称号及び氏名

博士(応用生命科学) Dikdik Kurnia

学位授与の日付

平成２０年３月３１日

論「Studies on Bioactive Compounds from Indonesian Medicinal文

名Plants and Mushrooms(インドネシア産薬用植物およびキノコに含まれる生理活性物質に関する研究）」
論文審査委員

主査

林英雄

副査

宮武和孝

副査

川口剛司

論文要旨
Chapter 1.

Introduction

For thousands of years, natural products derived from plants andmicroorganisms have played an important role throughout the world in treating andpreventing human diseases. The medicinal plants and mushrooms which grow inIndonesian rain forest are a rich source of natural products with a great diversity ofmolecular structures which can be used as lead compounds for development of new
drugs or agrochemicals, but their chemical constituents have not yet been completelyrecognized. From this viewpoint, the research was conducted to find novel bioactive
compounds from Indonesian medicinal plants and mushrooms.
Chapter 2.

Screening of Indonesian medicinal plants and mushrooms forbiologicalactivity

In order to select the active samples, a total of 278 methanol extracts of wildmushrooms and 150 methanol extracts of medicinal plants were tested against brine
shrimp (Artemia salina). As a result, 167 samples of mushrooms exhibited the mortality
rate of 80-100% and one sample showed immotile activity, and 10 samples of medicinal
plants exhibited the mortality rate of 80-100%. The mushroom of Microporellus
subsessilis showing immotile activity and the medicinal plant of Phaleria macrocarpa

(Scheff.) Boerl. were selected for further purification of the active constituents. This
study deals with the isolation and structural determination of active principles and theirbioactivities are also evaluated.
Chapter 3.

10-Phenyl-[11]-cytochalasans isolated from mushroom, Microporellussubsessilis

The methanol extract of M. subsessilis was purified by using the immotile
activity to follow the separations with combination of column chromatography on
Wakogel C-200, Silica G-60, Chromatorex ODS and preparative HPLC on Inertsil
ODS-3 to afford four active compounds of 10-phenyl-[11]-cytochalasans (1-3 and 6)
together with two inactive compounds (4 and 5). Compounds 1-3 were identified asknown compounds, while compounds 4 and 5 were determined to be new members of
10-phenyl-[11]-cytochalasans. In compound 4, the six-membered ring of 3 was
modified with a double bond newly introduced between C-6 and C-7 and ahydroxylmethyl was located at C-6. On the other hand, new compound 5 wasdetermined as a 6,7-epoxide of 3. In new compound 6, the hydroxyl at C-19 of 1 was
acylated with a C17 fatty acid containing two double bonds and a branched methyl. The
structure of fatty acid moiety was determined as a novel fatty acid. To our best
knowledge, compound 6 is the first member of cytochalasin family which contains along chain fatty acid moiety.

Chapter 4.

Biological activities of cytochalasin compounds

The immotile activities of 1-6 were evaluated against the brine shrimp.
10-Phenyl-[11]-cytochalasans 1, 2, 3 and 6 induced 72%, 78%, 64%, and 63%
immotility against the brine shrimp at 10 ppm, respectively. On the other hand,compounds 4 and 5 were inactive. These facts indicated that the hydroxyl group at C-7
and the exomethylene at C-6 were components essential for exhibiting immotility. The
fatty acid moiety as well as the substituents at C-19 in compound 6 was not significant
because the activities of compounds 1, 2, 3, and 6 were almost equal. These results werein good agreement with structure activity relationship studies of cytochalasins whichshowed the important role of the hydroxyl group at C-7 in exhibiting biological activity
on mammalian cell.
Chapter 5.

29-Norcucurbitacins isolated from medicinal plant, Phaleria
macrocarpa (Scheff.) Boerl.

The methanol extract of P. macrocarpa was purified by a combination of column

chromatography on Wakogel C-200, Silica G-60, and Chromatorex ODS to afford four
active compounds of 29-norcucurbitacin derivatives (7-10). Compounds 7, 9 and 10
were identified as fevicordin A, fevicordin A glucoside and fevicordin D glucoside,
respectively, which have been isolated from Fevillea cordifolia. Comparison of the 1Hand 13C-NMR spectra of 8 with those of fevicordin A (7) showed that the acetyl signals
attached at C-25 of fevicordin A disappeared in 8, thus all the spectral data wereconsistent with those of the proposed structure of 8 for fevicordin A derivative.
Consequently, compound 8 was determined to be a new member of norcucurbitacin, andwas named desacetylfevicordin A.
Chapter 6.

Biological activities of norcucurbitacin compounds

The bioactivity of the compounds 7, 8, 9, and 10 was evaluated using lethality
bioassay against the brine shrimp. After 24h observation, compounds 7, 8, 9, and 10
exhibited cytotoxic activity with LD50 values of 5 ppm, 3 ppm, 12 ppm and 6 ppm,respectively. The different functional groups attached at C-25 in compounds 7 and 8
showed no significant effect on their LD50 values indicating that both of acetyl andhydroxyl groups were not important in exhibiting activity.
Compounds 9 and 10 have the different functional groups at C-25 as well as in
7 and 8, and compound 10 was also different with compound 9 in the absent of (E)olefinic protons H-23 and H-24. Comparison of their LD50 values indicated thatcompound 10 was more active than 9. This fact suggested that the methylene groups atCH2-23 and CH2-24 in compound 10 increased the level of activity against the brine
shrimp.

Chapter 7.

