Florin Gheorghe Barla

称号及び氏名博士(応用生命科学)

学位授与の日付

平成２２年９月３０日

論Study on natural and synthesized polyphenolic compounds
possessing hyaluronidase and collagenase inhibitory effects（ヒアルロニダーゼおよびコラゲナーゼ阻害作用を有する天然および合成ポリフェノール化合物に関する研究）

文

名

論文審査委員

主査
副査
副査

乾 博
林
英雄
宮武 和孝

論文要旨

The inhibition of enzyme activity may be as significant as the activity itself in theregulation of biological processes. A lot of enzyme inhibitors, which are found from natural
sources or synthesized artificially, are used as therapeutically important tools in several clinicalareas. In addition to the medical use, inhibitors against certain enzymes, such as hyaluronidase,collagenase, and urease, are regarded to be useful as functional ingredients of foods andcosmetics.
Hyaluronic acid is found in the extracellular matrix of soft connective tissues, such as theumbilical cord, skin, synovial fluid, and the vitreous humor. This polysaccharide is involvedin cell adhesion and protection, formation of skin tissue, water retention in tissues, andmaintenance of flexibility in the skin due to its unique hydroscopic, rheologic, and viscoelastic
properties. Hyaluronidase is a mucopolysaccharase that hydrolyzes glycosaminoglycans,including hyaluronic acid, in the extracellular matrix during tissue remodeling. When the levelof hyaluronic acid decreases under conditions in which the hyaluronidase activity is increased,
the moisture and tension of the skin is reduced. Thus, hyaluronidase inhibitors are usefulcosmeceutical ingredients as they have antiwrinkle and antiaging effects on the skin. Inaddition, hyaluronidase induces histamine release from mast cells during inflammatory
reactions, and some hyaluronidase inhibitors, such as disodium cromoglycate, have been used tosuppress allergies and inflammation.
Collagen is the major fibrous component of the extracellular matrix in the skin, and thecollagen content in the skin decreases greatly during the aging process and due to long-term
exposure to ultraviolet radiation. Collagenase (matrix-metalloproteinase) plays an importantrole in unbalanced turn over or rapid breakdown of collagen in human
inflamed/ultraviolet-irradiated skin.
Therefore, collagenase inhibitors, in addition tohyaluronidase inhibitors, have useful functions as cosmetic materials.
Helicobacter pylori, which is thought to cause gastritis, peptic ulcer disease, gastricadenocarcinoma, and gastric lymphoma, is able to survive in the stomach by releasing anenzyme, urease. Urease converts urea into ammonia, which then counters the stomach acid.
This creates a neutralizing environment for protecting H. pylori from the acid in the stomach.
It is thus suggested that urease inhibitors are useful as therapeutic agents for H. pylori infection.
The aim of this study is to search biologically active ingredients useful for functional foods
and cosmeceutical products, from natural sources or synthesized compounds. With this intent,preliminarily, I have carried out the screening of some plants found in Okinawa Prefecture forinhibitors against hyaluronidase, collagenase and urease. I found that the leaf extract ofBischofia javanica (called “akagi” in Japanese) exhibited potent inhibitory effects against theseenzymes, compared with those of the leaf and bark extracts of Mallotus japonicus (called“akamegashiwa” in Japanese). Both B. javanica and M. japonicus are involved in the familyof Euphorbiacease, and distributed over Polynesia, Australia, and tropical zone of Asia. M.
japonicus is one of well-known Chinese medical plants, and its bark, which is commerciallyavailable in Japan, is fused and used for the prevention of gastric and duodenal ulcer, and gastrichyperacidity. In contrast, B. javanica is meagerly used for folk remedy in the limited area ofPolynesia.
The Bischofia leaf extract inhibited hyaluronidase with an IC50 value of 370 µg/ml, which
was comparable to that of disodium cromoglycate (230 µg/ml), which is a well-knownhyaluronidase inhibitor used as an antiinflammation and antiallergy agent. In addition, thecollagenase inhibitory activity of the Bischofia leaf extract was slightly lower than that of EDTA,
the most popular collagenase inhibitor (IC50 value: 590 µg/ml and 170 µg/ml, respectively).
