitening effects have been reported for low molecular weight systems such as plant extracts and sphingolipids, little knowledge has been found that white polysaccharides have a whitening action. .
本開示の多糖体は、後記実施例に示すように、Ｂ１６メラノーマ細胞におけるメラニンの蓄積抑制作用が認められている。よって、本開示の多糖体は、メラニン産生抑制作用及び美白作用を有する。
さらに、本開示の多糖体は、メラニン産生抑制作用を有することで、メラニン蓄積やメラニン産生量増加等に起因する疾患や症状の予防、改善又は治療が可能と考えられる。
本開示のメラニン産生量増加等に起因する各種疾患や症状として、例えば、皮膚がん、皮膚の色黒化（皮膚色素沈着症、悪性黒色腫）等が挙げられる。
よって、本開示の多糖体は、上述のような、メラニン産生抑制作用及び美白作用等のために使用してもよく、また、メラニン産生抑制剤、美白剤等の上述のような使用を目的とした各種製剤に使用することができ、これら各種製剤を製造するために使用することができる。
The polysaccharide of the present disclosure has been shown to inhibit melanin accumulation in B16 melanoma cells, as shown in Examples below. Therefore, the polysaccharide of the present disclosure has a melanin production inhibitory action and a whitening action.
Furthermore, it is considered that the polysaccharide of the present disclosure can prevent, improve, or treat diseases and symptoms caused by melanin accumulation, melanin production increase, and the like by having a melanin production inhibitory action.
Examples of various diseases and symptoms resulting from an increase in the melanin production amount of the present disclosure include skin cancer, skin darkening (skin pigmentation, malignant melanoma), and the like.
Therefore, the polysaccharide of the present disclosure may be used for the melanin production inhibitory action and the whitening action as described above, and is intended for the use as described above such as a melanin production inhibitor and a whitening agent. And can be used to produce these various formulations.
また、本開示の多糖体は、ヒトを含む動物に摂取、投与又は接触させて、上述のような、線維芽細胞増殖賦活作用、ＦＧＦ−７産生促進作用、皮膚の抗老化防止作用、シワ改善作用、血管内皮増殖賦活作用、ＶＥＧＦ産生促進作用、育毛作用、メラニン産生抑制作用及び美白作用等を図るために、使用することができる物質である。
従って、本開示の多糖体は、上述のような、線維芽細胞増殖賦活、ＦＧＦ−７産生促進、皮膚の抗老化防止、シワ改善、血管内皮増殖賦活、ＶＥＧＦ産生促進、育毛、メラニン産生抑制及び美白等のために、皮膚外用剤、化粧品、医薬品、医薬部外品、食品や機能性食品（例えば特定保健用食品等）等に配合することが可能であり、本開示の育毛剤、美白剤等は、これら皮膚外用剤、化粧品等として有用である。
In addition, the polysaccharide of the present disclosure is ingested, administered or brought into contact with animals including humans, as described above, fibroblast proliferation activation action, FGF-7 production promotion action, skin anti-aging prevention action, wrinkle improvement It is a substance that can be used to achieve an action, a vascular endothelial growth activation action, a VEGF production promotion action, a hair growth action, a melanin production inhibition action, a whitening action, and the like.
Therefore, the polysaccharide of the present disclosure has the above-described fibroblast proliferation activation, FGF-7 production promotion, skin anti-aging prevention, wrinkle improvement, vascular endothelial growth activation, VEGF production promotion, hair growth, melanin production inhibition and For whitening, etc., it can be added to skin external preparations, cosmetics, pharmaceuticals, quasi-drugs, foods and functional foods (for example, foods for specified health use), etc. Are useful as these external preparations for skin, cosmetics and the like.
本開示の多糖体は、特に皮膚外用剤、化粧品、医薬部外品に用いるのが好ましく、皮膚に塗布などに接触させる製剤が好適である。
前記育毛剤、美白剤等の製剤中における多糖体の含有量は、好ましくは０．００１〜０．０１質量％、より好ましくは０．０１〜０．１質量％であり、さらに好ましくは０．１〜１質量％である。
The polysaccharide of the present disclosure is particularly preferably used for an external preparation for skin, cosmetics, and quasi drugs, and a preparation that is brought into contact with the skin for application or the like is suitable.
The content of the polysaccharide in the preparation such as the hair restorer or whitening agent is preferably 0.001 to 0.01% by mass, more preferably 0.01 to 0.1% by mass, and still more preferably 0.00. It is 1-1 mass%.
なお、前記育毛剤及び美白剤等には、本開示の多糖体に、必要に応じて、任意の成分を組み合わせて使用してもよい。好ましい他の成分としては、薬学的に又は食品的に許容される成分であればよく、例えば、細胞賦活剤、抗酸化剤、保湿剤、紫外線防止剤、溶剤（水、アルコール類等）、油剤、界面活性剤、増粘剤、粉体、キレート剤、ｐＨ調整剤、乳化剤、安定化剤、着色剤、光沢剤、矯味剤、矯臭剤、賦形剤、結合剤、崩壊剤、滑沢剤、希釈剤、浸透圧調整剤、香料等が挙げられ、これらを目的とする製剤に応じて配合すればよい。
また、前記育毛剤及び美白剤等の形態は、特に限定されず、液状、ペースト状、ゲル状、固形状、粉末状等の何れの形態でもよい。
In addition, you may use for the said hair restorer, whitening agent, etc., combining the arbitrary component with the polysaccharide of this indication as needed. Other preferable components may be pharmaceutically or food-acceptable components. For example, cell activators, antioxidants, moisturizers, UV inhibitors, solvents (water, alcohols, etc.), oil agents , Surfactant, thickener, powder, chelating agent, pH adjuster, emulsifier, stabilizer, colorant, brightener, flavoring agent, flavoring agent, excipient, binder, disintegrant, lubricant , Diluents, osmotic pressure adjusting agents, fragrances and the like, and these may be blended according to the intended preparation.
The form of the hair-restoring agent and whitening agent is not particularly limited, and may be any form such as liquid, paste, gel, solid, and powder.
本技術は、以下の構成を採用することが可能である。
〔１〕 多糖の基本構造の糖残基が少なくともガラクトースとマンノースで構成されており、当該ガラクトースにフラノース型が存在する多糖体。
〔２〕前記ガラクトースの含有量が、全中性糖中、５０〜７９質量％である前記〔１〕記載の多糖体。
〔３〕前記多糖体の分子量が、ＧＰＣ測定において３〜９×１０４である前記〔１〕又は〔２〕記載の多糖体。
〔４〕 前記ガラクトースとマンノースの糖残基の比率が２．５〜３．５：１である前記〔１〕〜〔３〕の何れか１項記載の多糖体。
〔５〕 パラクロレラ由来の細胞外多糖体である前記〔１〕〜〔４〕の何れか１項記載の多糖体。好適にはパラクロレラ属ケッセリ（Parachlorella kessreli）由来の多糖体である。また、前記多糖体は水溶性であるのが好適である。
The present technology can employ the following configurations.
[1] A polysaccharide in which the sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type.
[2] The polysaccharide according to [1], wherein the content of the galactose is 50 to 79% by mass in the total neutral sugar.
[3] The polysaccharide according to [1] or [2], wherein the polysaccharide has a molecular weight of 3 to 9 × 10 4 in GPC measurement.
[4] The polysaccharide according to any one of [1] to [3], wherein a ratio of sugar residues of the galactose and mannose is 2.5 to 3.5: 1.
[5] The polysaccharide according to any one of [1] to [4], which is an extracellular polysaccharide derived from parachlorella. Preferred is a polysaccharide derived from Parachlorella kessreli. In addition, the polysaccharide is preferably water-soluble.
