JPH11147834A - Serine protease inhibitor - Google Patents
Serine protease inhibitorInfo
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- JPH11147834A JPH11147834A JP9335108A JP33510897A JPH11147834A JP H11147834 A JPH11147834 A JP H11147834A JP 9335108 A JP9335108 A JP 9335108A JP 33510897 A JP33510897 A JP 33510897A JP H11147834 A JPH11147834 A JP H11147834A
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- Prior art keywords
- morus
- solvent
- extract
- plants
- plant
- Prior art date
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、セージ(Salvia of
ficinalis L.)及びクワ(Morus)属から選択される１種又
は２種以上の植物の抽出物を有効成分とするセリンプロ
テアーゼ阻害剤に関し、更に詳しくは、膵炎治療剤，急
性動脈炎，肺気腫，動脈硬化，関節リュウマチ，癌の転
移・浸潤等の治療剤として、また、皮膚のはり・弾力を
回復維持することで皮膚の老化を防止し、若々しい肌の
状態を維持する効果が期待されるセリンプロテアーゼ阻
害剤、及びセリンプロテアーゼの一種であるエラスター
ゼ，トリプシン，キモトリプシン，プラスミン阻害剤に
関する。TECHNICAL FIELD The present invention relates to a sage ( Salvia of Salvia).
ficinalis L.) and a serine protease inhibitor comprising an extract of one or more plants selected from the genus Morus as an active ingredient, and more specifically, a therapeutic agent for pancreatitis, acute arteritis, emphysema, It is expected to be effective as a therapeutic agent for arteriosclerosis, rheumatoid arthritis, metastasis and invasion of cancer, and to prevent skin aging by restoring and maintaining skin elasticity and elasticity, and to maintain a youthful skin condition. The present invention relates to a serine protease inhibitor and an inhibitor of elastase, trypsin, chymotrypsin and plasmin, which are a kind of serine protease.
【０００２】[0002]
【従来の技術】生体内には、トリプシン，キモトリプシ
ン，トロンビン，プラスミン，エラスターゼ等種々のセ
リンプロテアーゼが存在し、それらの酵素が何らかの要
因により異常に活性化されると、炎症，疼痛，アレルギ
ー，血液異常，組織破壊などの疾患を引き起こすと考え
られている。例えば、急性膵炎或いは慢性膵炎の急性病
変期には、種々の要因により活性化した膵蛋白分解酵素
による膵臓組織の破壊と、活性化した酵素の血中への逸
脱による全身の組織、臓器障害など重篤な症状が発現す
ることが知られている。その原因として膵セリンプロテ
アーゼ、特にキーエンザイムとしてトリプシンの関与が
重視されており、近年上記の状態を改善する治療薬とし
て幾つかの合成抗トリプシン剤が開発上市されている。
また、紫外線曝露や加齢、種々の炎症刺激などにより、
エラスチン破壊酵素であるエラスターゼが過剰発現する
ことによって、エラスチンが変性・破壊されることが、
皮膚の弾力性低下につながると考えられており、エラス
ターゼの働きを抑て、皮膚に弾力やハリを与えるエラス
チンの変性・破壊を防止することが皮膚の老化防止に重
要である。更に、好中球エラスターゼ阻害剤は、急性動
脈炎，肺気腫，動脈硬化，関節リュウマチ，癌の転移・
浸潤などの治療薬としての使用が期待されている。2. Description of the Related Art Various serine proteases such as trypsin, chymotrypsin, thrombin, plasmin, and elastase are present in a living body, and when these enzymes are abnormally activated by some factor, inflammation, pain, allergy, blood, etc. It is thought to cause diseases such as abnormalities and tissue destruction. For example, during acute pancreatitis or the acute lesion stage of chronic pancreatitis, destruction of pancreatic tissue by pancreatic protease activated by various factors, and systemic tissue and organ damage due to escape of the activated enzyme into the blood. It is known that severe symptoms develop. The involvement of trypsin as a pancreatic serine protease, particularly as a key enzyme, has been emphasized as the cause, and in recent years, several synthetic antitrypsin agents have been developed and marketed as therapeutic agents for improving the above-mentioned conditions.
In addition, due to UV exposure and aging, various inflammatory stimuli,
Due to overexpression of elastin, an elastin-disrupting enzyme, elastin is denatured and destroyed.
It is thought to lead to a decrease in the elasticity of the skin, and it is important to prevent elastin from degenerating and destroying, which gives the skin elasticity and firmness, by suppressing the action of elastase. In addition, neutrophil elastase inhibitors are used for acute arteritis, emphysema, arteriosclerosis, rheumatoid arthritis, metastasis of cancer,
It is expected to be used as a therapeutic agent for invasion and the like.
【０００３】かかるセリンプロテアーゼ阻害剤として
は、数多く知られているが、その大部分は活性中心のセ
リンの水酸基と不可逆的に結合して失活させる不可逆的
阻害剤であり、酵素を再生させることが出来ないため副
作用が懸念される。一方可逆的な阻害剤としては基質類
似のアルデヒド，ケトン，ボロニックアシッド等が知ら
れている。[0003] Many such serine protease inhibitors are known, but most of them are irreversible inhibitors that irreversibly bind to and deactivate the hydroxyl group of serine in the active center. Is not possible, so there are concerns about side effects. On the other hand, as reversible inhibitors, aldehydes, ketones, boronic acids and the like similar to substrates are known.
