JPH08268905A - Colloidal silk fibroin and composition containing the same - Google Patents
Colloidal silk fibroin and composition containing the sameInfo
- Publication number
- JPH08268905A JPH08268905A JP7112255A JP11225595A JPH08268905A JP H08268905 A JPH08268905 A JP H08268905A JP 7112255 A JP7112255 A JP 7112255A JP 11225595 A JP11225595 A JP 11225595A JP H08268905 A JPH08268905 A JP H08268905A
- Authority
- JP
- Japan
- Prior art keywords
- silk fibroin
- colloidal
- silk
- water
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、医学、美容、衛生及び
食品など、様々な分野で有用な、コロイド状絹フィブロ
イン、その製造方法及びそれを含有する組成物に関す
る。FIELD OF THE INVENTION The present invention relates to colloidal silk fibroin useful for various fields such as medicine, beauty, hygiene and food, a method for producing the same, and a composition containing the same.
【０００２】[0002]
【従来技術と発明が解決すべき課題】従来、絹の用途と
しては衣料の原料としての利用以外に、絹の酸加水分解
物又は主要な構成蛋白質であるフィブロインを含有す
る、水溶性絹溶解物の利用が知られている。水溶性フィ
ブロインや酸加水分解物には保湿性があることから、主
にファンデーシヨン、リンス、シャンプなどの化粧品の
分野で使用されている （特開平０１−６１４１２号公報、特開平０１−１４４
４６９号公報、特開平０３−７７８０６号公報、特開平
０４−６６５１５号公報、特開平０４−７４１０９号公
報、特開平０４−８９４２１号公報）。しかしながら、
これら水溶性の絹溶解物は安定性に欠け、容易に凝固し
てしまうという欠点を有しており、酸加水分解物はアミ
ノ酸の混合物であり、必ずしも効果的とはいえなかっ
た、このように、従来の水溶性絹溶解物は、絹の有効利
用という点で多くの問題を有していた。しかも、これら
水溶性絹溶解物の製造には、絹の酸加水分解、酸の中
和、生成する塩の除去などの繁雑な工程が要求され、非
効率的であった。2. Description of the Related Art Conventionally, as a use of silk, a water-soluble silk melt containing an acid hydrolyzate of silk or fibroin, which is a main constituent protein, has been used in addition to the use as a raw material for clothing. The use of is known. Since water-soluble fibroin and acid hydrolysates have moisturizing properties, they are mainly used in the field of cosmetics such as foundations, rinses and shamps (JP-A 01-61412 and JP-A 01-144).
469, JP-A-03-77806, JP-A-04-66515, JP-A-04-74109, and JP-A-04-89421). However,
These water-soluble silk melts have the drawback of lacking stability and easily coagulating, and the acid hydrolyzate is a mixture of amino acids, which was not always effective. Conventional water-soluble silk melts have many problems in terms of effective utilization of silk. Moreover, the production of these water-soluble silk melts is inefficient because it requires complicated steps such as acid hydrolysis of silk, neutralization of acid, and removal of salts produced.
【０００３】[0003]
【課題を解決するための手段】本発明者らは、上記の課
題を解決し、絹の特性をより広範な分野で十分に活用す
るために、安定で有用な絹由来の製品を提供することを
目的として鋭意検討した結果、絹の無機塩類の水溶液と
水混和性の有機溶剤の混合液中の溶液を水に対して透析
することにより得られるコロイド状のフィブロイン含有
画分が、そのような目的にかなうことを見い出し、本発
明を完成させるに至った。即ち、本発明は、絹フィブロ
イン蛋白質からなり、微粒ゲル構造を有することを特徴
とするコロイド状絹フィブロインを提供するものであ
る。本発明のコロイド状絹フィブロインは、以下の理化
学的特性を有する。 １）ゲル濾過法により測定した場合、分子量は約３５万
〜３７万であり； ２）粒子径約０．１〜０．２μｍの球形であり、 ３）溶液はコロイド溶液特性の強いチンダル現象を示
し、 ４）２００〜３００ｎｍの範囲に紫外部吸収を有し、最
大吸収は２８０ｎｍにあり、 ５）等電点はｐＨ３．９である。[Means for Solving the Problems] The present inventors have provided a stable and useful silk-derived product in order to solve the above problems and fully utilize the properties of silk in a wider range of fields. As a result of intensive studies for the purpose, a colloidal fibroin-containing fraction obtained by dialysis of a solution in a mixture of an aqueous solution of silk inorganic salt and a water-miscible organic solvent against water is The inventors have found that they meet the purpose, and have completed the present invention. That is, the present invention provides a colloidal silk fibroin characterized by comprising a silk fibroin protein and having a fine particle gel structure. The colloidal silk fibroin of the present invention has the following physicochemical properties. 1) When measured by a gel filtration method, the molecular weight is about 350,000 to 370,000; 2) It is spherical with a particle size of about 0.1 to 0.2 μm; 3) The solution exhibits a strong Tyndall phenomenon with colloidal solution characteristics. 4) has an ultraviolet absorption in the range of 200 to 300 nm, the maximum absorption is at 280 nm, and 5) the isoelectric point is pH 3.9.
