薬物代謝工学分野 Division
o f M e t a b o l i c E n g i n e e r i n g
教授 服部 征雄 Professor
M a s a o , H a t t o r i ( P h . D . )
助教授 横津 隆子 AssociateP r o f e s s o r Takako Yokozawa ( P h . D . )
助手 宮代 博継 As凶antｷ P r o f e s s o r H i r o t s u g u M i y a s h i r o ( P h . D . )
技官 中村 憲夫 Research
A s s o c i a t e N o r i o Nakamura ( P h . D . )
薬物代謝工学分野は和漢薬の薬効，毒性発現に関与する代謝系の分子生物学的研究を発展させる
ことを設置目的とし，①和漢薬の薬効発現に関与する腸内細菌の役割，②薬物代謝に関する腸内 細菌遺伝子の解明，③腎毒性物質産生機構の分子生物学的解明とその制御に関する研究を課題とし て取りあげ，和漢薬の薬効発現機構、生体へのレスポンスなどの基礎的研究を通じて，和漢薬の科 学的評価や臨床応用をはかることを目指している。主な研究題目を以下に示す。
1. 天然物のバイオトランスフォーメイション
2. 和漢薬の薬効発現に関与する腸内細菌，酵素およびその遺伝子の解析
, 3 . AIDS,
C型肝炎の予防および治療薬の開発4. 腎疾患における病態の解明と腎臓病治療薬の開発
5. 糖尿病性腎症の治療戦略 本年度の主な研究を列挙すると：
1 .
ヒト腸内細菌による杜仲の樹皮に多量に含まれる pinoresinol diglucoside のエストロゲン様物質（エンテロジオール，エンテロラクトン）への変換を検討し，この化合物が加水分解，エー
テル環の開裂，脱メチル化，脱水酸基化反応を経て変換されることを明らかにした。また， こ
れらの代謝物のエストロゲン様作用，抗工ストロゲン様作用を検討した。
2. 霊芝に含まれる苦味成分の HPLC による分析法を確立し，霊芝製品の品質評価を行なった。ま た台湾産樟芝から 5 種の新規化合物を単離し，その細胞毒性を検討した。
3. 中国少数民族薬物の C 型肝炎ウイルス由来の RNA ポリメラーゼ阻害活性を探索した。
4. 蛋白・糖修飾を surrogate マーカーとして利用し，腎不全並びに糖尿病性腎症における病態把 握に着手した。また新たな治療手段を探索するために，温牌湯， epicatechin 3-0-gallate，緑 茶ポリフェノール，桂枝夜苓丸，八昧地黄丸，芥子菜，韓国産生薬を中心に検討した。
く〉著書 Book
1 ）楊秀偉，斜美栄，服部征雄．：『中約成分代謝分析』中国医菊科技出版．，北京，（2003).
2 ）横津隆子：『長寿科学事典』医学書院，東京， 2003,
p p . 9 1 9 ‑ : ‑ 9 2 2 .
。原著 Original
paper
1)
Abdel・HafezA. A . , Hosny A.H. E . , Jo M . , Kur
Nakamura N . , and H a t t o r i M.: S y n t h e s i s and e v a l u a t i o n of
anti-I主IV-1m l l l d anti‑HSV‑1 a c t i v i ｭ t i e s of 4H-[1,2,4 ]’ triazolo[l,5 ’ a]
pyrimidin-5圃oned e r i v a t i v e s .
Arzneim。 1Forsch.D r u g . R e s . ,
52:833圃839,
2 0 0 2 .
I n a o n e p o t p r o c e d u r e , 1 8 compounds o f
7・（substituted phenyl)-2-substi旬ted-6,7-dihydro-4H-[ 1 , 2 , 4 ] t r i a z o l o [ 1 , 5 ‑ a ]
pyrimidin・5-oned e r i v a t i v e s
(16・33)h a v e b e e n s y n t h e s i z e d .
3(5)-Amino-5(3)-substituted-l,2,4・triazoled e r i v a ｭ t i v e s
(7・ 12)w e r e u s e d a s s y n t h o n e s w h i c h w e r e c y c l o c o n d e n s e d by f u s i o n w i t h s u b s t i t u t e d m e t h y l c i n n a m a t e e s t e r s ( 1 3 ‑ 1 5 ) t o a f f o r d t h e t a r g e t compounds ( 1 6 ‑ 3 3 ) . I n a n e f f o r t t o d e v e l o p new n o n ‑ n u c l e o s i d e a n t i v i r a l a g e n t s , comｭ p o u n d s 1 6 ‑ 3 3 w e r e e v a l u a t e d f o r t h e i r a n t i ‑ H I V ‑ 1 a n d a n t i ‑ H S V ‑ 1 a c t i v i t i e s . C o m p l e t e i n h i b i t i o n o f t h e p r o l i f e r a t i o n o f H I V ‑ 1 v i r u s e s was a c h i e v e d by compounds 2 2 , 2 3 a n d 2 4 a t c o n c e n t r a t i o n s o f 2 5 , 2 5 a n d 5 0 ｵ g / m l , r e s p e c t i v e l y .
7-Phenyl-2・（n-pentyl）幽：6,7・dihydro-4H-[1 ム4]
t r i a z o l o [ 1 , 5 ‑ a ]
pyrimidinふone·( 1 9 ) e x h i b i t e d p o t e n t i a l a c t i v i t y a g a i n s t
HSV・ 1
w i t h 88% r e d u c t i o n i n t h e v i r a l p l a q u e s . The s u g g e s t e d marked s p e c i f i c i t y o f t h i s c l a s s o f compounds a s a n t i ｭ
HIV・ 1
a g e n t s i s d i s c u s s e d .