【０００４】しかしながら、これまでのセリンプロテア
ーゼ阻害剤の多くは、可逆的なものであっても、その体
内動態，副作用などの問題が解決されていなかったり、
作用効果が不十分であったり、安定性が悪かったりし
て、有効な効果を得るにはかなりの量を含有させなけれ
ばならないものも存在していた。[0004] However, many of the conventional serine protease inhibitors are reversible, but their pharmacokinetics and side effects have not been solved.
In some cases, the action and effect were insufficient or the stability was poor, and a considerable amount had to be contained in order to obtain an effective effect.
【０００５】[0005]
【発明が解決しようとする課題】そこで、本発明におい
ては、連用しても副作用などの問題がなく、高い安全性
を有するセリンプロテアーゼ阻害剤を提供することを目
的とした。SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a serine protease inhibitor which is free from problems such as side effects even when used continuously and has high safety.
【０００６】[0006]
【課題を解決するための手段】本発明者等は広く種々の
天然物についてセリンプロテアーゼ阻害作用を調べた結
果、セージ(Salvia officinalis L.)及びクワ属(Morus)
植物の抽出物が、優れたセリンプロテアーゼ阻害作用を
有し、しかも内服，外用にかかわらず、副作用の問題が
なく、安全性が高いことを見いだし、本発明を完成する
に至った。DISCLOSURE OF THE INVENTION The present inventors have studied the serine protease inhibitory activity of a wide variety of natural products and found that sage ( Salvia officinalis L.) and mulberry ( Morus )
The present inventors have found that a plant extract has an excellent serine protease inhibitory action, has no side effects regardless of whether it is taken internally or is topically used, and has high safety, and has completed the present invention.
【０００７】セージ(Salvia officinalis L.)は、シソ
科(Labiatae)アキギリ属(Salvia)の植物の一種で、高さ
３０〜７０ｃｍ，全草に白い軟毛が密生する常緑多年草
であり、ヨーロッパでは、古くから香草及び民間薬とし
て利用されてきた。このセージ(Salvia officinalis
L.)の抽出物の生理作用に関しては、抗菌作用（特開平
７−２６７８７３号公報，特開平８−１１９８７２号公
報等）、抗酸化作用（特開平３−９９８４号公報）、皮
膚過酸化脂質生成抑制作用（特開昭６１−２４５２２号
公報）、抗炎症作用（特開平１−８３０２２号公報，特
開昭６０−１５６６１８号公報等）、ヒアルロニダーゼ
阻害作用（特開平１−１２８９３３号公報）、微生物由
来のプロテアーゼ阻害作用（特開平１−１２８９３４号
公報）等が開示されている。しかしながら、セージ抽出
物が、セリンプロテアーゼ、特にエラスターゼ，トリプ
シン，キモトリプシン及びプラスミンに対し、高い阻害
活性を示すことはこれまで知られていなかった。[0007] sage (Salvia officinalis L.) is a kind of plant of the mint family (Labiatae) salvia (Salvia), a height 30~70cm, evergreen perennial white fluff in all the grass is dense, in Europe, It has been used as a herb and folk medicine since ancient times. This sage ( Salvia officinalis
L.) extract, the antibacterial action (JP-A-7-267873, JP-A-8-119872, etc.), the antioxidant action (JP-A-3-9984), skin lipid peroxide Production inhibitory action (JP-A-61-24522), anti-inflammatory action (JP-A-1-83022, JP-A-60-156618, etc.), hyaluronidase inhibitory action (JP-A-1-128933), A protease inhibitory action derived from microorganisms (Japanese Patent Laid-Open No. 1-128934) is disclosed. However, it has not been known until now that sage extracts exhibit high inhibitory activity against serine proteases, especially elastase, trypsin, chymotrypsin and plasmin.