【０００４】本発明のコロイド状絹フィブロインは、絹
を無機塩類の水溶液と水混和性の有機溶剤との混合液に
溶解させ、得られた溶液を水に対して透析することによ
り製造されるものであり、起源は、家蚕又は野蚕のいず
れであってもよい。無機塩類としては、絹の可溶化に有
用な水易溶性の任意の無機塩が使用可能であるが、一般
式：ＭｅＸ（式中、Ｍｅはアルカリ金属又はアルカリ土
類金属、Ｘはハロゲン、硫酸根又は硝酸根を表す）で示
される塩類が本発明には好ましい。特に好ましいのは、
アルカリ金属又はアルカリ土類金属のハロゲン化物であ
り、例えば、塩化カルシウム、臭化リチウム、塩化マグ
ネシウムなどを例示することができる。使用する塩類の
濃度は、絹が可溶化される濃度であればよく、特に限定
されないが、通常、３０〜６０％程度の、飽和濃度に近
い状態で用いることができる。しかしながら、用いる塩
に応じて適宜調節可能である。The colloidal silk fibroin of the present invention is produced by dissolving silk in a mixed solution of an aqueous solution of an inorganic salt and a water-miscible organic solvent, and dialyzing the resulting solution against water. The origin may be either domestic silkworms or wild silkworms. As the inorganic salt, any readily water-soluble inorganic salt useful for solubilizing silk can be used, but a general formula: MeX (in the formula, Me is an alkali metal or alkaline earth metal, X is a halogen, sulfuric acid). (Representing root or nitrate root) are preferred for the present invention. Particularly preferred is
It is a halide of an alkali metal or an alkaline earth metal, and examples thereof include calcium chloride, lithium bromide, magnesium chloride and the like. The concentration of the salt used is not particularly limited as long as it is a concentration capable of solubilizing silk, but it can be used in a state of about 30 to 60%, which is close to the saturated concentration. However, it can be adjusted appropriately depending on the salt used.
【０００５】水混和性の有機溶剤としては、当該技術分
野で水と容易に混ざり合うことが知られている広範な溶
剤を用いることができ、例えば、アルコール、アセト
ン、ジオキサン及びピリジンを挙げることができる。し
かしながら、本発明の目的には、アルコール系溶媒やア
セトンが好ましく、中でも低級アルコール、とりわけエ
タノールやメタノールが好ましい。無機塩類の水溶液と
有機溶剤との混合比は、１〜１０：１、好ましくは約
５：１であり、絹１００ｇに対して、混合液を約０．１
〜３．０Ｌ、好ましくは約０．５〜２．０Ｌより好まし
くは約０．５〜１．０Ｌ用いる、通常、絹を含有する混
合溶液を室温〜９５℃、好ましくは約６０〜９５℃で約
１分〜３日間、好ましくは約１〜５分間加熱処理する
と、絹が可溶化され、絹溶液が得られる。As the water-miscible organic solvent, a wide variety of solvents known in the art to easily mix with water can be used, and examples thereof include alcohol, acetone, dioxane and pyridine. it can. However, for the purposes of the present invention, alcoholic solvents and acetone are preferred, with lower alcohols being preferred, especially ethanol and methanol. The mixing ratio of the aqueous solution of the inorganic salt and the organic solvent is 1 to 10: 1, preferably about 5: 1, and the mixed solution is about 0.1 to 100 g of silk.
-3.0 L, preferably about 0.5-2.0 L, more preferably about 0.5-1.0 L, usually a silk-containing mixed solution at room temperature to 95 ° C, preferably about 60-95 ° C. Heat treatment for about 1 minute to 3 days, preferably about 1 to 5 minutes, solubilizes silk and obtains a silk solution.
【０００６】絹溶液の透析は、有機化学や生化学の分野
で一般的な透析膜と水を用い、通常の方法で行う。電気
透析器等を使用してもよい。流水（流速約５０〜２００
ｍｌ／分、好ましくは約１００ｍｌ／分、総水量約３０
０〜１０００Ｌに相当〕を用いることが好ましいが、溜
め水でも可能である。本発明方法に使用可能な透析膜と
して、例えば、セロファン膜（シームレスセルロースチ
ューブ：三光純薬株式会社）、膀胱膜等を挙げることが
できる。透析は、使用した塩類が透析液中に検出されな
くなるまで行う。通常、流水を用いる場合、約２〜５
日、好ましくは３〜４日間であり、透析終了時には、内
液として目的のコロイド状絹フィブロイン溶液が得られ
る、溜め水を用いる場合は、絹溶液の５〜２０倍、好ま
しくは７〜１０倍の容量の水を用い、通常、約２４時間
毎に透析液を交換して、２〜５日間、好ましくは３〜４
日間透析すればよい。透析液中の塩類の検出は、通常の
定性分析法を用いて行うことができ、例えば、塩化カル
シウムの場合、蓚酸アンモニウム溶液による白色沈殿の
生成の有無に基いて判定される。いずれかの方法で得ら
れたコロイド溶液はそのままで、或いは、凍結乾燥した
後粉末として用いることができる。The dialysis of the silk solution is carried out by a usual method using a dialysis membrane and water which are common in the fields of organic chemistry and biochemistry. An electrodialyzer or the like may be used. Running water (flow velocity about 50-200
ml / min, preferably about 100 ml / min, total water volume about 30
It is preferable to use [corresponding to 0 to 1000 L], but it is also possible to use stored water. Examples of the dialysis membrane that can be used in the method of the present invention include cellophane membrane (seamless cellulose tube: Sanko Junyaku Co., Ltd.) and bladder membrane. Dialysis is performed until the salt used cannot be detected in the dialysate. Usually, when using running water, it is about 2-5
The day, preferably 3 to 4 days, at the end of dialysis, the desired colloidal silk fibroin solution is obtained as an internal solution. When using reservoir water, it is 5 to 20 times, preferably 7 to 10 times that of the silk solution. The amount of water used is usually about 2 to 5 days, preferably 3 to 4 with the dialysate changed every 24 hours.