Conclusion

In the course of our search for novel bioactive compounds from Indonesian
medicinal plants and mushrooms, their methanol extracts were tested using primary
screening system with the brine shrimp (Artemia salina) to detect their bioactiveconstituents. During the present bioassay-guided investigation of methanol extracts, six
cytocahalasins (1-6) and four 29-norcucurbitacin family (7-10) have been isolated frommushroom of M. subsessillis and medicinal plant of P. macrocarpa, respectively.
The important result in the current study suggested that the primary screening
system using the brine shrimp is a useful tool to guide a separation and purification
process of the active constituents from pharmaceutically valuable natural sources, andthat the Indonesian biodiversity including medicinal plants and fungi is an importantand promising natural sources in the discovery programs of drugs and agrochemicals.

12OH
H

H
RHN
O
(1) R = OH
(2) R = H
(3) R = OCH 3O

78OH

H

78O

OH

H
H

H3CO H

HN

19OO

H

H3CO H

HN

191266HH

CH 2OH

19O

O

OH

(5)

(4)

OH
H

H

19HN
O

O

H
OH

O
(6)
O

OH

O

OH
25O
HHOOH

2423OROH

β- D-Glc-O

2HOO
25OROH2(7) R = Ac
(8) R = H

HO(9) R = Ac
(10) R = H 23,24-dihydro審査結果の要旨
インドネシアの熱帯雨林地帯は植物やキノコ類の多様性に恵まれており，そこでは多くの植物が薬用として種々の疾病の治療や予防のために用いられている．しかしながらそれらの植物に含まれる有効成分の化学的解明はほとんど進んでいないのが現状である．そこ
で，本申請者はインドネシア産植物および野生キノコから新規な生理活性物質あるいは新たな薬剤の開発におけるリード化合物となる物質を見出すことを目的として研究を行い，以下の成果を得た．
まず，インドネシア産野生キノコ 278 種および薬用植物 150 種から得られたメタノール抽出物について，ブラインシュリンプ（Artemia salina）幼生に対する生理活性を検索した．その結果， 167 種のキノコおよび 10 種の植物が 1 mg/ml の濃度で 80% 以上の致死作用を示した．さらに興味あることに，キノコの 1 種（Microporellus subsessilis）はブラインシュリンプに対し、運動停止活性を示した．そこで，本研究では Microporellus

subsessilis および顕著な致死活性の認められた植物 Phaleria macrocarpa に含まれる活性物質の化学構造の解明を行った．

M. subsessilis のメタノール抽出物をヘキサンおよび酢酸エチルで順次抽出した．活性の認められた酢酸エチル抽出画分を各種クロマトグラフィーを用いて精製し，4 種の運動停止活性サイトカラシン類（1，2，3，6）および 2 種の関連化合物（4、5）を単離した．化合 物 1 は 7,18,19-trihydroxy-16,18-dimethyl-10-phenyl-[11]-cytocharasa-6(12),13(E)diene-1,2-dione であり，化合物 2 と 3 はいずれも既知の 10-phenyl-[11]- cytocharasan類であった．また，新規化合物 4 および 5 は各種スペクトルデータの解析からそれぞれの構 造 を

12,18-dihydroxy-19-methoxy-16,18-dimethyl-10-phenyl-[11]-cytocharasa-6(7),13(E)-diene-1,21-dione お よ び 6,7-epoxy-18-hydroxy-19-methoxy-16,18-dimethyl10-phenyl-[11]-cytocharasa-13(E)- ene-1,21-dione と決定した．さらに、化合物 ６ はス
ペクトルデータから化合物 １ の 19 位水酸基が長鎖脂肪酸でアシル化された化合物であることが推定された．そこで化合物 6 から側鎖脂肪酸のメチルエステルを誘導し，構造解析を試みた．しかしながら，得られるメチルエステルが極微量であったため，同じ脂肪酸を M. subsessilis メタノール抽出物のヘキサン相から単離し，これを用いて構造解析を行った．その結果，側鎖脂肪酸は 4-methyl- 6(E),8(E)-hexadecadienoic acid であることが明らかとなった．
化合物 1 および 2，3，6 は 10 μg/ml の濃度でブラインシュリンプの運動をそれぞれ
72％ および 78％，64％，63％ 停止した．一方，化合物 4 と 5 は活性を示さなかった．
これらのことから活性発現には 6(12) 位の二重結合と 7 位の水酸基が重要であることが示唆された．
次に，薬用植物である P. macrocarpa に含まれる活性物質の解明を行った．種子粉末をメタノールで抽出し，得られた抽出物からブラインシュリンプに対する致死作用を指標として活性物質の精製を行い，4 種の 29-ノルククルビタシン類（7，8，9，10）を単離した．
各種スペクトルデータを解析し，化合物 7 および 9，10 をそれぞれ fevicordin A およびfevicordin A glucoside，fevicordin D glucoside と同定した．化合物 8 はスペクトルデータから新規物質である desacetylfevicordin A であることが推定され，化合物 7 のデスアセチル体を誘導することにより化合物 8 の構造を確認した．
化合物 7 および 8，9，10 のブラインシュリンプに対する半数致死濃度はそれぞれ5 μg/ml および 3 μg/ml，12 μg/ml，6 μg/ml であった．すなわち，化合物 9 と 10 に存在するグルコース部分および化合物 7 と 8 に存在するアセチル基は活性発現に関与していないことが示唆された．
以上のように，インドネシア産薬用植物およびキノコから多くの生理活性物質を明らかにしたことから，熱帯産植物が有用天然物の供給源として多いに期待されることが示された．また，ブラインシュリンプを用いる簡便な生物検定系が生理活性物質を検索する際の指標として用い得ることが示された．これらの成果は天然物化学および生物有機化学の両分野に多大の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．