【０００８】クワ属(Morus)属は、クワ科(Moraceae)植
物の一種で、果実を食用にすること、また葉を蚕の飼料
にするために数種が広く栽培されている。また、クワ属
植物の根皮を桑白皮、葉を桑葉、果実を桑たいと呼び、
それぞれ生薬として利用されてきた。例えば、桑白皮
は、消炎性利尿剤，緩下剤として利用されてきており、
その抽出物に対しては、チロシナーゼ阻害作用（特開昭
５０−１３５２３６号公報他），微生物由来のプロテア
ーゼ阻害作用（特開平６−２５０００号公報），抗菌作
用（特開平８−１５１３２５号公報他）などが開示され
ている。桑葉はペントサン，ガラクタン，グルコース，
カロチン，タンニンなどを含み、中国で駆風薬として、
日本では民間で桑茶として利用されており、桑葉抽出物
の抗酸化作用（特開昭６０−４２４８５号公報），活性
酸素消去作用（特開平８−１４３４６６号公報）等が開
示されている。さらに、桑たい中には、有機酸，粘液
質，色素，糖分などを含有し、利尿，鎮咳に効果がある
といわれ、生食したり、醸酒用にも利用される。しかし
ながら、クワ属植物が、セリンプロテアーゼ、特にエラ
スターゼ，トリプシン，キモトリプシン及びプラスミン
に対し、高い阻害活性を示すことはこれまで知られてい
なかった。The genus Morus is a kind of plant of the mulberry family (Moraceae), and several species are widely cultivated in order to feed fruits and feed on leaves of silkworms. Also, the root bark of the mulberry plant is called mulberry bark, the leaves are mulberry leaves, and the fruit is mulberry,
Each has been used as a crude drug. For example, mulberry bark has been used as an anti-inflammatory diuretic and laxative,
The extract has a tyrosinase inhibitory action (JP-A-50-135236 and others), a microorganism-derived protease inhibitory action (JP-A-6-25000), and an antibacterial action (JP-A-8-151325 and others). ) Are disclosed. Mulberry leaves are pentosan, galactan, glucose,
Contains carotene, tannin, etc. as a carminative in China
In Japan, it is used as mulberry tea in the private sector, and the antioxidant action of mulberry leaf extract (JP-A-60-42485), the active oxygen scavenging action (JP-A-8-143466) and the like are disclosed. . Furthermore, mulberry contains organic acids, mucus, pigments, sugars, etc., and is said to have an effect on diuresis and antitussive, and is also used for raw eating and sake brewing. However, it has not been known that mulberry plants exhibit a high inhibitory activity against serine proteases, particularly elastase, trypsin, chymotrypsin and plasmin.
【０００９】[0009]
【００１０】本発明においてセージ(Salvia officinali
s L.)の抽出物を得る際、抽出に供する部位は特に限定
されないが、葉，花及び全草を生のまま若しくは乾燥さ
せて用いることができる。In the present invention, sage ( Salvia officinali) is used.
s L.), the site to be subjected to extraction is not particularly limited, and leaves, flowers, and whole plants can be used as they are or dried.
【００１１】本発明において用いられるクワ(Morus)属
植物としては、クワ（ヤマグワ，ノグワ）(Morus bomby
cis Koidz. , Morus japonica L.H.Bailey non Sieb. ,
Morus alba L.var.stylosa Bur.)、マグワ（カラヤマ
グワ，トウグワ）(Morus albaL. , Morus atropurpure
Roxb.)、ロソウ（ログワ，マルバグワ，モチグワ）(Mor
us multicaulis Perr. , Morus latifolia(Bur.)Poir.
, Morus alba L.var.latifolia Bur. , Morus alba L.
var.multicaulis Loud.)、オガサワラグワ(Morus bonin
ensis Koidz.)、イチベイ(Morus argutidens Koidz.)、
シマグワ(Morusaustralis Poir. , Morus acidosa Grif
f)、ハマグワ(Morus bombycis Koidz. var.maritima Ko
idz.)、ハチジョウグワ(Morus kagayamae Koidz.)、モ
ウコグワ(Morus mongolica(Bur.)Schneid , Morus alba
L.var.mongolica Bur.)、クロミグワ(Morus nigra
L.)、アカミグワ(Morus rubra L.)、ノグワ（ケグワ）
(Morustiliaefolia Makino)等が例示されるが、特に限
定されない。これらのクワ属植物の抽出物を得る際に抽
出に供する部位は特に限定されないが、根皮，樹皮，
葉，実，花から選択される１種又は２種以上の部位の抽
出物を得るのが好ましく、さらには、葉からの抽出物
が、セリンプロテアーゼ活性の点から最も好ましい。The mulberry ( Morus ) genus plant used in the present invention includes mulberry (Yamaguwa, Nogwa) ( Morus bomby ).
cis Koidz., Morus japonica LHBailey non Sieb.,
Morus alba L.var. Stylosa Bur.) , Morus alba (Karayamaguwa, white mulberry) (Morus alba L., Morus atropurpure
Roxb.), Rosou (Logwa, Marbagwa, Mochiwa) ( Mor
us multicaulis Perr., Morus latifolia (Bur.) Poir.
, Morus alba L.var. Latifolia Bur. , Morus alba L.
var. multicaulis Loud.), Ogasawara Harrow (Morus bonin
ensis Koidz.), Ichibei (Morus argutidens Koidz.),
Shimaguwa ( Morusaustralis Poir., Morus acidosa Grif
f), Hamaguwa ( Morus bombycis Koidz.var. maritima Ko
idz.), Hachijogwa ( Morus kagayamae Koidz.), Mokogwa ( Morus mongolica (Bur.) Schneid, Morus alba
L.var. Mongolica Bur.), Kuromiguwa (Morus nigra
L.), Akamigua ( Morus rubra L.), Nogwa (Kegwa)
( Morustiliaefolia Makino) and the like, but are not particularly limited. The site to be subjected to extraction when obtaining the extract of the mulberry plant is not particularly limited.
It is preferable to obtain an extract of one or more sites selected from leaves, fruits, and flowers, and an extract from leaves is most preferable in terms of serine protease activity.