〔７〕 多糖の基本構造の糖残基が少なくともガラクトースとマンノースで構成されており、当該ガラクトースにフラノース型が存在する多糖体を含有するＦＧＦ−７産生促進剤、ＶＥＧＦ産生促進剤、線維芽細胞増殖賦活剤、血管内皮増殖賦活剤、細胞増殖賦活剤、育毛剤、メラニン産生抑制剤、美白剤、シワ改善剤、皮膚の抗老化剤、血管新生促進剤。
〔８〕 前記〔２〕〜〔５〕の何れか１項記載のパラクロレラ細胞外多糖体を含有するＦＧＦ−７産生促進剤、ＶＥＧＦ産生促進剤、線維芽細胞増殖賦活剤、血管内皮増殖賦活剤、細胞増殖賦活剤、育毛剤、メラニン産生抑制剤、美白剤、シワ改善剤、皮膚の抗老化剤、又は血管新生促進剤。
〔９〕 前記細胞増殖賦活が、毛乳頭細胞増殖賦活である前記〔７〕又は〔８〕記載の細胞増殖賦活剤。
〔１０〕 多糖の基本構造の糖残基が少なくともガラクトースとマンノースで構成されており、当該ガラクトースにフラノース型が存在する多糖体を含有する皮膚外用剤。
〔１１〕好適には、前記〔７〕〜〔１３〕に記載の各種製剤の多糖体は、前記〔２〕〜〔５〕の何れか１項記載の多糖体である。
[7] FGF-7 production promoter, VEGF production promoter, fibroblast, wherein the sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and contains a polysaccharide having a furanose type in the galactose Growth activator, vascular endothelial growth activator, cell proliferation activator, hair restorer, melanin production inhibitor, whitening agent, wrinkle improving agent, skin anti-aging agent, angiogenesis promoter.
[8] FGF-7 production promoter, VEGF production promoter, fibroblast proliferation activator, vascular endothelial proliferation activator containing the parachlorella extracellular polysaccharide according to any one of [2] to [5] Agent, cell proliferation activator, hair restorer, melanin production inhibitor, whitening agent, wrinkle improving agent, skin anti-aging agent, or angiogenesis promoter.
[9] The cell proliferation activator according to [7] or [8], wherein the cell proliferation activation is hair papilla cell proliferation activation.
[10] A skin external preparation containing a polysaccharide in which a sugar residue having a basic structure of a polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type.
[11] Preferably, the polysaccharide of the various preparations according to [7] to [13] is the polysaccharide according to any one of [2] to [5].
〔１２〕 ＦＧＦ−７産生促進剤、ＶＥＧＦ産生促進剤、線維芽細胞増殖賦活剤、血管内皮増殖賦活剤、細胞増殖賦活剤、育毛剤、メラニン産生抑制剤、美白剤、シワ改善剤、皮膚の抗老化剤又は血管新生促進剤の製造のための、多糖の基本構造の糖残基が少なくともガラクトースとマンノースで構成されており、当該ガラクトースにフラノース型が存在する多糖体の使用。
好適には、前記〔２〕〜〔５〕の何れか１項記載の多糖体である。
〔１３〕 多糖の基本構造の糖残基が少なくともガラクトースとマンノースで構成されており、当該ガラクトースにフラノース型が存在する多糖体の、ＦＧＦ−７産生促進剤、ＶＥＧＦ産生促進剤、線維芽細胞増殖賦活剤、血管内皮増殖賦活剤、細胞増殖賦活剤、育毛剤、メラニン産生抑制剤、美白剤、シワ改善剤、皮膚の抗老化剤又は血管新生促進剤への使用。
好適には、前記〔２〕〜〔５〕の何れか１項記載の多糖体である。
〔１４〕 ＦＧＦ−７産生量減少、線維芽細胞増殖能の低減、毛母細胞増殖能の低減、線維芽細胞増殖能の低減、ＶＥＧＦ産生量減少、血管内皮増殖抑制、毛母細胞活性低下、及びメラニン産生量増加等に起因する疾患の、予防、改善又は治療のための、〕多糖の基本構造の糖残基が少なくともガラクトースとマンノースで構成されており、当該ガラクトースにフラノース型が存在する多糖体。
好適には、前記〔２〕〜〔５〕の何れか１項記載の多糖体である。
[12] FGF-7 production promoter, VEGF production promoter, fibroblast proliferation activator, vascular endothelial growth activator, cell proliferation activator, hair restorer, melanin production inhibitor, whitening agent, wrinkle improving agent, skin Use of a polysaccharide in which the sugar residue of the basic structure of a polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type, for the production of an anti-aging agent or angiogenesis-promoting agent.
The polysaccharide according to any one of [2] to [5] is preferable.
[13] FGF-7 production promoter, VEGF production promoter, fibroblast proliferation of polysaccharides in which the sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type Use as an activator, vascular endothelial growth activator, cell proliferation activator, hair restorer, melanin production inhibitor, whitening agent, wrinkle improving agent, skin anti-aging agent or angiogenesis promoter.
The polysaccharide according to any one of [2] to [5] is preferable.
[14] Reduction of FGF-7 production, reduction of fibroblast proliferation ability, reduction of hair matrix proliferation ability, reduction of fibroblast proliferation ability, reduction of VEGF production, inhibition of vascular endothelial growth, reduction of hair matrix activity, Polysaccharides in which the sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type for prevention, improvement or treatment of diseases caused by increased production of melanin, etc. body.
The polysaccharide according to any one of [2] to [5] is preferable.
また、本技術は、以下の構成を採用することが可能である。
（１） パラクロレラ属単細胞藻類又は改変体を炭素源含有培養培地で好気的な条件下で従属培養し、生産される多糖体を回収する多糖体の製造方法。
（２） 前記多糖体が、前記〔１〕多糖の基本構造の糖残基が少なくともガラクトースとマンノースで構成されており、当該ガラクトースにフラノース型が存在する多糖体である、前記（１）〜（１０）の何れか１項記載の多糖体の製造方法。好適には前記〔２〕〜〔５〕の多糖体である。
（３）パラクロレラ属単細胞藻類が、パラクロレラ属ケッセリ（Parachlorella kessreli）である前記（１）又は（２）記載の多糖体の製造方法。
（４）前記炭素源の培地中の濃度が、０．１〜１０質量％である前記（１）〜（３）の何れか１項記載の多糖体の製造方法。
（５） 前記炭素原が、グルコース、果糖及びショ糖から選ばれる１種又は２種以上のものである前記（１）〜（４）記載の多糖体の製造方法。
（６） 前記培養が、通気条件下の従属的培養である前記（１）〜（５）の何れか１項記載の多糖体の製造方法。
（７） 前記培養の溶存酸素濃度が、３〜１３ｐｐｍである前記（１）〜（６）の何れか１項記載の多糖体の製造方法。
（８） 前記培養の温度が、５〜４０℃である前記（１）〜（７）の何れか１項記載の多糖体の製造方法。
（９） 前記培養のｐＨが、４〜９である前記（１）〜（８）の何れか１項記載の多糖体の製造方法。
（１０） 前記培養後、遠心分離及びろ過を単独で又は２種組み合わせて藻体を除去する前記（１）〜（９）の何れか１項記載の多糖体の製造方法。
（１１） 前記藻体を除去した後に凍結乾燥し、さらに水溶液に溶解後、水溶性画分を回収する前記（１）〜（１０）の何れか１項記載の多糖体の製造方法。
（１２） 前記（１）〜（１１）の何れか１項記載の多糖体の製造方法にて得られた多糖体。
（１３）Parachlorella kessleri-PNC1と命名され、FERM BP-11493として寄託された藻体である前記（１）〜（１２）の何れか１項記載の多糖体の製造方法。
（１４） Parachlorella kessleri-PNC1と命名され、FERM BP-11493として寄託された藻体。
Further, the present technology can employ the following configurations.
(1) A method for producing a polysaccharide, comprising subjecting Parachlorella unicellular algae or a modified body to subculture under aerobic conditions in a carbon source-containing culture medium and recovering the produced polysaccharide.