Furthermore, the leaf extract also exhibited the urease inhibitory effect with the almost the samepotential as acetohydroxamic acid, which is reported to suppress H. pylori-induced gastritis byinhibiting urease (IC50 value: 4.9 µg and 2.8 µg/ml, respectively). These results suggest thatthe Bischofia leaf extract has potential as a novel ingredient for use in a wide range offunctional foods and cosmeceutical products.
From 570 g of the crude Bischofia leaf extract, 11.8 mg white powder of compound 1 was
purified as a hyaluronidase inhibitor by Diaion HP-20 and Chromatorex column
chromatographies followed by repeated HPLC using a Cosmosil C18 column. This compoundwas identified as 1,2,3,4,6-penta-O-galloyl-b-D-glucose by comparison of 1H- and 13C-NMR
spectral data with those reported in the literatures. The IC50 value of this compound forhyaluronidase was estimated to be 21.6 µM, which was substantially lower than that of
disodium cromoglycate (450 µM). Kinetic analyses indicated that this compound acted as an
uncompetitive inhibitor with a Ki value of 10.5 µM.
1,2,3,4,6-Penta-O-galloyl-b-D-glucose (compound 1) also exhibited a collagenase
inhibitory effect with an IC50 value of 76 µM, which was also substantially lower than that of
EDTA (580 µM). In addition, three collagenase inhibitors, compounds 2 (3.3 mg), 3 (3.1 mg)
and 4 (3.3 mg), were isolated from the Bischofia leaf extract (570 g). Compounds 2, 3 and 4
were identified as fisetin, 3,4,8,9,10-pentahydroxy-6H-dibenzo[b,d]pyran-6-one and gallic acid,respectively, on the basis of following 1H- and 13C-NMR spectral data by comparison with thosereported in literatures. The IC50 values of these compounds were estimated to be 205 µM, 150µM and 2.1 mM, respectively.
Gallic acid is a phenolic compound that is found as a constituent of hydrolyzable tannins,
including 1,2,3,4,6-penta-O-galloyl-b-D-glucose, in many plants. Gallic acid and hydrolyzabletannins have many biological activities, including the hyaluronidase and collagenase inhibition
and antioxidative effects. The gallate esters of n-alkanols also act as a potent antioxidant, andpropyl, octyl and dodecyl (lauryl) gallates are permitted additives for antioxidation in foods in
the United States, although they are not natural compounds. To determine whether n-alkyl
gallates are also useful as active ingredients of functional foods and cosmeceutical products, I
identified their inhibitory activities against hyaluronidase and collagenase.
Gallic acid and its esters of n-alkanols with different chain length (C1-C12) were examined
as to their hyaluronidase inhibitory activity, and their IC50 values were estimated. Methyl,ethyl, propyl, butyl and dodecyl gallates as well as gallic acid exhibited virtually no inhibitory
activity against hyaluronidase. In contrast, hexyl, heptyl, octyl, nonyl and decyl gallates
exhibited potent hyaluronidase inhibitory activities. It is hence thought that the hyaluronidase
inhibitory activity associated with the length of the hydrophobic alkyl chain to a large extent.
Octyl gallate was the most potent inhibitor among the alkyl gallates examined, and its IC50 value(106 µM) was substantially lower than that of disodium cromoglycate. Kinetic analyses
indicated that octyl gallate acted as an uncompetitive inhibitor with a Ki value of 45 µM.
Octyl 3,5-dihydroxybenzoate was a potent inhibitor of hyaluronidase, with an IC50 value of 113µM; however, other derivatives of octyl benzoate (octyl 3,4-dihydroxybeozoate, octyl3-hydroxybenzoate and octyl 4-hydroxybenzoate) exhibited virtually no inhibitory activity.
These results suggest that the two hydroxy groups at the 3 and 5 positions in the benzoate
moiety are essential for hyaluronidase inhibition. This is in contrast with the catechol structurenecessary for antioxidative effects.
Collagenase was inhibited by gallic acid, but gallic acid esters of n-alkanols with shortchain lengths (C1-C4) did not inhibit the enzyme. These results suggest that the carboxyl groupin gallic acid is essential for collagenase inhibition. However, similar to the case of thehyaluronidase inhibition, the collagenase reaction was strongly inhibited by gallate esters of