You can dialyze for days. The detection of salts in the dialysate can be carried out by using an ordinary qualitative analysis method. For example, in the case of calcium chloride, it is judged based on the presence or absence of white precipitate produced by the ammonium oxalate solution. The colloidal solution obtained by either method can be used as it is, or can be used as a powder after freeze-drying.
【０００７】本発明により得られたコロイド溶液は極め
て安定であり、それを構成するフィブロイン蛋白質は塩
類で溶解後、ゲル濾過法で測定した結果、分子量約３５
〜３７万で、粒子径は約０．１〜０．２μｍの球形であ
る、また、コロイド溶液特性の強いチンダル現象を示
し、２００〜３００ｎｍの範囲に紫外部吸収を有し、最
大吸収は２８０ｎｍにある。しかし等電点（ｐＨ３．
９）において、２５℃で３日目に変性して強固なゲルを
形成する。本発明のコロイド状絹フィブロインは、以下
の試験例に示すように、予想外に優れた保湿作用、動脈
硬化の原因とされるＬＤＬ−コレステロール低下作用、
静菌作用、紫外線吸収作用等、広範な分野で有用な様々
な活性を有する多機能性物質であり、従来の水溶性フィ
ブロインとは別物質である。The colloidal solution obtained by the present invention is extremely stable, and the fibroin protein constituting it is dissolved in salts and then measured by gel filtration. As a result, the molecular weight is about 35.
It has a spherical shape with a particle size of about 370,000 and a particle size of about 0.1 to 0.2 μm, exhibits a strong Tyndall phenomenon of colloidal solution characteristics, has an ultraviolet absorption in the range of 200 to 300 nm, and has a maximum absorption of 280 nm. It is in. However, the isoelectric point (pH 3.
In 9), it denatures at 25 ° C. on the third day to form a firm gel. As shown in the following test examples, the colloidal silk fibroin of the present invention has an unexpectedly excellent moisturizing action, LDL-cholesterol lowering action which causes arteriosclerosis,
It is a multifunctional substance having various activities useful in a wide range of fields such as bacteriostatic action and ultraviolet absorbing action, and is a substance different from conventional water-soluble fibroin.
【０００８】従って、本発明はまた、コロイド状絹フィ
ブロインを含有する組成物を提供するものである。その
ような組成物の例として、保湿及び／又は紫外線吸収作
用を利用した化粧品、ＬＤＬ−コレステロール低下作用
に基き血管系疾患の治療及び／又は予防に有用な医薬組
成物、静菌作用を利用した医薬部外品などが挙げられ
る。さらに、静菌作用を利用し、食品への添加剤として
も有用である。本発明のコロイド状絹フィブロインを含
有する組成物は、コロイド溶液又は凍結乾燥品の形のフ
ィブロインと、当該技術分野で通常用いられる担体、賦
形剤、希釈剤、基剤等を用い、常法に従って製造され
る、経口又は非経口投与のための医薬、食品、化粧品、
医薬部外品などであってよい。例えば、錠剤、粉末、カ
プセル剤、ペレットなどの固体、懸濁液等の液体、クリ
ーム状、又はスプレー等、任意の形をとることができ
る。Accordingly, the present invention also provides a composition containing colloidal silk fibroin. As examples of such compositions, cosmetics utilizing moisturizing and / or ultraviolet absorbing action, pharmaceutical compositions useful for treatment and / or prevention of vascular diseases based on LDL-cholesterol lowering action, and bacteriostatic action were used. Examples include quasi drugs. Furthermore, it is also useful as an additive to foods by utilizing the bacteriostatic effect. The composition containing the colloidal silk fibroin of the present invention comprises a fibroin in the form of a colloidal solution or a lyophilized product, and a carrier, an excipient, a diluent, a base and the like usually used in the art, and a conventional method. Pharmaceuticals, foods, cosmetics for oral or parenteral administration manufactured according to
It may be a quasi drug or the like. For example, it can take any form such as a solid such as tablet, powder, capsule, pellet, liquid such as suspension, cream, or spray.