【００１２】本発明において用いられるセージ及びクワ
属植物の抽出物を得る際の抽出溶媒としては、精製水、
エタノール，メタノール，イソプロパノール，イソブタ
ノール，n-ヘキサノール，メチルアミルアルコール，2-
エチルブタノール，n-オクチルアルコールなどのアルコ
ール類、グリセリン，エチレングリコール，エチレング
リコールモノメチルエーテル，エチレングリコールモノ
エチルエーテル，プロピレングリコール，プロピレング
リコールモノメチルエーテル，プロピレングリコールモ
ノエチルエーテル，トリエチレングリコール，1,3-ブチ
レングリコール，ヘキシレングリコール等の多価アルコ
ール又はその誘導体、アセトン，メチルエチルケトン，
メチルイソブチルケトン，メチル-n-プロピルケトンな
どのケトン類、酢酸エチル，酢酸イソプロピルなどのエ
ステル類、エチルエーテル，イソプロピルエーテル，n-
ブチルエーテル等のエーテル類などの極性溶媒から選択
される１種又は２種以上の混合溶媒が好適に使用でき、
また、リン酸緩衝生理食塩水等の無機塩類を添加した溶
媒をも用いることができるが、特に限定はされない。本
発明の目的には、セリンプロテアーゼ阻害作用の点か
ら、極性溶媒が好ましく、さらには、メタノール，エタ
ノール，1,3-ブチレングリコール，プロピレングリコー
ル，精製水から選択される１種又は２種以上の混合溶
媒、特にエタノール水溶液を溶媒とすることが好まし
い。The extraction solvent used for obtaining the extract of the sage and mulberry plants used in the present invention includes purified water,
Ethanol, methanol, isopropanol, isobutanol, n-hexanol, methyl amyl alcohol, 2-
Alcohols such as ethylbutanol and n-octyl alcohol, glycerin, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, propylene glycol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, triethylene glycol, 1,3- Polyhydric alcohols such as butylene glycol and hexylene glycol or derivatives thereof, acetone, methyl ethyl ketone,
Ketones such as methyl isobutyl ketone and methyl-n-propyl ketone, esters such as ethyl acetate and isopropyl acetate, ethyl ether, isopropyl ether, n-
One or more mixed solvents selected from polar solvents such as ethers such as butyl ether can be suitably used,
In addition, a solvent to which an inorganic salt such as a phosphate buffered saline has been added can also be used, but is not particularly limited. For the purpose of the present invention, a polar solvent is preferred from the viewpoint of serine protease inhibitory action, and one or more kinds selected from methanol, ethanol, 1,3-butylene glycol, propylene glycol and purified water are further preferred. It is preferable to use a mixed solvent, particularly an aqueous ethanol solution, as the solvent.
【００１３】抽出方法としては、室温，冷却又は加温し
た状態で浸漬して抽出する方法、水蒸気蒸留等の蒸留法
を用いて抽出する方法、生の植物から圧搾して抽出物を
得る圧搾法等が例示され、これらの方法を単独で、又は
２種以上を組み合わせて抽出を行う。[0013] The extraction method includes a method of immersion and extraction at room temperature, in a cooled or heated state, a method of extraction using a distillation method such as steam distillation, and a method of squeezing raw plants to obtain an extract. And the like, and these methods are used alone or in combination of two or more.
【００１４】抽出の際の植物と溶媒との比率は特に限定
されるものではないが、植物１に対して溶媒０．５〜１
０００重量倍、特に抽出操作、効率の点で０．５〜１０
０重量倍が好ましい。また、抽出温度は、常圧下で室温
から溶剤の沸点以下の範囲とするのが便利であり、抽出
時間は抽出温度などによって異なるが、２時間〜２週間
の範囲とするのが好ましい。The ratio of the plant to the solvent at the time of extraction is not particularly limited.
000 weight times, especially 0.5 to 10 in terms of extraction operation and efficiency.
0 times by weight is preferred. The extraction temperature is conveniently in the range from room temperature to the boiling point of the solvent under normal pressure, and the extraction time varies depending on the extraction temperature and the like, but is preferably in the range of 2 hours to 2 weeks.
【００１５】また、このようにして得られたセージ及び
クワ属植物の抽出物は、抽出物をそのまま用いることも
できるが、セリンプロテアーゼ阻害作用を失わない範囲
内で脱臭，脱色，濃縮等の精製操作を加えたり、さらに
はカラムクロマトグラフィー等を用いて分画物として用
いてもよい。これらの抽出物や精製物，分画物は、これ
らから溶媒を除去することによって乾固物とすることも
でき、さらにアルコールなどの溶媒に可溶化した形態、
或いは乳剤の形態で提供することができる。The extract of the sage and mulberry plants thus obtained can be used as it is, but it is purified by deodorization, decolorization, concentration, etc. within a range that does not lose the serine protease inhibitory action. It may be used as a fraction by performing additional operations or using column chromatography. These extracts, purified products, and fractionated products can be dried by removing the solvent from them, and can be further solubilized in a solvent such as alcohol.
Alternatively, it can be provided in the form of an emulsion.