(2) The above-mentioned (1) to (1), wherein the polysaccharide is a polysaccharide in which the sugar residue of the basic structure of [1] polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type. 10. The method for producing a polysaccharide according to any one of 10). The polysaccharides [2] to [5] are preferred.
(3) The method for producing a polysaccharide according to (1) or (2) above, wherein the unicellular algae of Parachlorella is Parachlorella kessreli.
(4) The method for producing a polysaccharide according to any one of (1) to (3), wherein the concentration of the carbon source in the medium is 0.1 to 10% by mass.
(5) The method for producing a polysaccharide according to (1) to (4), wherein the carbon source is one or more selected from glucose, fructose and sucrose.
(6) The method for producing a polysaccharide according to any one of (1) to (5), wherein the culture is subordinate culture under aerated conditions.
(7) The method for producing a polysaccharide according to any one of (1) to (6), wherein the dissolved oxygen concentration in the culture is 3 to 13 ppm.
(8) The method for producing a polysaccharide according to any one of (1) to (7), wherein the culture temperature is 5 to 40 ° C.
(9) The method for producing a polysaccharide according to any one of (1) to (8), wherein the pH of the culture is 4 to 9.
(10) The method for producing a polysaccharide according to any one of (1) to (9), wherein the algal bodies are removed by centrifuging and filtration singly or in combination after the culturing.
(11) The method for producing a polysaccharide according to any one of (1) to (10), wherein the algal bodies are removed, freeze-dried, and further dissolved in an aqueous solution, and then the water-soluble fraction is collected.
(12) A polysaccharide obtained by the method for producing a polysaccharide according to any one of (1) to (11).
(13) The method for producing a polysaccharide according to any one of (1) to (12) above, which is an algal body named Parachlorella kessleri- PNC1 and deposited as FERM BP-11493.
(14) Algae named Parachlorella kessleri-PNC1 and deposited as FERM BP-11493.
以下、本発明（本技術）を具体的に説明するために実施例及び比較例等を挙げるが、本
発明（本技術）は実施例等に限定されるものではない。
Hereinafter, examples and comparative examples will be given to specifically describe the present invention (present technique), but the present invention (present technique) is not limited to the examples.
独立行政法人 国立環境研究所 微生物系統保存施設（NIESコレクション）から、NIES-2152〜NIES-2162、2177、2178、2179の14株を、パナック株式会社新規事業開発第二部が入手した。各１株を複数に分けて、表１に示す培地にて継代培養しながら、これらの中から、光学顕微鏡において、細胞体の細胞外に墨汁に染まらない部分が非常に多く認められるものを、細胞外多糖体の生産性が高い株として探索した。
ここで、多糖体が細胞外に多く生産されること；藻体と多糖体との分離が容易であると、多糖体の回収が容易であること；藻体が多糖体生産に再利用可能なものであること；従属培養に適していると、大型タンクでの培養も可能であることが、工業的生産に適している藻体と考える。
継代培養を繰り返しながら、これらの中から、細胞外多糖体の生産性が高く、かつ従属培養に適した株を見出し、これをPNC1株とした。
Panac Co., Ltd. New Business Development Department 2 obtained 14 strains of NIES-2152 to NIES-2162, 2177, 2178, and 2179 from the National Institute for Environmental Studies Microbial System Storage Facility (NIES Collection). Dividing each strain into a plurality of subcultures in the medium shown in Table 1, and from these, in the optical microscope, those that do not stain the ink body outside the cell body of the cell body very much The strain was searched for as a high productivity of extracellular polysaccharides.
Here, a large amount of polysaccharides are produced extracellularly; the separation of alga bodies and polysaccharides is easy and the polysaccharides can be easily recovered; the alga bodies can be reused for polysaccharide production. Being suitable for subculture, it is possible to cultivate in a large tank, and it is considered an algal body suitable for industrial production.
While repeating subculture, a strain having high productivity of extracellular polysaccharide and suitable for subculture was found out of these, and this strain was designated as PNC1 strain.
藻体PNC1株を、表１に示す培地にて従属培養を行い、光学顕微鏡において、墨汁染色を行った。細胞体の細胞外に墨汁に染まらない部分が認められた。その墨汁に染まらない部分について、以下の製造例１〜３に示す方法にて多糖体を分離し、分析を行った結果、藻体の細胞外に多糖体が生産されていることを見出した（図１）。
さらに、前記PNC1株に関する形態観察及びrRNA遺伝子配列に基づく分子系統樹により（図２）、パラクロレラ属ケッセリ（Parachlorella kessreli）単細胞緑藻類と判断し、Parachlorella kessleri-PNC1株と命名した。独立行政法人 製品評価技術基盤機構 特許生物寄託センター（NITE-IPOD）に、Parachlorella kessleri-PNC1株(FERM BP-11493)として寄託した。
The algal PNC1 strain was subcultured in the medium shown in Table 1 and stained with ink using an optical microscope. A portion of the cell body that was not stained with ink was found outside the cell body. As a result of separating and analyzing the polysaccharides by the methods shown in the following Production Examples 1 to 3 for the portion not stained with the ink, it was found that the polysaccharides were produced outside the algal cells ( FIG. 1).
Furthermore, by morphological observation and molecular phylogenetic tree based on the rRNA gene sequence (Fig. 2), the PNC1 strain was judged to be a Parachlorella kessreli unicellular green algae and named Parachlorella kessleri-PNC1 strain. Deposited as Parachlorella kessleri-PNC1 strain (FERM BP-11493) at the National Center for Biological Biology (NITE-IPOD).
製造例１：本開示の多糖体の製造方法
Parachlorella kessleri-PNC1株(FERM BP-11493)を含むスラント状培地（５０ｍｌ）を、グルコース含有培養培地２５０ｍｌを含む３Ｌ容バッフル付き三角フラスコに添加し、照度８０００〜１００００ｌｕｘ、２８℃で、１６０ｒｐｍにて４日間前培養した。なお、前培養前に、予備的に藻体数を増やしてもよい。
なお、グルコース含有培養培地は、表１の基礎培地１Ｌ当たりにグルコース１０ｇを含有させたものである。
前培養物２４０ｍｌを３０Ｌ容のジャーファメンターに移して１２日間本培養を行い、これを本培養物とした。このときの培養条件は、３０℃、２３０ｒｐｍ、通気０．５６ｖｖｍ、内圧０．３ｋｇｆ／ｃｍ２、ｐＨ７．２の好気的な条件下であり、光照射は行わなかった。
本培養物を、遠心分離（７０００ｒｐｍ（６５００Ｇ）、２５℃）し、目的とする多糖体を含有する上清液と藻体とに分離した。さらに分画分子量６０００のＵＦ膜（ＳＩＰ１０１３）を用いて、原液を２Ｌまで濃縮した。濃縮物（２Ｌ）に、陰イオン交換樹脂２００ｍＬを添加し、ブリックス０．１まで回収し、ＵＦ膜ろ過にて、５００ｍＬ濃縮し、凍結乾燥して、パラクロレラ由来の多糖体を得た。収率は、培養液１Ｌ当たり、０．２〜０．４ｇであった。
Production Example 1: Production method of polysaccharide of the present disclosure
A slant medium (50 ml) containing Parachlorella kessleri-PNC1 strain (FERM BP-11493) was added to a 3 L baffled Erlenmeyer flask containing 250 ml of glucose-containing culture medium, and an illuminance of 8000 to 10,000 lux at 28 ° C. and 160 rpm. Pre-cultured for 4 days. In addition, you may increase the number of algal bodies preliminarily before preculture.
The glucose-containing culture medium contains 10 g of glucose per liter of the basal medium shown in Table 1.