【０００９】医薬組成物として用いる場合、本発明のコ
ロイド状絹フィブロインの１日あたりの投与量は経口投
与に際しては、通常、約１００〜５０００ｍｇ、好まし
くは約５００〜２０００ｍｇ、非経口投与に際しては約
１０〜５００ｍｇ、好ましくは約５０〜２００ｍｇの範
囲とすることができる。また、食品に添加する場合、食
品５０〜２００ｇ中に約５〜２５０ｍｇ、好ましくは約
２５〜１００ｍｇを添加すればよい。更に、ファンデー
シヨン、リンス、シャンプ等の化粧品へ添加する場合、
現在使用されている水溶性フィブロインと同様に用いる
ことができ、医薬部外品への添加量もこれらの使用量に
準じて適宜決定される。しかしながら、コロイド状フィ
ブロインの投与量又は配合量は、患者の状態、体重又は
年齢、或いは製品の形態又は使用方法等の様々な条件を
考慮して調節されねばならず、上記の数値に限定される
ものではない。When used as a pharmaceutical composition, the daily dose of the colloidal silk fibroin of the present invention is usually about 100 to 5000 mg, preferably about 500 to 2000 mg for oral administration, and about 0.1 for parenteral administration. It can range from 10 to 500 mg, preferably about 50 to 200 mg. When it is added to foods, about 5-250 mg, preferably about 25-100 mg, may be added to 50-200 g of foods. Furthermore, when added to cosmetics such as foundation, rinse, shampoo,
It can be used in the same manner as the water-soluble fibroin currently used, and the amount added to quasi drugs is also appropriately determined according to the amount used. However, the dosage or amount of colloidal fibroin must be adjusted in consideration of various conditions such as the condition of the patient, weight or age, or form of the product or method of use, and is limited to the above numerical values. Not a thing.
【００１０】[0010]
【実施例】以下に実施例を挙げて本発明を説明するが、
これらは本発明を制限するものではない。実施例１ 絹５ｇを塩化カルシウム４０ｇ、水４０ｍｌ及びエタノ
ール１０ｍｌに温度９５℃で５分間加温して溶解する。
得られた溶液を、３日間流水（流速１００ｍｌ／分）に
て透析し、コロイド状絹フィブロイン溶液約５０ｍｌを
得る。これを凍結乾燥すると白色の極めて微細な粉末約
５ｇが得られた。The present invention will be described below with reference to examples.
These do not limit the invention. Example 1 5 g of silk is dissolved in 40 g of calcium chloride, 40 ml of water and 10 ml of ethanol by heating at a temperature of 95 ° C. for 5 minutes.
The obtained solution is dialyzed against running water (flow rate 100 ml / min) for 3 days to obtain about 50 ml of colloidal silk fibroin solution. This was freeze-dried to obtain about 5 g of a white extremely fine powder.
【００１１】実施例２ 絹７．５ｇを臭化リチウム４０ｇ、水４０ｍｌ及びエタ
ノール１０ｍｌに温度９５℃で５分間加温して溶解す
る。得られた溶液を実施例１と同様に処理してコロイド
状絹フィブロイン溶液を得、凍結乾燥して白色粉末７．
５ｇを得た。 Example 2 7.5 g of silk is dissolved in 40 g of lithium bromide, 40 ml of water and 10 ml of ethanol by heating at a temperature of 95 ° C. for 5 minutes. The obtained solution was treated in the same manner as in Example 1 to obtain a colloidal silk fibroin solution, which was freeze-dried to give a white powder.
5 g was obtained.
【００１２】実施例３ 絹５ｇを塩化カルシウム４０ｇ、水４０ｍｌ及びメタノ
ール１０ｍｌに温度２５℃で７２時間加温して溶解す
る。得られた溶液を実施例１と同様に処理してコロイド
状絹フィブロイン溶液を得、凍結乾燥して白色粉末約５
ｇを得た。 Example 3 5 g of silk is dissolved in 40 g of calcium chloride, 40 ml of water and 10 ml of methanol by heating at a temperature of 25 ° C. for 72 hours. The obtained solution was treated in the same manner as in Example 1 to obtain a colloidal silk fibroin solution, which was freeze-dried to give a white powder of about 5
g was obtained.
【００１３】実施例４ 保湿・紫外線吸収作用をもつ化
粧用クリームの調製 実施例１で得られたコロイド状絹フィブロインを含有す
る化粧用クリームを、以下の成分から調製する。 凍結乾燥粉末 １．０ｇ ステアリルアルコール ６．０ｍｌ ステアリン酸 １．０ｍｌ 水添ラノリン ５．０ｍｌ オクチルドデシルアルコール １０．０ｍｌ ポリオキシエチレン（２０モル）モノセチル アルコールエーテル ４．０ｍｌ グリセリンモノステアリン酸エステル ２．０ｍｌ プロピレングリコール ５．０ｍｌ 精製水 ６６．０ｍｌ 香料 適量 上記成分を良く混合し、適当な容器に充填する。 Example 4 Preparation of Cosmetic Cream Having Moisturizing / Ultraviolet Absorbing Effect A cosmetic cream containing the colloidal silk fibroin obtained in Example 1 is prepared from the following ingredients. Lyophilized powder 1.0 g Stearyl alcohol 6.0 ml Stearic acid 1.0 ml Hydrogenated lanolin 5.0 ml Octyldodecyl alcohol 10.0 ml Polyoxyethylene (20 mol) monocetyl alcohol ether 4.0 ml Glycerine monostearate 2.0 ml Propylene Glycol 5.0 ml Purified water 66.0 ml Perfume Suitable amount The above ingredients are mixed well and filled in a suitable container.