【００１６】本発明のセリンプロテアーゼ阻害剤は、当
分野で公知の化合物と混合し、非経口投与，経口投与又
は外部投与に適した、医薬品，医薬部外品，化粧品，食
品の形で使用することができる。食品においては、油脂
製品や乳化製品、清涼飲料等に添加することができる。
医薬品では経口剤，外用剤，注射剤，吸入剤，点鼻・点
眼剤等に添加することができ、これらの使用方法に応じ
て、錠剤，液剤，注射剤，軟膏，クリーム，ローショ
ン，エアゾール剤，座剤等の所望の剤型にすることがで
きる。また、必要に応じて賦形剤，基剤，乳化剤，安定
剤，溶解助剤，矯味剤，保存剤，芳香剤，着色剤，コー
ティング剤などを適宜配合することができる。医薬部外
品・化粧品としては、化粧水，乳液，クリーム等に添加
することができ、必要に応じて油分，保湿剤，紫外線吸
収剤，水溶性高分子，酸化防止剤，界面活性剤，金属イ
オン封鎖剤，抗菌防腐剤等が配合できる。The serine protease inhibitor of the present invention is mixed with a compound known in the art and used in the form of a drug, quasi-drug, cosmetic, or food suitable for parenteral administration, oral administration or external administration. be able to. In foods, it can be added to oils and fats products, emulsified products, soft drinks and the like.
Pharmaceuticals can be added to oral preparations, external preparations, injections, inhalants, nasal drops, eye drops, etc. Depending on the method of use, tablets, liquids, injections, ointments, creams, lotions, aerosols , Suppositories and the like. In addition, excipients, bases, emulsifiers, stabilizers, dissolution aids, flavoring agents, preservatives, fragrances, coloring agents, coating agents, and the like can be appropriately added as needed. As quasi-drugs / cosmetics, it can be added to lotions, emulsions, creams, etc. If necessary, oils, humectants, ultraviolet absorbers, water-soluble polymers, antioxidants, surfactants, metals An ion sequestering agent, an antibacterial preservative and the like can be blended.
【００１７】医薬品として利用する場合の植物抽出物の
投与量は、使用する植物の種類，抽出溶媒，精製の程度
や、患者の年齢，症状等により大きく変動するが、一般
には、経口投与の場合、乾燥重量として５〜５００mg/
日の範囲である。食品や化粧品に配合する場合は、その
効果や添加した際の香り、色調の点から考え、０．００
１〜５重量％の濃度範囲とすることが望ましい。The dosage of a plant extract when used as a medicament varies greatly depending on the type of plant used, the extraction solvent, the degree of purification, the age and symptoms of the patient, etc., but in general, the dosage is oral. 5 to 500 mg / dry weight
Range of days. When blended in foods and cosmetics, consider the effect, the scent when added, and the color tones.
It is desirable to set the concentration range of 1 to 5% by weight.
【００１８】[0018]
【実施例】さらに本発明の特徴について、実施例により
詳細に説明する。EXAMPLES Further, the features of the present invention will be described in detail with reference to examples.
【００１９】実施例１〜実施例６ 表１に示した植物の各部５００ｇを、５０容量％エタノ
ール水溶液５０００ｍｌに浸漬し、室温で一週間静置す
ることにより抽出した。その後、植物体を濾別除去し、
溶媒を減圧留去した後、得られた固形分を５０容量％エ
タノール水溶液にて再溶解し５０ｍｌとし、実施例１〜
６を得た。Examples 1 to 6 500 g of each part of the plant shown in Table 1 was immersed in 5000 ml of a 50% by volume aqueous ethanol solution, and extracted by leaving it to stand at room temperature for one week. Then, the plant body is removed by filtration,
After distilling off the solvent under reduced pressure, the obtained solid was redissolved in a 50% by volume aqueous ethanol solution to make up to 50 ml.
6 was obtained.
【００２０】[0020]
【表１】 [Table 1]
【００２１】実施例のセリンプロテアーゼ阻害作用を、
好中球エラスターゼ，トリプシン，α-キモトリプシ
ン，プラスミンを用いて検討した。結果を表２にまとめ
て示す。The serine protease inhibitory action of the Examples
Neutrophil elastase, trypsin, α-chymotrypsin, and plasmin were examined. The results are summarized in Table 2.
【００２２】好中球エラスターゼ活性阻害 実施例１〜実施例６を用いて、好中球エラスターゼ活性
阻害率を測定した。好中球エラスターゼ活性は、サクシ
ニル（Ｏ−メチル）−アラニル−アラニル−プロリル−
バリル−４−メチル−クマリル−７−アミド（９μＭ濃
度，ペプチド化学研究所社製）を基質として、３７℃に
て１時間ヒト好中球由来のエラスターゼ（１μg/ml，Ｓ
ｉｇｍａ社製）と反応させた後、分解生成物である7-ア
ミノ-4-メチルクマリンの生成量を、励起波長３５５n
m，蛍光波長４６０nmで蛍光強度を測定することにより
評価した。実施例をそれぞれ０．１mg/ml添加した場
合、及び実施例未添加の場合について酵素活性を測定
し、下記の式（１）を用いて好中球エラスターゼ活性阻
害率を算出した。Inhibition of neutrophil elastase activity Using Examples 1 to 6, the inhibition rate of neutrophil elastase activity was measured. Neutrophil elastase activity was determined to be succinyl (O-methyl) -alanyl-alanyl-prolyl-
Human neutrophil-derived elastase (1 μg / ml, S) was used at 37 ° C. for 1 hour using valyl-4-methyl-cumaryl-7-amide (9 μM concentration, manufactured by Peptide Chemical Laboratories) as a substrate.