240 ml of the preculture was transferred to a 30 L jar fermenter and main culture was carried out for 12 days, which was designated as the main culture. The culture conditions at this time were aerobic conditions of 30 ° C., 230 rpm, aeration 0.56 vvm, internal pressure 0.3 kgf / cm 2 , pH 7.2, and no light irradiation was performed.
The main culture was centrifuged (7000 rpm (6500 G), 25 ° C.) and separated into a supernatant liquid containing the target polysaccharide and algal bodies. Further, the stock solution was concentrated to 2 L using a UF membrane (SIP1013) with a molecular weight cut off of 6000. 200 mL of an anion exchange resin was added to the concentrate (2 L), recovered to Brix 0.1, concentrated to 500 mL by UF membrane filtration, and freeze-dried to obtain a parachlorella-derived polysaccharide. The yield was 0.2 to 0.4 g per liter of the culture solution.
製造例２：多糖体の精製
パラクロレラ由来の多糖体１ｇに蒸留水４０ｍＬを添加して混合撹拌し、懸濁液を得た。この懸濁液を４℃にて一晩放置し、この懸濁液を遠心分離（７０００ｒｐｍ、６５００ｇ、５分間）し、上清と沈殿部とに分けた。上清について凍結乾燥し、水溶性のパラクロレラ由来の多糖体（以下、「水溶性パラクロレラ多糖」という）を得た（０．４〜０．５ｇ）。
Production Example 2: Purification of polysaccharide 40 mL of distilled water was added to 1 g of a polysaccharide derived from Parachlorella and mixed and stirred to obtain a suspension. This suspension was allowed to stand at 4 ° C. overnight, and this suspension was centrifuged (7000 rpm, 6500 g, 5 minutes) to separate into a supernatant and a precipitate. The supernatant was freeze-dried to obtain a water-soluble parachlorella-derived polysaccharide (hereinafter referred to as “water-soluble parachlorella polysaccharide”) (0.4 to 0.5 g).
実施例１：水溶性パラクロレラ多糖体の構造解析
〔多糖体の分子量〕
水溶性パラクロレラ多糖体の分子量（Ｍｗ）を、ＧＰＣ分析にて測定した結果、６．２×１０４、Ｍｗ（重量平均分子量）／Ｍｎ（数平均分子量）＝１．５あった（図３のＰ１参照）。また、上述の製造例１及び２と同様にして得られた水溶性パラクロレラ多糖体の分子量（Ｍｗ）を、ＧＰＣ分析にて測定した結果、６．７×１０４、Ｍｗ（重量平均分子量）／Ｍｎ（数平均分子量）＝１．５あった（図３のＰ２参照）。
＜ＧＰＣ分析＞
測定カラム：TSKgel GMPWXL(7.6mmID x 300 mm：東ソー株式会社)の2本直列
測定温度：40℃
移動相：0.1 M NaNO3
HPLCシステム：島津 LC-20システム
検出器：示差屈折計 LaboSystem RI-2000
標準物質：プルラン（昭和電工 P-52）
使用ソフト（GPC計算）：LC Solutions, GPC software（島津）
Example 1: Structural analysis of water-soluble parachlorella polysaccharide [molecular weight of polysaccharide]
As a result of measuring the molecular weight (Mw) of the water-soluble parachlorella polysaccharide by GPC analysis, it was 6.2 × 10 4 , Mw (weight average molecular weight) / Mn (number average molecular weight) = 1.5 (FIG. 3). P1). Moreover, as a result of measuring the molecular weight (Mw) of the water-soluble parachlorella polysaccharide obtained by carrying out similarly to the above-mentioned manufacture examples 1 and 2 by GPC analysis, 6.7 * 10 < 4 >, Mw (weight average molecular weight) / Mn (number average molecular weight) = 1.5 (see P2 in FIG. 3).
<GPC analysis>
Measurement column: TSKgel GMPW XL (7.6mmID x 300 mm: Tosoh Corporation) in series Measurement temperature: 40 ° C
Mobile phase: 0.1 M NaNO 3
HPLC system: Shimadzu LC-20 system Detector: Differential refractometer LaboSystem RI-2000
Reference material: Pullulan (Showa Denko P-52)
Software used (GPC calculation): LC Solutions, GPC software (Shimadzu)
〔多糖体の組成〕
水溶性パラクロレラ多糖体を、２Ｍ−ＴＦＡ（トリフルオロ酢酸）、１２０℃、２時間で加水分解した。この加水分解物を、室温、ＮａＢＨ４にて還元し、さらに（無水酢酸：１−メチルイミダゾール ９：１）にてアセテート化し、アルジトールアセテート誘導体を得た。アルジトールアセテート誘導体は、ＧＣで分析を行った。
ＧＣ測定は、GL Science GC-353ガスクロマトグラフ（カラム SP-2330 (Spelco)；検出器 FID；220 ℃の恒温分析）にて、行った。
アラビノース（４．７％）、ラムノース（４．０％）、キシロース（１．７％）、マンノース（２２．５％）、ガラクトース（６７．１％）であった。
[Polysaccharide composition]
The water-soluble parachlorella polysaccharide was hydrolyzed with 2M-TFA (trifluoroacetic acid) at 120 ° C. for 2 hours. This hydrolyzate was reduced with NaBH 4 at room temperature and further acetated with (acetic anhydride: 1-methylimidazole 9: 1) to obtain an alditol acetate derivative. The alditol acetate derivative was analyzed by GC.
GC measurement was performed with a GL Science GC-353 gas chromatograph (column SP-2330 (Spelco); detector FID; constant temperature analysis at 220 ° C.).
They were arabinose (4.7%), rhamnose (4.0%), xylose (1.7%), mannose (22.5%) and galactose (67.1%).
また、水溶性パラクロレラ多糖体について、メチル化分析を行った。
多糖試料をＤＭＳＯに溶解後、粉末ＮａＯＨを加え撹拌した後、ＣＨ３Ｉを加え多糖をメチル化した。メチル化多糖を回収後、２Ｍ−ＴＦＡ中１２０℃で２時間加水分解し、ＮａＢＤ４で還元後、無水酢酸と１−メチルイミダゾールでアセチル化した。得られた誘導体はＧＣ−ＭＳで分析した。
GC-MSは、Shimadzu QP-5000 GC-MS ガスクロマトグラフ質量分析装置〔カラム：SPB-50 (0.32 x 30 m)〕を行った。
本多糖体のマンノース残基として、非還元末端、１，２−結合、１，３−結合した残基が認められた。また、本多糖体のガラクトース残基の大部分はフラノースである、また本多糖体のガラクトース残基として、非還元末端、１，２−結合フラノース、１，５−結合フラノース、１，６−結合フラノース， １，２，６−結合フラノース、そして１，３−結合ピラノースが認められた。
Moreover, methylation analysis was performed about water-soluble parachlorella polysaccharide.
The polysaccharide sample was dissolved in DMSO, powdered NaOH was added and stirred, and then CH 3 I was added to methylate the polysaccharide. After recovering the methylated polysaccharide, it was hydrolyzed in 2M-TFA at 120 ° C. for 2 hours, reduced with NaBD 4 and then acetylated with acetic anhydride and 1-methylimidazole. The obtained derivative was analyzed by GC-MS.
GC-MS was performed using Shimadzu QP-5000 GC-MS gas chromatograph mass spectrometer [column: SPB-50 (0.32 × 30 m)].
As the mannose residue of the polysaccharide, a non-reducing end, 1,2-linked, and 1,3-linked residues were observed. Further, most of the galactose residues of the polysaccharide are furanose, and as the galactose residues of the polysaccharide, non-reducing ends, 1,2-linked furanose, 1,5-linked furanose, 1,6-linked Furanose, 1,2,6-linked furanose, and 1,3-linked pyranose were observed.