【００１４】実施例５ 錠剤 成 分 重 量 （ｍｇ／錠） 実施例１で得たコロイド状絹フィブロイン末 １８６ ポリビニルピロリドン １３．５ ポリソルベート８０ ４．５ 乳糖無水物 ２６０ 架橋結合したポリビニルピロリドン ３０ ステアリン酸 ４．５ ステアリン酸マグネシウム １．５ コロイド状絹フィブロイン、乳糖、及び架橋結合ポリビ
ニルピロリドンの一部をポリビニルピロリドン及びポリ
ソルベート８０の水溶液で造粒する。顆粒を乾燥し、適
当な大きさに減じ、ステアリン酸、ステアリン酸マグネ
シウム、及び残りの架橋結合ポリビニルピロリドンと混
合する。混合物をそれぞれ５００ｍｇの錠剤に打錠す
る。 Example 5 Tablet Composition Weight ( mg / tablet) Colloidal silk fibroin powder obtained in Example 1 186 Polyvinylpyrrolidone 13.5 Polysorbate 80 4.5 Lactose anhydride 260 Crosslinked polyvinylpyrrolidone 30 Stearin Acid 4.5 Magnesium stearate 1.5 Colloidal silk fibroin, lactose, and part of the crosslinked polyvinylpyrrolidone are granulated with an aqueous solution of polyvinylpyrrolidone and polysorbate 80. The granules are dried, reduced to size and mixed with stearic acid, magnesium stearate, and the rest of the crosslinked polyvinylpyrrolidone. The mixture is compressed into tablets of 500 mg each.
【００１５】実施例６ カプセル剤 成 分 重量（ｍｇ／カプセル） 実施例１で得たコロイド状絹フィブロイン末 １７０ 乳糖、含水スプレー乾燥 ２３０ ラウリル硫酸ナトリウム ８ 架橋結合したポリビニルピロリドン １５ ヒドロキシプロピルメチルセルロース ９ コロイド状二酸化ケイ素 ３ コロイド状絹フィブロイン、スプレー乾燥した含水乳糖
及び架橋結合ポリビニルピロリドンの混合物をラウリル
硫酸ナトリウム及びヒドロキシプロピルメチルセルロー
スの水溶液で造粒する。顆粒を乾燥し、適当な大きさに
減じ、コロイド状二酸化ケイ素と混合する。次いで、こ
の混合物を通常の封入機を用いて、サイズ３のゼラチン
硬カプセル充填し、それぞれ、最終混合物４３５ｍｇを
含有するゼラチン硬カプセル剤を得る。[0015] Example 6 Capsules Ingredient Weight (mg / capsule) Example 1 obtained in colloidal silk fibroin powder 170 Lactose, polyvinylpyrrolidone 15 hydroxypropylmethylcellulose 9 colloidally and hydrous spray-dried 230 sodium lauryl sulfate 8 crosslinks A mixture of silicon dioxide 3 colloidal silk fibroin, spray dried hydrous lactose and cross-linked polyvinylpyrrolidone is granulated with an aqueous solution of sodium lauryl sulphate and hydroxypropyl methylcellulose. The granules are dried, reduced to size and mixed with colloidal silicon dioxide. This mixture is then filled into size 3 gelatin hard capsules using a conventional encapsulation machine to give gelatin hard capsules, each containing 435 mg of the final mixture.
【００１６】実施例で得たコロイド状絹フィブロインの
ＬＤＬ−コレステロール低下作用、保湿作用、紫外線吸
収作用及び防黴作用について試験した。試験例１ ＬＤＬ−コレステロール低下作用 凍結乾燥したコロイド状絹フィブロインのＬＤＬ−コレ
ステロール低下作用を動物実験により評価した。あらか
じめコレステロールを多く含む飼料で７日間飼育した血
中コレステロールの高いラット８尾（総コレステロール
１８０ｍｇ／ｄｌ）を、実施例で得たコロイド状絹フィ
ブロイン末を５％含む栄養評価用の飼料（ＡＩＮ７６、
東京実験動物株式会社）により７日間飼育した。次い
で、各ラットの血中総コレステロール、ＨＤＬ−及びＬ
ＤＬ−コレステロールを側定し、統計処理して平均値と
誤差を求めた。対照として、栄養評価用の飼料のみで飼
育したラットについても同様に処理した。結果を表１に
示す。The colloidal silk fibroin obtained in the examples was tested for LDL-cholesterol lowering action, moisturizing action, ultraviolet absorbing action and antifungal action. Test Example 1 LDL-cholesterol lowering action The LDL-cholesterol lowering action of freeze-dried colloidal silk fibroin was evaluated by an animal experiment. Eight rats with high blood cholesterol (total cholesterol 180 mg / dl), which had been previously bred for 7 days with a diet rich in cholesterol, were fed with a diet for nutritional evaluation (AIN76, containing 5% of the colloidal silk fibroin powder obtained in Example).
The animals were bred for 7 days by Tokyo Experimental Animal Co., Ltd. Then, total blood cholesterol, HDL- and L of each rat
DL-cholesterol was measured and statistically processed to obtain an average value and an error. As a control, the rats fed only the diet for nutritional evaluation were treated in the same manner. The results are shown in Table 1.