After the reaction with Sigma Co., Ltd.), the amount of 7-amino-4-methylcoumarin, which is a decomposition product, was measured at an excitation wavelength of 355 n.
The evaluation was performed by measuring the fluorescence intensity at a fluorescence wavelength of 460 nm. The enzymatic activity was measured for each of the examples where 0.1 mg / ml was added and for the case where no examples were added, and the neutrophil elastase activity inhibition rate was calculated using the following formula (1).
【００２３】[0023]
【数１】 (Equation 1)
【００２４】トリプシン活性阻害 トリプシン活性は、０．１Ｍリン酸緩衝液（ｐＨ８．
０）０．３mlに、ブタ膵臓由来トリプシン（４０００〜
５０００USPunit/mg，和光純薬社製）溶液（４０μg/m
l）０．０３mlを添加して３０℃で５分間インキュベー
トを行い、基質としてＢＡＰＡ（N-α-ベンゾイル-DL-
アルギニン-p-ニトロアニリド塩酸塩）０．０２mlを加
えて更に３０℃で３０分間インキュベートした後、２０
％酢酸溶液を０．３ml添加して反応を停止し、４０５nm
の吸光度を測定することにより評価した。実施例をそれ
ぞれ０．１mg/ml添加した場合、及び実施例未添加の場
合について酵素活性を測定し、式（２）を用いてトリプ
シン活性阻害率を算出した。Inhibition of trypsin activity Trypsin activity was measured using 0.1 M phosphate buffer (pH 8.
0) Porcine pancreatic trypsin (4000-4000) was added to 0.3 ml.
5000 USPunit / mg, Wako Pure Chemical Industries, Ltd.) solution (40 μg / m
l) Add 0.03 ml, incubate at 30 ° C for 5 minutes, and use BAPA (N-α-benzoyl-DL-
After adding 0.02 ml of arginine-p-nitroanilide hydrochloride) and further incubating at 30 ° C. for 30 minutes,
The reaction was stopped by adding 0.3 ml of a acetic acid solution at 405 nm.
Was evaluated by measuring the absorbance. The enzymatic activity was measured for each of the examples where 0.1 mg / ml was added and for the case where no examples were added, and the trypsin activity inhibition rate was calculated using equation (2).
【００２５】[0025]
【数２】 (Equation 2)
【００２６】α-キモトリプシン活性阻害 α-キモトリプシン活性は、０．１Ｍリン酸緩衝液（ｐ
Ｈ８．０）０．４mlに、α-キモトリプシンタイプ７溶
液（０．６５μg/ml０．１Ｍリン酸緩衝液，Ｓｉｇｍａ
社製）０．０５mlを加え、３７℃で５分間インキュベー
トを行い、基質としてサクシニル−アラニル−アラニル
−プロリル−フェニルアラニン−p-ニトロアニリド塩酸
塩溶液（３．０ｍＭ ５０％ＤＭＳＯ溶液）０．０２ml
を加えて、更に３７℃で３０分間インキュベートした
後、２０％酢酸溶液を０．３ml添加して反応を停止し、
４０５nmの吸光度を測定することにより評価した。実施
例をそれぞれ０．１mg/ml添加した場合、及び実施例未
添加の場合について酵素活性を測定し、式（２）を用い
てα-キモトリプシン活性阻害率を算出した。Inhibition of α-chymotrypsin activity α-chymotrypsin activity was measured using a 0.1 M phosphate buffer (p
H8.0) in 0.4 ml of an α-chymotrypsin type 7 solution (0.65 μg / ml 0.1 M phosphate buffer, Sigma)
0.05 ml), incubated at 37 ° C. for 5 minutes, and 0.02 ml of a succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide hydrochloride solution (3.0 mM 50% DMSO solution) as a substrate.
And further incubated at 37 ° C. for 30 minutes. Then, the reaction was stopped by adding 0.3 ml of a 20% acetic acid solution,
It was evaluated by measuring the absorbance at 405 nm. The enzymatic activity was measured for each of the examples where 0.1 mg / ml was added and for the case where no examples were added, and the α-chymotrypsin activity inhibition rate was calculated using equation (2).
【００２７】プラスミン活性阻害 プラスミン活性は、直径９cmのシャーレにプラスミノー
ゲン除去フィブリノーゲンタイプ２の０．６％水溶液４
mlを入れ、ｐＨ７．４の０．１Ｍリン酸緩衝液４mlを加
えて攪拌し、トロンビン（１０unit/ml）０．１mlを滴
下し、ゆっくりと混和し３０分間静置し、フィブリンゲ
ルを調製した。プラスミン溶液（１０unit/ml）をシャ
ーレのゲル上に添加し、３７℃で２時間インキュベート
し、フィブリンゲルの溶解した面積を測定した。実施例
をそれぞれ０．１mg/ml添加した場合、及び実施例未添
加の場合について酵素活性を測定し、式（３）を用いて
プラスミン活性阻害率を算出した。Inhibition of plasmin activity Plasmin activity was determined by adding a 0.6% aqueous solution of plasminogen-removed fibrinogen type 2 to a Petri dish having a diameter of 9 cm.