また、１Ｈ-ＮＭＲ分析（Varian Unity plus 500、１Ｈ：５００ＭＨｚ，１３Ｃ：１２５ＭＨｚ、〔溶媒：重水（Ｄ２Ｏ）、測定温度は３０℃、内部標準：ＤＳＳ〕）により、水溶性パラクロレラ多糖体のガラクトース残基の多くは、フラノース型で存在することが認められた。
水溶性パラクロレラ多糖体のタンパク質含量（ローリー法）及びウロン酸含量（カルバゾール−硫酸法）にて測定を行った結果、タンパク質及びウロン酸の存在は、多量ではないが、認められた。
In addition, by 1 H-NMR analysis (Varian Unity plus 500, 1H: 500 MHz, 13 C: 125 MHz, [solvent: heavy water (D 2 O), measurement temperature is 30 ° C., internal standard: DSS]), water-soluble parachlorella polysaccharide Many of the body's galactose residues were found to exist in the furanose form.
As a result of measuring the protein content (Raleigh method) and uronic acid content (carbazole-sulfuric acid method) of the water-soluble parachlorella polysaccharide, the presence of protein and uronic acid was recognized, although not in large quantities.
水溶性パラクロレラ多糖体に存在するガラクトース及びマンノースのＤＬを調べた。
水溶性パラクロレラ多糖体の試料を２Ｍ ＴＦＡ中、１２０℃で２時間加水分解した後、酸を窒素気流下で除去した。
加水分解物に（Ｓ）−（＋）−２−ｂｕｔａｎｏｌ（１００μＬ）と塩化アセチル（７．５μＬ）を加え、６０℃で一晩加熱し、ブチルグリコシドとした。試薬を窒素気流下で除去後、トリメチルシリル化し得られた誘導体をＧＣで分析した。このとき、既知のＤ−Ｇａｌ，Ｌ−Ｇａｌ，Ｄ−Ｍａｎ及びＬ−Ｍａｎも同様に誘導体を調製し、それぞれの保持時間との比較により、構成糖の絶対配置を確認した。Ｄ−ガラクトース及びＤ−マンノースが認められた。
＜ＧＣ条件＞
Ｃｏｌｕｍｎ： ＳＰＢ−１ （Spelco, 0.32 mm x 30 m）
ＧＣ： ＧＬ Ｓｃｉｅｎｃｅ ＧＣ−３５３ガスクロマトグラフ（検出器 ＦＩＤ）
キャリアガス：窒素
オーブン温度： １３５−２００℃（１℃／ｍｉｎ）
The DL of galactose and mannose present in the water-soluble parachlorella polysaccharide was examined.
A sample of the water-soluble parachlorella polysaccharide was hydrolyzed in 2M TFA at 120 ° C. for 2 hours, and then the acid was removed under a stream of nitrogen.
(S)-(+)-2-butanol (100 μL) and acetyl chloride (7.5 μL) were added to the hydrolyzate and heated at 60 ° C. overnight to form butyl glycoside. After removing the reagent under a nitrogen stream, the derivative obtained by trimethylsilylation was analyzed by GC. At this time, derivatives of known D-Gal, L-Gal, D-Man and L-Man were prepared in the same manner, and the absolute configuration of the constituent sugars was confirmed by comparison with the respective retention times. D-galactose and D-mannose were observed.
<GC conditions>
Column: SPB-1 (Spelco, 0.32 mm x 30 m)
GC: GL Science GC-353 gas chromatograph (detector FID)
Carrier gas: Nitrogen Oven temperature: 135-200 ° C (1 ° C / min)
このことから、本開示の多糖の基本構造の糖残基は、少なくともガラクトースとマンノースを主体として構成されており、当該ガラクトースのほとんどがフラノース型で存在するものと推定した。 From this, it was estimated that the sugar residue of the basic structure of the polysaccharide of the present disclosure is mainly composed of at least galactose and mannose, and most of the galactose exists in the furanose type.
実施例２：毛乳頭細胞賦活試験
〔ヒト正常毛乳頭細胞〕
凍結保存ヒト正常毛乳頭細胞（TOYOBO, Code No.CA60205a）の起眠時、継代培養、必要細胞数までの増殖には、毛乳頭細胞専用培地を使用した。培養はインキュベーター（５％ＣＯ２、３７℃）内で行った。
Example 2: Hair papilla cell activation test [human normal hair papilla cells]
For the preservation of cryopreserved human normal hair papilla cells (TOYOBO, Code No. CA60205a) during sleep, subculture, and growth to the required number of cells, a medium dedicated to hair papilla cells was used. The culture was performed in an incubator (5% CO 2 , 37 ° C.).
なお、実施例２及び後述の実施例３で使用する試薬等について、以下に示す。
＜試薬＞
DMEM 培地 （Nacalai tesque, 14242-65）
FBS （Invitrogen, Cat. No. 10091-148）
Penicillin-streptmycin solution （Nacalai tesque, Cat.No.26253-84）
0.25% trypsin-EDTA solution （Nacalai tesque、32777-44）
PBS （日水製薬株式会社, Code No. 05913）
Melanin （Sigma, Cat. No. M8631-100MG）
Phenyl-１-thiourea (PTU) （Wako, Cat. No. 166-13702）
Dimethyl sulfoxide （Nacalai tesque Cat. No. 13445-74）
Theophyline （Wako, Cat. No. 209-09932）
毛乳頭細胞増殖培地（TOYOBO, Code No.TMTPGM-250）添加剤含
毛乳頭細胞専用サブカルチャーセット（TOYOBO, Code No.CA090K）
生細胞数測定試薬ＳＦ（nacalai tesque, Cat.No. 07553）
Adenosine (Wako, Cat. No. 016-10493)
＜その他＞
Minisart 0.2 μm （Sartorius, Code No. 16534）
96 well culture plate （SUMILON, Cat. No. MS-8096F）
24 well cell culture cluster （TPP, Code No. 92424）
15 ml centrifuge tube （Corning, Code No. 430766）
Precision microplate reader （Molecular Devices）
Thermo Alumi Bath (IWAKI, ALB-121)
＜標品等＞
［１］メラニン生成を促進させるためテオフィリンを使用した。テオフィリンはＤＭＳＯに溶解・濾過滅菌し、３６ｍｇ／ｍｌ溶液を調製した。使用時は培地にて希釈し、９０μｇ／ｍＬに調整した。
［２］ Ａｄｅｎｏｓｉｎｅ
毛乳頭細胞賦活試験の陽性コントロールとしてアデノシンを使用した。アデノシンはＤＭＳＯに溶解・濾過滅菌し、１００ｍＭ溶液を調製した。使用時は培地にて希釈し、１００μＭに調整した。
In addition, it shows below about the reagent etc. which are used in Example 2 and Example 3 mentioned later.
<Reagent>
DMEM medium (Nacalai tesque, 14242-65)
FBS (Invitrogen, Cat. No. 10091-148)
Penicillin-streptmycin solution (Nacalai tesque, Cat.No.26253-84)
0.25% trypsin-EDTA solution (Nacalai tesque, 32777-44)
PBS (Nissui Pharmaceutical Co., Ltd., Code No. 05913)
Melanin (Sigma, Cat. No. M8631-100MG)
Phenyl-1-thiourea (PTU) (Wako, Cat. No. 166-13702)
Dimethyl sulfoxide (Nacalai tesque Cat. No. 13445-74)
Theophyline (Wako, Cat. No. 209-09932)
Hair papilla cell growth medium (TOYOBO, Code No.TMTPGM-250) additive-containing subculture set for hair papilla cells (TOYOBO, Code No. CA090K)
Viable cell count reagent SF (nacalai tesque, Cat. No. 07553)
Adenosine (Wako, Cat.No. 016-10493)
<Others>
Minisart 0.2 μm (Sartorius, Code No. 16534)
96 well culture plate (SUMILON, Cat. No. MS-8096F)
24 well cell culture cluster (TPP, Code No. 92424)
15 ml centrifuge tube (Corning, Code No. 430766)
Precision microplate reader (Molecular Devices)
Thermo Alumi Bath (IWAKI, ALB-121)
<Standard products>
[1] Theophylline was used to promote melanin production. Theophylline was dissolved in DMSO and sterilized by filtration to prepare a 36 mg / ml solution. At the time of use, it was diluted with a medium and adjusted to 90 μg / mL.