【表１】 表から、実施例で得たコロイド状絹フィブロイン末は動
脈硬化の原因となるＬＤＬ−コレステロールを顕著に低
下することが明らかである。[Table 1] From the table, it is clear that the colloidal silk fibroin powder obtained in the Examples markedly reduces LDL-cholesterol which causes arteriosclerosis.
【００１７】試験例２ 保湿作用 実施例１で得たフィブロインのコロイド溶液の保湿作用
を文献（手塚ら；西日本皮膚科雑誌８０２頁 昭和５０
年）記載の方法で試験した。フィブロインのコロイド溶
液（０．５、１．０、２．０％溶液）と従来の方法で得
られるフィブロイン水溶液（０．５、１．０、２．０％
溶液）の保湿作用を比較した。新鮮な牛の鼻及びひずめ
を横浜食肉衛生検査所より入手し、皮下組織をできるだ
け除去した後、直ちに約１ｃｍ２の大きさに切り、凍結
切片法にて１００μの厚さにスライスした。次いで、上
記の各濃度の検体溶液に室温で１８時間浸漬した後、２
５゜Ｃ、相対湿度７９％のデシケータ中に７２時間放置
し、切片の保水量（ｍｇ／１００ｍｇ）を求めた。結果
を表２に示す。 Test Example 2 Moisturizing Effect The moisturizing effect of the colloidal solution of fibroin obtained in Example 1 was described in the literature (Tezuka et al .; West Japan Dermatological Magazine, page 802, 1975).
(Year) was tested by the method described. Fibroin colloidal solution (0.5, 1.0, 2.0% solution) and fibroin aqueous solution obtained by conventional method (0.5, 1.0, 2.0%)
The moisturizing effects of the solutions) were compared. Fresh beef nose and hoof were obtained from Yokohama Meat Inspection Institute, and after removing subcutaneous tissue as much as possible, they were immediately cut into a size of about 1 cm 2 and sliced into a thickness of 100 μ by a frozen section method. Then, after soaking in the sample solution of each concentration described above at room temperature for 18 hours, 2
The piece was left in a desiccator at 5 ° C and a relative humidity of 79% for 72 hours, and the water retention amount (mg / 100 mg) of the section was determined. Table 2 shows the results.
【表２】 表から、本願発明のコロイド状絹フィブロインは既存の
保湿剤である水溶性フィブロインを凌駕する保湿効果を
有することが明らかである。[Table 2] From the table, it is clear that the colloidal silk fibroin of the present invention has a moisturizing effect that surpasses the existing moisturizing agent, water-soluble fibroin.
【００１８】試験例３ 静菌作用 米軍規格のＭＩＬ−Ｅ−５２７２Ｃに定められている以
下の４群： Ａ：Ｍｙｒｏｔｈｅｃｉｕｍ ｖｅｒｒｕｃａｒｉａ； Ｂ：Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ； Ｃ：Ａｓｐｅｒｇｉｌｌｕｓ ｔｅｒｒｅｕｓ；及び Ｄ：Ｐｅｎｉｃｉｌｌｉｕｍ ｃｉｔｒｉｎｕｍ の試験菌に対する静菌効果を調べた。それぞれの菌種を
約１０ｍｌの蒸留水（ｐＨ６．０）を用いて胞子分散液
を調製し、撹拌して胞子を均一に分散させた後、ポテト
・デキストロース寒天培地に実施例で得たコロイド状絹
フィブロイン末を１％又は１０％添加した培地に噴霧
し、２８℃、湿度約９５％の条件下、２８日間培養し、
菌の増殖状況を評価した。対照として、無添加培地を用
いて同様に処理した。結果を表３に示す。 Test Example 3 Bacteriostatic Action The following four groups defined in US military standard MIL-E-5272C: A: Myrothecium verrucaria; B: Aspergillus terreus; C: Aspergillus terreus; and D: Penicillium citrinum. The bacteriostatic effect on the bacteria was investigated. A spore dispersion was prepared by using about 10 ml of distilled water (pH 6.0) for each bacterial strain, and the spores were uniformly dispersed by stirring, and then the spores were dispersed on a potato-dextrose agar medium in the colloidal form obtained in Example. The silk fibroin powder was sprayed on a medium containing 1% or 10%, and cultured for 28 days at 28 ° C. and a humidity of about 95%.
The growth status of the bacteria was evaluated. As a control, the same treatment was performed using a non-supplemented medium. The results are shown in Table 3.
【表３】 表中、＋は微生物繁殖、−は微生物繁殖抑制を表す。表
から、本発明のコロイド状絹フィブロインは、各種の菌
株の増殖を抑制し、静菌作用を有することが明らかであ
る[Table 3] In the table, + represents microbial growth, and-represents microbial growth inhibition. From the table, it is clear that the colloidal silk fibroin of the present invention suppresses the growth of various strains and has a bacteriostatic effect.