Then, 4 ml of a 0.1 M phosphate buffer having a pH of 7.4 was added, and the mixture was stirred. 0.1 ml of thrombin (10 unit / ml) was added dropwise, mixed gently, and allowed to stand for 30 minutes to prepare a fibrin gel. . A plasmin solution (10 unit / ml) was added to the petri dish gel, incubated at 37 ° C. for 2 hours, and the dissolved area of the fibrin gel was measured. The enzymatic activity was measured when 0.1 mg / ml was added to each of the examples and when no example was added, and the plasmin activity inhibition rate was calculated using the formula (3).
【００２８】[0028]
【数３】 (Equation 3)
【００２９】[0029]
【表２】 [Table 2]
【００３０】表２に示した結果より、本発明の実施例１
〜６は、好中球エラスターゼ，トリプシン，α-キモト
リプシン，プラスミンに対して、危険率１％で有意な活
性阻害作用を有することが示された。From the results shown in Table 2, Example 1 of the present invention was obtained.
~ 6 were shown to have a significant activity inhibitory effect on neutrophil elastase, trypsin, α-chymotrypsin, and plasmin at a 1% risk factor.
【００３１】続いて、本発明の各実施例について熱及び
光に対する安定性を評価した。各実施例を１００℃で１
０分間熱処理した場合、及び３カ月間露光保存した場合
のそれぞれについて、０．１ｍｇ／ｍｌ添加時の好中球
エラスターゼ活性阻害率を測定し、未処理の場合の好中
球エラスターゼ活性阻害率と比較して表３に示した。表
３より明らかなように、いずれの実施例も熱及び光に対
し非常に良好な安定性を示し、１００℃で１０分間の熱
処理及び３カ月間の露光保存によっても、好中球エラス
ターゼ活性阻害作用の低下はほとんど見られなかった。Subsequently, the stability to heat and light was evaluated for each example of the present invention. Each example was prepared at 100 ° C for 1
The neutrophil elastase activity inhibition rate at the time of addition of 0.1 mg / ml was measured for each of the case of heat treatment for 0 minute and the case of exposure and storage for 3 months. Table 3 shows a comparison. As is clear from Table 3, all the examples showed very good stability to heat and light, and the neutrophil elastase activity was inhibited by the heat treatment at 100 ° C. for 10 minutes and the exposure storage for 3 months. Little reduction in action was observed.
【００３２】[0032]
【表３】 [Table 3]
【００３３】また、本発明の各実施例について、培養ヒ
ト線維芽細胞に対する細胞毒性を評価した。ヒト由来線
維芽細胞を、１ウェル当たり２．０×１０4個となるよ
うに９６穴マイクロプレートに播種し、２４時間後に、
実施例のそれぞれを１．０ｍｇ／ｍｌ含有する１．０容
量％牛胎仔血清添加ダルベッコ修正基礎栄養培地培地に
て３７℃で２４時間さらに培養して、生細胞数を計測し
て細胞生存率を求め、５０％致死濃度（ＬＤ50）を算出
した。その結果、表３に示すように、いずれの実施例に
おいてもＬＤ50は１００．０ｍｇ／ｍｌ以上であり、試
験した濃度では細胞毒性は認められなかった。Further, for each of the examples of the present invention, cytotoxicity to cultured human fibroblasts was evaluated. Human-derived fibroblasts were seeded in a 96-well microplate at 2.0 × 10 4 cells / well, and 24 hours later,
Each of the examples was further cultured at 37 ° C. for 24 hours in Dulbecco's modified basal nutrient medium containing 1.0% by volume of fetal calf serum containing 1.0 mg / ml, and the number of viable cells was counted to determine the cell viability. The 50% lethal concentration (LD 50 ) was calculated. As a result, as shown in Table 3, in all Examples, the LD 50 was 100.0 mg / ml or more, and no cytotoxicity was observed at the tested concentrations.
【００３４】[0034]
【発明の効果】以上詳述したように、セージ(Salvia of
ficinalis L.)及びクワ属(Morus)植物の抽出物を含有す
る本発明のセリンプロテアーゼ阻害剤は、エラスター
ゼ，トリプシン，キモトリプシン，プラスミン等のセリ
ンプロテアーゼ阻害に対し優れた作用を示し、本発明に
かかるセリンプロテアーゼ阻害剤は、膵炎治療剤，急性
動脈炎，肺気腫，動脈硬化，関節リュウマチ，癌の転移
・浸潤等の治療剤として、また、皮膚のはり・弾力を回
復維持することで皮膚の老化を防止し、若々しい肌の状
態を維持する効果が期待される。As described in detail above, sage ( Salvia of Salvia)
ficinalis L.) and the serine protease inhibitor of the present invention containing an extract of a mulberry plant ( Morus ) show an excellent action on inhibiting serine proteases such as elastase, trypsin, chymotrypsin, and plasmin, and are related to the present invention. Serine protease inhibitors are therapeutic agents for pancreatitis, acute arteritis, emphysema, arteriosclerosis, rheumatoid arthritis, metastasis and invasion of cancer, etc., and restore skin aging by restoring and maintaining skin elasticity and elasticity. The effect is expected to prevent and maintain youthful skin condition.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 ＦＩ Ａ６１Ｋ 35/78 ＡＣＪ Ａ６１Ｋ 35/78 ＡＣＪＤ ＡＤＡ ＡＤＡ ＡＤＵ ＡＤＵ ＡＧＺ ＡＧＺ (72)発明者 岡野 由利 滋賀県八日市市岡田町字野上112−１ 株 式会社ノエビア基礎研究所内 (72)発明者 正木 仁 滋賀県八日市市岡田町字野上112−１ 株 式会社ノエビア基礎研究所内──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. 6 Identification code FI A61K 35/78 ACJ A61K 35/78 ACJD ADA ADA ADU ADU AGU AGZ AGZ (72) Inventor Yuri Okano 112 Nogami, Okada-cho, Yokaichi, Shiga Prefecture -1 Within Noevir Basic Research Laboratory, Inc. (72) Inventor Hitoshi Masaki 112-1, Nogami, Okada-cho, Yokaichi, Shiga Prefecture Within Noevir Basic Research Laboratory, Inc.