[2] Adenosine
Adenosine was used as a positive control for the dermal papilla cell activation test. Adenosine was dissolved in DMSO and sterilized by filtration to prepare a 100 mM solution. At the time of use, it was diluted with a medium and adjusted to 100 μM.
〔毛乳頭毒性試験〕
毛乳頭細胞を１．４ｘ１０４個／１００μＬ／ｗｅｌｌの濃度で９６ウェルプレートに播種し、翌日（８０％コンフルエント）、各濃度（０．０１、０．１、１質量％）の水溶性パラクロレラ多糖体を含む試験培地に交換し、２４時間培養した。
細胞の増殖性を、生細胞数測定試薬ＳＦにて、測定した。生細胞数測定試薬を１０％含む増殖培地を１００μＬ／ｗｅｌｌで添加し、３７℃、５％ＣＯ２でインキュベートした。３０、９０分後にマイクロプレートリーダーを用いて反応培地の吸光度を測定した（測定波長：４５０ｎｍ、対照波長：５９５ｎｍ）。試験数３にて行った。
水溶性パラクロレラ多糖体の０．０１、０．１及び１質量％の何れの濃度でも、毛乳頭細胞に対して細胞毒性がほとんど認められなかった。
[Hair nipple toxicity test]
Papilla cells are seeded in a 96-well plate at a concentration of 1.4 × 10 4 cells / 100 μL / well, and the following day (80% confluent), each concentration (0.01, 0.1, 1% by mass) of water-soluble parachlorella The test medium was replaced with a polysaccharide and cultured for 24 hours.
Cell proliferation was measured with a living cell number measuring reagent SF. A growth medium containing 10% of a viable cell count measurement reagent was added at 100 μL / well, and incubated at 37 ° C. and 5% CO 2 . After 30 and 90 minutes, the absorbance of the reaction medium was measured using a microplate reader (measurement wavelength: 450 nm, control wavelength: 595 nm). The number of tests was three.
At any concentration of the water-soluble parachlorella polysaccharide of 0.01, 0.1 and 1% by mass, almost no cytotoxicity was observed on the hair papilla cells.
〔毛乳頭細胞・細胞賦活試験〕
毛乳頭細胞を５ｘ１０３個／１００μＬ／ｗｅｌｌの濃度で９６ウェルプレートに播種し、翌日（５０％コンフルエント）、各濃度（０．００００１、０．００１、０．１質量％）の被験物質を含む試験培地に交換し、２４時間、７２時間培養した。細胞増殖性を、生細胞数測定試薬ＳＦを用いて測定した。生細胞数測定試薬を１０％含む増殖培地を１００μＬ／ｗｅｌｌで添加し、３７℃、５％ＣＯ２でインキュベートした。３０、９０分後にマイクロプレートリーダーを用いて反応培地の吸光度を測定した（測定波長：４５０ｎｍ、対照波長：５９５ｎｍ）。試験数５にて行った。
表２に示すように、水溶性パラクロレラ多糖体の０．００００１質量％以上の濃度で、優れた毛乳頭細胞増殖賦活作用が認められた。
＜有意差検定＞
比較試験区間では有意差検定を行った。検定はＳｔｕｄｅｎｔ Ｔ−ｔｅｓｔとして行いＰ＜０．０５（帰無仮説が５％未満）のものを有意差ありと判断した。
[Papilla cells / cell activation test]
Papilla cells are seeded in a 96-well plate at a concentration of 5 × 10 3 cells / 100 μL / well and contain the test substance at each concentration (0.00001, 0.001, 0.1% by mass) the next day (50% confluent). The culture medium was changed to a test medium and cultured for 24 hours and 72 hours. Cell proliferation was measured using a living cell count reagent SF. A growth medium containing 10% of a viable cell count measurement reagent was added at 100 μL / well, and incubated at 37 ° C. and 5% CO 2 . After 30 and 90 minutes, the absorbance of the reaction medium was measured using a microplate reader (measurement wavelength: 450 nm, control wavelength: 595 nm). The number of tests was 5.
As shown in Table 2, an excellent hair papillary cell proliferation activating effect was observed at a concentration of 0.00001% by mass or more of the water-soluble parachlorella polysaccharide.
<Significant difference test>
A significant difference test was performed in the comparative test section. The test was performed as Student T-test, and P <0.05 (the null hypothesis was less than 5%) was judged to be significant.
一般に細胞増殖過程では分裂が盛んな指数増殖期と分裂を停止し安定的に細胞を維持する維持期に大別できる。本試験において各時期における水溶性パラクロレラ多糖の効果を検証するため細胞密度（コンフルエント）が４０〜６０％の状態で水溶性パラクロレラ多糖を添加し、２４時間培養後の生細胞数を測定した。また、さらに４８時間培養（合計３日間）により維持期における細胞へ与える影響を検証した。
指数増殖期（培養１日）、維持期（培養３日）において水溶性クロレラ多糖体の最低濃度区において無添加区と比較して約１．１〜１．４倍の増殖促進を示した。これは被験物質に細胞分裂促進効果と維持促進効果があることが考えられる。
一方、陽性対照であるアデノシンは指数増殖期において増殖促進作用を示したものの、維持期では無添加区と同程度の生細胞数であった。これはアデノシンが細胞分裂を促進するものの細胞密度が１００％となった後では細胞維持には作用しないことが考えられる。
よって、本開示の多糖体は、優れた細胞増殖賦活作用及び優れた毛乳頭細胞増殖賦活作用を有することが認められた。
In general, the cell growth process can be broadly divided into an exponential growth phase in which division is active and a maintenance phase in which division is stopped and cells are stably maintained. In this test, in order to verify the effect of water-soluble parachlorella polysaccharide at each stage, water-soluble parachlorella polysaccharide was added at a cell density (confluent) of 40 to 60%, and the number of living cells after 24 hours of culture was measured. . Further, the effect on the cells in the maintenance phase was verified by further 48 hours of culture (3 days in total).
In the exponential growth phase (1 day of culture) and the maintenance phase (3 days of culture), the lowest concentration group of the water-soluble chlorella polysaccharide showed about 1.1 to 1.4 times the growth promotion as compared to the non-added group. This may be because the test substance has a cell division promoting effect and a maintenance promoting effect.
On the other hand, adenosine, which is a positive control, showed a growth promoting effect in the exponential growth phase, but in the maintenance phase, the number of living cells was the same as that in the non-added group. This may be because adenosine promotes cell division but does not act on cell maintenance after the cell density reaches 100%.
Therefore, it was confirmed that the polysaccharide of the present disclosure has an excellent cell proliferation activating effect and an excellent hair papillary cell proliferation activating effect.