【００１９】試験例４ 紫外線吸収作用 実施例５で調製したクリーム約０．５ｇを２名の健常人
の右上腕部約３ｃｍ２に塗布した。塗布面を１ｃｍ２の
円形の穴を開けた黒色の紙で覆い、紫外線殺菌灯（ナシ
ョナル１５Ｗ）を距離３０ｃｍで１０分間照射した。ク
リームを塗布しない場合、２名とも照射部の皮膚に発赤
が生じたが、クリーム塗布の場合には皮膚に変化が認め
られなかった。これは、コロイド状絹フィブロイン自身
の紫外線吸収能と、コロイド粒子による紫外線の遮断に
よると考えられる。また、この試験で両名にアレルギー
反応などの副作用は認められなかった。 Test Example 4 Ultraviolet Absorption Action About 0.5 g of the cream prepared in Example 5 was applied to about 3 cm 2 of the upper right arm of two healthy persons. The coated surface was covered with black paper having a circular hole of 1 cm 2 and was irradiated with an ultraviolet germicidal lamp (National 15W) at a distance of 30 cm for 10 minutes. When the cream was not applied, redness occurred on the skin of the irradiated part in both cases, but no change was observed in the skin when the cream was applied. It is considered that this is due to the UV absorbing ability of the colloidal silk fibroin itself and the blocking of UV rays by the colloidal particles. In addition, no side effects such as allergic reaction were observed in both subjects in this test.
【００２０】[0020]
【発明の効果】本発明のコロイド状絹フィブロインは、
優れた保湿作用、ＬＤＬ−コレステロール低下作用、静
菌作用、紫外線吸収作用等、極めて広範な分野で有用な
様々な活性を有しており、医学、美容、衛生及び食品な
どの主成分あるいは添加剤として有用である。また、本
発明方法によれば、上記の多機能性蛋白質を容易に製造
することができ、絹の有効利用に貢献できる。The colloidal silk fibroin of the present invention is
It has various activities useful in a very wide range of fields such as excellent moisturizing action, LDL-cholesterol lowering action, bacteriostatic action, ultraviolet absorbing action, etc., and is a main component or additive for medicine, beauty, hygiene and food. Is useful as Further, according to the method of the present invention, the above-mentioned multifunctional protein can be easily produced, which can contribute to the effective utilization of silk.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ａ６１Ｋ 7/48 8517−4Ｈ Ｃ０７Ｋ 1/14 Ｃ０７Ｋ 1/14 8517−4Ｈ 14/435 14/435 Ｃ０９Ｋ 3/00 １０４Ｚ Ｃ０９Ｋ 3/00 １０４ Ａ６１Ｋ 37/12 ＡＤＢ ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Internal reference number FI Technical display location A61K 7/48 8517-4H C07K 1/14 C07K 1/14 8517-4H 14/435 14/435 C09K 3/00 104Z C09K 3/00 104 A61K 37/12 ADB
Claims (10)
ル構造を有することを特徴とするコロイド状絹フィブロ
イン。1. A colloidal silk fibroin comprising a silk fibroin protein and having a fine-grained gel structure.
イド状絹フィブロイン： １）ゲル濾過法により側定した場合、分子量は約３５〜
３７万であり、 ２）粒子径約０．１〜０．２μｍの球形であり、 ３）溶液はコロイド溶液特性の強いチンダル現象を示
し、 ４）２００〜３００ｎｍの範囲に紫外部吸収を有し、最
大吸収は２８０ｎｍにあり、 ５）等電点はｐＨ３．９である。2. The colloidal silk fibroin according to claim 1, which has the above characteristics: 1) When determined by gel filtration, the molecular weight is about 35-35.
370,000, 2) spherical with a particle size of about 0.1 to 0.2 μm, 3) the solution exhibits a strong Tyndall phenomenon with colloidal solution characteristics, and 4) has an ultraviolet absorption in the range of 200 to 300 nm. , The maximum absorption is at 280 nm, and 5) the isoelectric point is pH 3.9.
和性の有機溶剤との混合溶液に溶解し、得られた溶液を
水に対して透析することからなる請求項１記載のコロイ
ド状絹フィブロインの製造方法。3. The colloid according to claim 1, which comprises dissolving silk in a mixed solution of an aqueous solution of a water-soluble inorganic salt and a water-miscible organic solvent, and dialyzing the resulting solution against water. Of producing silk fibroin in the shape of a filament.
アルカリ金属又はアルカリ土類金属、Ｘはハロゲン、硫
酸根又は硝酸根を表す〕で示される化合物である請求項
３記載の方法。4. The method according to claim 3, wherein the inorganic salt is a compound represented by the formula: MeX (wherein Me represents an alkali metal or an alkaline earth metal, and X represents a halogen, a sulfate group or a nitrate group). .
トン、ジオキサン、ピリジンから選択される溶剤である
請求項３記載の方法。5. The method according to claim 3, wherein the water-miscible organic solvent is a solvent selected from alcohol, acetone, dioxane and pyridine.
ィブロインを含有する組成物。6. A composition containing the colloidal silk fibroin according to claim 1 or 2.
る請求項６記載の組成物。7. The composition according to claim 6, which has an LDL-cholesterol lowering action.
物。8. The composition according to claim 6, which has a moisturizing effect.
組成物。9. The composition according to claim 6, which has an ultraviolet absorbing effect.