n-alkanols when the alkyl chain length ranged between C6 and C10, and the most potent inhibitor
was octyl gallate. The IC50 value of octyl gallate (1.08 mM) was lower than that of gallic acid.
Octyl 3,4-dihydroxybenzoate as well as octyl gallate inhibited collagenase, whereas octyl
3,5-dihydroxybenzoate did not. This result is in contrast with that in the case of thehyaluronidase inhibition.
Thus, the gallate esters of n-alkanols with alkyl chain lengths between C6 and C10, in
particular octyl gallate, might be useful ingredients of functional foods and cosmeceuticalproducts to improve inflammation and allergic reactions, and to prevent wrinkles and skinaging.

審査結果の要旨

一般に、酵素活性を阻害する化合物は医薬品として様々な疾患の治療に応用されている。同様に、酵素阻害剤は機能性食品や薬用化粧品の活性成分としても利用されている。
本研究は、機能性食品や薬用化粧品の活性成分として有用なヒアルロニダーゼやコラゲナーゼなどに阻害作用を有する新規なポリフェノール化合物の検索することを目的とした。なお、ヒアルロニダーゼ阻害剤は抗炎症・抗アレルギー作用を有する機能性食品素材として有用であり、またコラゲナーゼ阻害剤はヒアルロニダーゼ阻害剤とともに皮膚の保湿等に有効な化粧品素材となりうる。
本研究を行うにあたり最初に、沖縄に自生する様々な植物から調製した抽出物について機能性食品や薬用化粧品として有用な酵素阻害作用を検討した。その結果、アカギ（Bischofia javanica）に強いヒアルロニダーゼ、コラゲナーゼ阻害活性を見いだした。
アカギは東南アジアやオセアニアに広く分布するトウダイグサ科に分類される常緑高木であり、抗炎症作用を有する民間薬としてポリネシアの限定された地域で僅かであるが利用されている。一方、同様にトウダイグサ科には樹皮や葉が漢方薬として胃潰瘍などの消化器疾患に用いられるアカメガシワ（Mallotus japonicus）がよく知られている。
そこで、両者を比較したが、アカギ葉抽出物は、アカメガシワ（葉、樹皮）抽出物に比べ、どちらの酵素に対しても強い阻害活性を示した。特に、ヒアルロニダーゼ阻害の50%阻害濃度（IC50）は 370 µg/ml であり、ヒアルロニダーゼ阻害物質であり抗炎症・抗アレルギー剤として利用されている薬剤である disodium cromoglycate（IC50：230µg/ml; 450 µM）と比較しても遜色のないレベルであった。また、アカギ葉抽出物には胃炎や胃がんの原因となるピロリ菌の増殖抑制に有用なウレアーゼ阻害作用があり、その阻害活性はピロリ菌に起因する胃炎を抑制することが in vivo で証明されているacetohydroxamic acid と同レベルであった。
アカギ葉抽出物からヒアルロニダーゼ阻害物質を単離・精製したところ、1,2,3,4,6penta-O-galloyl-b-D-glucose が得られた。本化合物の IC50 値は 76 µM であり、disodium
cromoglycate に比べて明らかに低値であった。なお、阻害様式は不競合阻害であり、Ki 値は 10.5 µM であった。

1,2,3,4,6-penta-O-galloyl-b-D-glucose にはヒアルロニダーゼ阻害活性とともにコラゲナーゼ阻害作用が見いだされた。その IC50 値は 76 µM であり、よく知られたコラゲナーゼ阻害剤である EDTA（IC50：580 µM）より強い阻害作用を示すことがわかった。
さらに、アカギ葉抽出物からコラゲナーゼ阻害剤として fisetin（IC50 ：205 µM）、
3,4,8,9,10-pentahydroxy-6H- dibenzo[b,d]pyran-6-one（IC50：150 µM）および gallicacid（IC50：2.1 mM）の３種の化合物が単離された。
様々な n-alkyl gallate を合成し、ヒアルロニダーゼおよびコラゲナーゼ阻害活性を調べた。その結果、両阻害作用ともに alkyl 基の鎖長によって大きく影響を受け、どちらも octyl gallate に最も強い阻害活性が見いだされた。なお、ヒアルロニダーゼ阻害のIC50 値は 106 µM であり、コラゲナーゼ阻害に対しては 1.08 mM であった。
次に galloyl基の水酸基の特異性について検討した。その結果、ヒアルロニダーゼ阻害についてはoctyl gallate 同様に octyl 3,5-dihydroxybenzoate に強い活性が見られたが、コラゲナーゼ阻害では octyl 3,4-dihydroxybenzoate に強い活性が現れた。以上の結果から、octylgallate は米国では抗酸化作用を有する食品添加物として利用されているが、さらに機能性食品や薬用化粧品の活性成分としても有用であることが明らかになった。
以上、本研究では、沖縄や東南アジアなどに自生するアカギには強いヒアルロニダーゼ、コラゲナーゼさらにウレアーゼ阻害作用があり、機能性食品素材や化粧品素材として有用であることを明らかにした。さらに、様々な n-alkyl gallate を合成し、そのなかで食品の抗酸化剤として利用されている octyl gallate に強いヒアルロニダーゼ、コラゲナーゼ阻害活性を見いだした。これらの成果は、食品科学や天然物化学などの分野に多大な貢献をするものと考えられ、最終試験の結果と併せて、博士（応用生命科学）の学位を授与することを適当と認める。