実施例３：美白作用（メラニン産生抑制）試験
〔細胞培養〕
Ｂ１６ ｍｅｌａｎｏｍａ細胞を３ｘ１０４個／ｍｌ／ｗｅｌｌで２４ウェルプレートに播種し、翌日、各濃度（０．０１、０．１質量％）の水溶性パラクロレラ多糖体を含む試験培地１ｍｌに交換し、４８時間培養した。その後、全細胞の細胞内メラニン量を測定した。試験数５にて行った。
〔細胞内メラニンの測定〕
培地上清を除去後、ウェルに接着している細胞をＰＢＳ、１ｍｌで洗浄する。細胞に０．２５％ ｔｒｙｐｓｉｎ−ＥＤＴＡ ｓｏｌｕｔｉｏｎを１００μｌを加え３７℃、５％ＣＯ２条件下で１分間静置する。トリプシン処理によって剥離した細胞をＰＢＳ１ｍｌにて回収し、１．５ｍｌチューブに移した。その後、１，０００ｒｐｍ、４℃で５分間遠心し、上清を除去後、ＰＢＳにて細胞ペレットを２回洗浄した。細胞懸濁液を３，０００ｒｐｍで５分間遠心した後、ＰＢＳを除去し、１Ｎ水酸化ナトリウム溶液１１０μｌに細胞を懸濁した。剥離した細胞を含むすべてのメラニン量を測定するため、培地上清、洗浄後のＰＢＳを回収し３，０００ｒｐｍで５分間遠心した後、得られた沈殿と細胞懸濁液を混ぜ、１００℃で３０分インキュベートして細胞を溶解し、この溶解液８０μｌを９６ウェルプレートに移し、４０５ｎｍにおける吸光度をマイクロプレートリーダーにて測定し、細胞内のメラニン量を算出した。メラニンの標準品として、市販合成メラニンを用いた。１Ｎ水酸化ナトリウム溶液にて２．５〜１６０μｇ／ｍｌ溶液を調製し、それぞれの吸光度から検量線を作成した。この標準検量線をもとに、各検体におけるメラニン量を算出した。
メラニン産生抑制試験の結果を表４に示す。水溶性パラクロレラ多糖体にてメラニン産生抑制を有意に示した。
よって、本開示の多糖体は、優れたメラニン産生抑制作用及び優れた美白作用を有することが認められた。
Example 3: Whitening action (melanin production inhibition) test [cell culture]
B16 melanoma cells were seeded in a 24-well plate at 3 × 10 4 cells / ml / well, and the following day, the test medium was replaced with 1 ml of a test medium containing water-soluble parachlorella polysaccharides of each concentration (0.01, 0.1% by mass). Cultured for 48 hours. Thereafter, the amount of intracellular melanin in all cells was measured. The number of tests was 5.
[Measurement of intracellular melanin]
After removing the culture supernatant, the cells adhering to the wells are washed with 1 ml of PBS. 100 μl of 0.25% trypsin-EDTA solution is added to the cells, and the cells are allowed to stand at 37 ° C. under 5% CO 2 for 1 minute. Cells detached by trypsin treatment were collected with 1 ml of PBS and transferred to a 1.5 ml tube. Then, it centrifuged at 1,000 rpm and 4 degreeC for 5 minute (s), the supernatant was removed, and the cell pellet was wash | cleaned twice by PBS. After centrifuging the cell suspension at 3,000 rpm for 5 minutes, PBS was removed and the cells were suspended in 110 μl of 1N sodium hydroxide solution. In order to measure the total amount of melanin including detached cells, the culture medium supernatant and the washed PBS were collected and centrifuged at 3,000 rpm for 5 minutes, and the resulting precipitate and the cell suspension were mixed at 100 ° C. Cells were lysed by incubating for 30 minutes, 80 μl of this lysate was transferred to a 96-well plate, the absorbance at 405 nm was measured with a microplate reader, and the amount of intracellular melanin was calculated. Commercially available synthetic melanin was used as a standard product of melanin. A 2.5 to 160 μg / ml solution was prepared with a 1N sodium hydroxide solution, and a calibration curve was prepared from each absorbance. Based on this standard calibration curve, the amount of melanin in each specimen was calculated.
The results of the melanin production inhibition test are shown in Table 4. The water-soluble parachlorella polysaccharide showed significant inhibition of melanin production.
Therefore, it was recognized that the polysaccharide of this indication has the outstanding melanin production inhibitory effect and the outstanding whitening effect | action.
実施例４：線維芽細胞増殖賦活試験
〔培養上清〕
毛乳頭細胞の培養条件及び被験物質処理条件などは、上述の実施例１及び２と同様に行った。培養上清は測定まで冷凍保存した。
〔試薬〕
FGF7 Human ELISA kit（abcam、Code No．ablOO519）
〔分析装置〕
Precision microplate reader（Molecular Devices）
〔試薬等の調製〕
ＦＧＦ−７測定において必要量１００μＬ、ＶＥＧＦ測定において必要量２００μＬである。
実施例２で得られた培養上清量が約９０μＬを、１×ＰＢＳを用いて約４倍に希釈しＦＧＦ−７測定に使用した。
〔培養上清中のＦＧＦ−７量の測定〕
［１］ＦＧＦ−７の測定はキットのプロトコールに順じて行った。以下にその詳細を述べる。
［２］ＦＧＦ−７標準原液（５０ｎｇ／ｍｌ）をもとに、反応緩衝液を用いて４００、１３３．３、４４．４４、１４．８１、４．９４、１．６５、０．５５ｐｇ／ｍｌのＦＧＦ−７標準溶液を調製する。
［３］ＦＧＦ−７標準溶液および検体（培養上清）１００μｌをＦＧＦ−７固相化マイクロプレートの各ウェルに添加し、室温にて２時間３０分反応した（一次反応）。
ウェル内の溶液を除去し、洗浄液で４回洗浄する。
［４］ビオチン標識抗ＦＧＦ−７抗体溶液を各ウェルに１００μｌずつ添加し、室温で６０分間反応させる（二次反応）。
［５］ウェル内の溶液を除去し、洗浄液で４回洗浄する。
［６］ストレプトアピジン溶液１００μｌを各ウェルに添加し、室温で４５分反応する。
［７］酵素基質溶液１００μｌを各ウェルに添加し、遮光・室温下で３０分間静置する（発色反応）。
［８］反応停止溶液を各ウェルに５０μｌずつ添加し、プレートミキサーで１分間混和後、プレートリーダーで各ウェルの吸光度を測定する（測定波長４５０ｎｍ）。
［９］標準曲線から、検体中のＦＧＦ−７濃度を算出する。
Example 4: Fibroblast proliferation activation test [culture supernatant]
The culture conditions of the dermal papilla cells and the test substance treatment conditions were the same as in Examples 1 and 2 above. The culture supernatant was stored frozen until measurement.
〔reagent〕
FGF7 Human ELISA kit (abcam, Code No. ablOO519)
〔Analysis equipment〕
Precision microplate reader (Molecular Devices)
[Preparation of reagents, etc.]
The required amount is 100 μL for FGF-7 measurement, and the required amount is 200 μL for VEGF measurement.
About 90 μL of the culture supernatant obtained in Example 2 was diluted about 4 times with 1 × PBS and used for FGF-7 measurement.
[Measurement of the amount of FGF-7 in the culture supernatant]
[1] FGF-7 was measured according to the protocol of the kit. Details are described below.
[2] Based on FGF-7 standard stock solution (50 ng / ml), 400, 133.3, 44.44, 14.81, 4.94, 1.65, 0.55 pg / Prepare ml FGF-7 standard solution.
[3] FGF-7 standard solution and 100 μl of specimen (culture supernatant) were added to each well of the FGF-7 solid-phased microplate and reacted at room temperature for 2 hours 30 minutes (primary reaction).
Remove the solution in the well and wash 4 times with the washing solution.
[4] 100 μl of biotin-labeled anti-FGF-7 antibody solution is added to each well and reacted at room temperature for 60 minutes (secondary reaction).
[5] The solution in the well is removed and the plate is washed 4 times with a washing solution.
[6] Add 100 μl of streptapidin solution to each well and react for 45 minutes at room temperature.
[7] Add 100 μl of the enzyme substrate solution to each well, and allow to stand at room temperature for 30 minutes in the dark (coloring reaction).
[8] Add 50 μl of the reaction stop solution to each well, mix with a plate mixer for 1 minute, and then measure the absorbance of each well with a plate reader (measurement wavelength: 450 nm).
[9] Calculate the FGF-7 concentration in the specimen from the standard curve.