物。10. The composition according to claim 6, which has a bacteriostatic effect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7112255A JPH08268905A (en) | 1995-03-31 | 1995-03-31 | Colloidal silk fibroin and composition containing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7112255A JPH08268905A (en) | 1995-03-31 | 1995-03-31 | Colloidal silk fibroin and composition containing the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH08268905A true JPH08268905A (en) | 1996-10-15 |
Family
ID=14582133
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7112255A Pending JPH08268905A (en) | 1995-03-31 | 1995-03-31 | Colloidal silk fibroin and composition containing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH08268905A (en) |
Cited By (17)
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|---|---|---|---|---|
| WO2000037581A1 (en) * | 1998-12-22 | 2000-06-29 | Marine Bio Co., Ltd. | Ultraviolet shielding agents |
| US6218357B1 (en) * | 1996-08-15 | 2001-04-17 | Seiji Terauchi | Fibroin fluid and process for the production thereof |
| WO2001042300A1 (en) * | 1999-12-09 | 2001-06-14 | National Institute Of Agrobiological Sciences | Process for producing functional silk fibroin and utilization of the same |
| JP2001278757A (en) * | 2000-03-30 | 2001-10-10 | Kanebo Ltd | Dentifrice composition |
| WO2002034885A1 (en) * | 2000-10-24 | 2002-05-02 | National Institute Of Agrobiological Sciences | Sericin-containing material, process for producing the same and method of using the same |
| EP1201245A1 (en) * | 1999-03-04 | 2002-05-02 | Seiren Co., Ltd. | Functional oral preparations |
| KR20020049165A (en) * | 2000-12-19 | 2002-06-26 | 히로시 오제키 | Composition having bactericidal action, and cosmetic material and uv blocking material comprising the composition |
| JP2002302499A (en) * | 2001-04-04 | 2002-10-18 | Kanebo Ltd | Granular silk fibroin and method for producing the same |
| KR100365291B1 (en) * | 2000-06-07 | 2002-12-18 | 주식회사 바이오썸 | An agent for preventing development of alcoholic fatty liver comprising an active substance derived from silk fibroin |
| WO2003045336A1 (en) * | 2001-11-29 | 2003-06-05 | National Institute Of Agrobiological Sciences | Emulsifier and process for producing the same |
| JP2005510268A (en) * | 2001-10-25 | 2005-04-21 | ユニヴァーシティー オブ コネティカット | Bioactive material, method for producing bioactive material and method of use thereof |
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- 1995-03-31 JP JP7112255A patent/JPH08268905A/en active Pending
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| US6218357B1 (en) * | 1996-08-15 | 2001-04-17 | Seiji Terauchi | Fibroin fluid and process for the production thereof |
| WO2000037581A1 (en) * | 1998-12-22 | 2000-06-29 | Marine Bio Co., Ltd. | Ultraviolet shielding agents |
| EP1201245A1 (en) * | 1999-03-04 | 2002-05-02 | Seiren Co., Ltd. | Functional oral preparations |
| EP1241178A4 (en) * | 1999-12-09 | 2004-09-08 | Nat Inst Of Agrobio Sciences | Process for producing functional silk fibroin and utilization of the same |
| WO2001042300A1 (en) * | 1999-12-09 | 2001-06-14 | National Institute Of Agrobiological Sciences | Process for producing functional silk fibroin and utilization of the same |
| JP2001278757A (en) * | 2000-03-30 | 2001-10-10 | Kanebo Ltd | Dentifrice composition |
| KR100365291B1 (en) * | 2000-06-07 | 2002-12-18 | 주식회사 바이오썸 | An agent for preventing development of alcoholic fatty liver comprising an active substance derived from silk fibroin |
| US6815427B2 (en) | 2000-10-24 | 2004-11-09 | National Institute Of Agrobiological Sciences | Sericin-containing material, process for producing the same method of using the same |
| WO2002034885A1 (en) * | 2000-10-24 | 2002-05-02 | National Institute Of Agrobiological Sciences | Sericin-containing material, process for producing the same and method of using the same |
| KR20020049165A (en) * | 2000-12-19 | 2002-06-26 | 히로시 오제키 | Composition having bactericidal action, and cosmetic material and uv blocking material comprising the composition |
| JP2002302499A (en) * | 2001-04-04 | 2002-10-18 | Kanebo Ltd | Granular silk fibroin and method for producing the same |
| JP2005510268A (en) * | 2001-10-25 | 2005-04-21 | ユニヴァーシティー オブ コネティカット | Bioactive material, method for producing bioactive material and method of use thereof |
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| EP1459728A4 (en) * | 2001-11-29 | 2005-04-20 | Nat Inst Of Agrobio Sciences | Emulsifier and process for producing the same |
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| JP2010100656A (en) * | 2002-06-25 | 2010-05-06 | Shiseido Co Ltd | Antiaging preparation |
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| WO2007082923A2 (en) * | 2006-01-20 | 2007-07-26 | Basf Se | Use of protein microbeads in cosmetics |
| WO2007082923A3 (en) * | 2006-01-20 | 2007-10-04 | Basf Ag | Use of protein microbeads in cosmetics |
| KR100847299B1 (en) * | 2007-11-29 | 2008-07-18 | 주식회사 펩트론 | Fraction of silk peptide with an excellent moisturizing effect and use thereof |
| JP2016069334A (en) * | 2014-09-30 | 2016-05-09 | 株式会社ダリヤ | Hair-growing cosmetics |
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