Claims (5)
ワ属(Morus)植物から選択される１種又は２種以上の植
物の抽出物を有効成分とするセリンプロテアーゼ阻害
剤。1. A serine protease inhibitor comprising as an active ingredient an extract of one or more plants selected from sage ( Salvia officinalis L.) and mulberry ( Morus ) plants.
ワ(Morus)属植物から選択される１種又は２種以上の植
物の抽出物を有効成分とするエラスターゼ阻害剤。2. An elastase inhibitor comprising as an active ingredient an extract of one or more plants selected from sage ( Salvia officinalis L.) and mulberry ( Morus ) plants.
ワ(Morus)属植物から選択される１種又は２種以上の植
物の抽出物を有効成分とするトリプシン阻害剤。3. A trypsin inhibitor comprising as an active ingredient an extract of one or more plants selected from sage ( Salvia officinalis L.) and mulberry ( Morus ) plants.
ワ(Morus)属植物から選択される１種又は２種以上の植
物の抽出物を有効成分とするキモトリプシン阻害剤。4. A chymotrypsin inhibitor comprising as an active ingredient an extract of one or more plants selected from sage ( Salvia officinalis L.) and mulberry ( Morus ) plants.
ワ(Morus)属植物から選択される１種又は２種以上の植
物の抽出物を有効成分とするプラスミン阻害剤。5. A plasmin inhibitor comprising as an active ingredient an extract of one or more plants selected from sage ( Salvia officinalis L.) and mulberry ( Morus ) plants.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33510897A JP3796340B2 (en) | 1997-11-18 | 1997-11-18 | Serine protease inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33510897A JP3796340B2 (en) | 1997-11-18 | 1997-11-18 | Serine protease inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH11147834A true JPH11147834A (en) | 1999-06-02 |
| JP3796340B2 JP3796340B2 (en) | 2006-07-12 |
Family
ID=18284868
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP33510897A Expired - Fee Related JP3796340B2 (en) | 1997-11-18 | 1997-11-18 | Serine protease inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3796340B2 (en) |
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|---|---|---|---|---|
| JP2003183122A (en) * | 2001-12-21 | 2003-07-03 | Ichimaru Pharcos Co Ltd | Agent for inhibiting activity of collagenase |
| KR100477896B1 (en) * | 2002-04-25 | 2005-03-18 | 한국생명공학연구원 | Active fractions showing inhibitory effects on heparinase activity and cancer metastasis from the root bark of Morus alba L. |
| JP2005529885A (en) * | 2002-04-25 | 2005-10-06 | ザ スクリプス リサーチ インスティテュート | Treatment and prevention of pulmonary conditions |
| KR100594567B1 (en) | 2004-08-30 | 2006-06-30 | 한국생명공학연구원 | Sangenon C and sangenon G inhibiting heparinase activity |
| US7485327B2 (en) * | 2000-12-12 | 2009-02-03 | Angiolab, Inc. | Composition comprising Melissa leaf extract for anti-angiogenic and matrix metalloproteinase inhibitory activity |
| JP2009508943A (en) * | 2005-09-22 | 2009-03-05 | トルービオ・アーベー | Stabilized protease composition |
| JP2010150231A (en) * | 2008-12-24 | 2010-07-08 | Medical & Pharmaceutical Industry Technology & Development Center | Antitussive composition and method for preparing the same |
| JP2011190200A (en) * | 2010-03-12 | 2011-09-29 | Saishunkan Seiyakusho:Kk | Expression inducer for heat shock protein |
| US8394372B2 (en) | 2004-09-21 | 2013-03-12 | Trobio Ab | Stabilized protease composition |
| CN104288733A (en) * | 2014-10-18 | 2015-01-21 | 宿州学院 | Formula and method for treating emphysema |
| JP2015042675A (en) * | 2014-11-13 | 2015-03-05 | 株式会社再春館製薬所 | Expression inducer for heat shock protein |
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| CN105079757A (en) * | 2015-05-17 | 2015-11-25 | 柳晖 | Traditional Chinese medicine composition for treating emphysema |
| CN113785933A (en) * | 2021-09-16 | 2021-12-14 | 陕西理工大学 | Method for eliminating activity of antinutritional factor serine protease inhibitor in folium Mori and its application |
| US11554153B2 (en) * | 2014-06-16 | 2023-01-17 | Unigen, Inc. | Compositions and methods for managing or improving bone disorders, joint disorders, cartilage disorders, or a combination thereof |
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