実施例２で回収した毛乳頭細胞培養上清中のＦＧＦ−７産生量を測定した。
表５及び６に示すように、毛乳頭細胞培養１日間では、陽性対照であるアデノシンにおいてＦＧＦ−７が未検出であった。しかし、水溶性パラクロレラ多糖体において、ＦＧＦ−７産生促進効果が認められた。さらに、３日間では、水溶性パラクロレラ多糖体の０．０１〜１質量％の濃度において、アデノシンと比較しても高いＦＧＦ−７産生促進効果が認められた。
よって、本開示の多糖体に非常に優れたＦＧＦ−７の産生促進作用、線維芽細胞増殖賦活作用を有することが認められた。
The amount of FGF-7 produced in the dermal papilla cell culture supernatant collected in Example 2 was measured.
As shown in Tables 5 and 6, FGF-7 was not detected in the positive control, adenosine, in the dermal papilla cell culture for 1 day. However, the FGF-7 production promoting effect was observed in the water-soluble parachlorella polysaccharide. Further, in 3 days, a high FGF-7 production promoting effect was observed even at a concentration of 0.01 to 1% by mass of the water-soluble parachlorella polysaccharide compared with adenosine.
Therefore, it was confirmed that the polysaccharide of the present disclosure has a very excellent FGF-7 production promoting action and fibroblast proliferation activating action.
実施例５：血管内皮増殖賦活試験
〔培養上清〕
毛乳頭細胞の培養条件及び被験物質処理条件などは、上述の実施例１及び２と同様に行った。培養上清は測定まで冷凍保存した。
〔試薬〕
Human VEGF Quantikine ELISA（R＆D Systems，Cat．No．DVEOO）
〔分析装置〕
Precision microplate reader（Molecular Devices）
〔培養上清中のVEGF量の測定〕
ＶＥＧＦの測定はキットのプロトコールに順じて行った。以下にその詳細を述べる。
［１］ＶＥＧＦ標準原液（２０００ｐｇ／ｍｌ）をもとに、反応緩衝液を用いて１０００、５００、２５０、１２５、６２．５、３１．２、１５．６ｐｇ／ｍｌのＶＥＧＦ標準溶液を調製する。
［２］ＶＥＧＦ標準溶液および検体（培養上清）２００μｌを５０μｌの希釈溶液を各ウェルに添加したＶＥＧＦ固相化マイクロプレートに添加し、室温にて２時間反応した（一次反応）。
［３］ウェル内の溶液を除去し、洗浄液で３回洗浄する。
［４］西洋ワサビベルオキシダーゼ結合ＶＥＧＦ抗体溶液を各ウェルに２００μｌずつ添加し、室温で２時間反応させる（二次反応）。
［５］ウェル内の溶液を除去し、洗浄液で３回洗浄する。
［６］基質溶液２００μＬを各ウェルに添加し、室温で２０分反応する。
［７］反応停止溶液を各ウェルに５０μｌずつ添加し、プレートミキサーで１分間混和後、プレートリーダーで各ウェルの吸光度を測定する（測定波長４５０ｎｍ）。
［８］標準曲線から、検体中のＶＥＧＦ濃度を算出する。
〔有意差検定〕
比較試験区間では有意差検定を行った。検定はＳｔｕｄｅｎｔＴ−ｔｅｓｔとして行いＰく０．０５（帰無仮説が５％未満）のものを有意差ありと判断した。
Example 5: Vascular endothelial growth activation test [culture supernatant]
The culture conditions of the dermal papilla cells and the test substance treatment conditions were the same as in Examples 1 and 2 above. The culture supernatant was stored frozen until measurement.
〔reagent〕
Human VEGF Quantikine ELISA (R & D Systems, Cat. No. DVEOO)
〔Analysis equipment〕
Precision microplate reader (Molecular Devices)
[Measurement of VEGF content in culture supernatant]
VEGF was measured according to the kit protocol. Details are described below.
[1] Based on VEGF standard stock solution (2000 pg / ml), prepare 1000, 500, 250, 125, 62.5, 31.2, 15.6 pg / ml VEGF standard solution using reaction buffer. .
[2] VEGF standard solution and 200 μl of specimen (culture supernatant) were added to a VEGF-immobilized microplate to which 50 μl of diluted solution was added to each well, and reacted at room temperature for 2 hours (primary reaction).
[3] The solution in the well is removed and washed with a washing solution three times.
[4] 200 μl of a horseradish oxidase-conjugated VEGF antibody solution is added to each well and reacted at room temperature for 2 hours (secondary reaction).
[5] The solution in the well is removed and washed with a washing solution three times.
[6] Add 200 μL of the substrate solution to each well and react at room temperature for 20 minutes.
[7] Add 50 μl of the reaction stop solution to each well, mix with a plate mixer for 1 minute, and then measure the absorbance of each well with a plate reader (measurement wavelength: 450 nm).
[8] Calculate the VEGF concentration in the specimen from the standard curve.
[Significant difference test]
A significant difference test was performed in the comparative test section. The test was performed as Student T-test, and a value of 0.05 (null hypothesis was less than 5%) was judged to be significant.
実施例２で回収した毛乳頭細胞培養上清中のＶＥＧＦ産生量を測定した。
表７及び８に示すように、陽性対照であるアデノシンのＶＥＧＦ産生促進効果はあまりなかった。しかし、水溶性パラクロレラ多糖体いずれも高濃度試験区でＶＥＧＦ産生促進効果が認められた。
よって、本開示の多糖体に非常に優れたＶＥＧＦの産生促進作用及び血管内皮増殖賦活作用を有することが認められた。
The amount of VEGF produced in the dermal papilla cell culture supernatant collected in Example 2 was measured.
As shown in Tables 7 and 8, the positive control, adenosine, had little VEGF production promoting effect. However, all the water-soluble parachlorella polysaccharides were found to promote VEGF production in the high concentration test section.
Therefore, it was confirmed that the polysaccharide of the present disclosure has a very excellent VEGF production promoting action and vascular endothelial growth activation action.
実際の毛髪組織では毛乳頭細胞は３次元的に増殖していることから、産生促進されたＦＧＦ−７及びＶＥＧＦが十分に機能すると考えられ、さらに毛乳頭細胞増殖賦活作用も認められているので、本開示の多糖体は、非常に優れた育毛作用を発揮すると考えられる。 In the actual hair tissue, dermal papilla cells are three-dimensionally proliferated. Therefore, it is considered that the production-promoted FGF-7 and VEGF function sufficiently, and further, the dermal papilla cell proliferation activation effect is also recognized. The polysaccharide of the present disclosure is considered to exhibit a very excellent hair-growth effect.
本開示の多糖体は、種々の効能が認められ、また大量生産も可能であるため、幅広い分野での応用が可能である。また、本開示の多糖体の製造方法にて、高付加価値の多糖体を効率良く生産することが可能であるため、製造コストの点や安定供給の点からも、産業上の利用可能性が非常に高い。 Since the polysaccharide of the present disclosure has various effects and can be mass-produced, it can be applied in a wide range of fields. In addition, since the polysaccharide manufacturing method of the present disclosure can efficiently produce a high-value-added polysaccharide, the industrial applicability can be achieved in terms of manufacturing cost and stable supply. Very expensive.
（１）Parachlorella kessleri-PNC1株(FERM BP-11493)（受託日：2012年 7月19日）
寄託先：〒305-8566 茨城県つくば市東1-1-1 つくばセンター中央第6、独立行政法人 製品評価技術基盤機構 特許生物寄託センター（NITE-IPOD）。
(1) Parachlorella kessleri-PNC1 strain (FERM BP-11493) (Contract date: July 19, 2012)
Deposit place: 1-1-1 Higashi Tsukuba City, Ibaraki 305-8566 Tsukuba Center Chuo No. 6, National Institute for Product Evaluation Technology Patent Biological Deposit Center (NITE-IPOD).
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