2) Takahashi K . , Ouyang X . , Komatsu K . , Nakamura N . , Hattori M . , B a l h > a A . , and Azuma J . : Sodium tanshinone H A s u l f o n a t e derived from Danshen ( S a l v i a m i l t i o n h i z a ) a t t e n u a t e s hyｭ
pe凶rophy
induced by a n g i o t e n s i n I I i n c u l t u r e d neonantal r a t c a r d i a c c t e U s . B i o c h e m . P h a r m . ,
64:745司750,
2 0 0 2 .
和漢薬研究所年報 29巻（2002年） p.88 参照．
3) Kuboyama K . , Tohda C . , Zhao J . , NakamuraｷN., H a t t o r i M . , and Komaltsu K . : Axon‑o i r denｭ
drite-predomina~t
outgrowth induced by c o n s t i t u e n t s from A s h w a g a m l l l h ! a . N e o u r o c h e m i s t r y ,
13:1715・1720,
2 0 0 2 .
和漢薬研究所年報 29巻（2002年） p.87 参照．
4 ) Xie L . , Ahn E . , . Akao T . ,
Abdel・HafezA. A . , Nakamura N . , and H a t t o r i M . : Transformation of a r c t i i n t o
e~trogenicand a n t i e s t r o g e n i c s u b s t a n c e s by human i n t e s l t i i n a l b a c t e r i a . Chem.
Pharm. B u l l . , 5 1 :
378・384,2 0 0 3 .
A f t e r a n a e r o b i c i n c u b a t i o n o f a r c t i i n ( 1 ) f r o m t h e s e e d s o f A r c t i u m Z a p p a w i t h a
huma~f e c a l s u s p e n s i o n , s i x m e t a b o ｭ l i t e s w e r e f o r m e d , a n d t h e i r s t r u c t u r e s w e r e i d e n t i f i e d a s
（十arctigenin( 2 ) ,
(2R,3R）ふ（3’，4'-dihydroxybe回yl)・3-(3”,4"-dimethoxybenzyl)butyrolactone ( 3 ) ,
(2R,3R)-2・（3 ’－hydroxybenzyl）・3・（3”，4”－dimethoxyben勾rl)butyrolactone( 4 ) ,
(2R,3R)-2・（3'-hydroxybenzyl)-3・（3”－hydroxy-4”－me出oxybenzyl)butyrolactone( 5 ) ,
(2R,3R)-2・（3’－hydroxybe回yl)-3-(3”,4”-dihydroxybenzyl)butyrolactone ( 6 ) , a n d
（ー）－enterolactone( 7 ) by v a r i o u s s p e c t r o s c o p i c means i n c l u d i n g t w o
d i m e n s i o n a l (2D)‑NMR, mass
spectrome甘y,a n d c i r c u l a r d i c h r o i s m . A p o s s i b l e m e t a b o l i c p a t h w a y was p r o p o s e d o n
t h e b a s i s o f t h e i r s t r u c t u r e s a n d t h e t i m e c o u r s e ‑ < : > f t h e ‑ t r a n s f o r m a t i o n . E n t e r o l a c t o n e s
obtained 仕omt h e
b i o t r a n s f o r m a t i o n o f a r c t i i n a n d s e c o i s o l a r i c i r e s i n o l d i g l u c o s i d e (SDG, f r o m t h e s e e d s o f L i n u m u s i t a t i s s i u m ) by
human i n t e s t i n a l b a c t e r i a w e r e p r o v e d t o b e e n a n t i o m e r s , w i t h t h e
（・）幽（2R,3R)a n d
（＋）ー（2S~3S)c o n f i g u r a t i o n s , r e s p e c ｭ
t i v e l y . Compound 6 showed t h e m o s t p o t e n t p r o l i f e r a t i v e e f f e c t on t h e g r o w t h o f MCF‑7 human b r e a s t c a n c e r ｷ c e l l s
i n c u l t u r e among 1 a n d s i x m e t a b o l i t e s , w h i l e i t showed i n h i b i t o r y a c t i v i t y on
es仕adiol-mediatedp r o l i f e r a t i o n o f
MCF‑7 c e l l s a t a c o n c e n t r a t i o n o f 1 0 ｵM. ｷ T h e s e r e s u l t s i n d i c a t e t h a t t h e t r a n s f o r m a t i o n o f ] ̲ by i n t e s t i n a l f l o r a m i g h t
b e e s s e n t i a l f o r t h e m a n i f e s t a t i o n o f t h e e s t r o g e n i c a n d a n t i e s t r o g e n i c a c t i v i t y o f 1 .
Me Me
‑Glc
H
田ーーー・’FArc t i i n ( ‑ ) ‑ A r c t i g e n i n
H H
‑Me
－ーーー”F －－一ー’P‑Me
OMe
AM3‑Ml F i g . 1
H
・Me
H
－ー－－’ー
AM2
H
~
OH
AM4
H
‑OH
－－－－’ー
（ー）εnterolactone OMe
AM3
5) Tewtrakul S . , Miyashiro H . , Nakamura N . , H a t t o r i M., Kawabata T . , Otake T . , Yoshinaga T . , Fujiwara T . , Supavita T . , Yuenyongawad S . , Rattanasuwon P . , and Dej‑Adisai S . :
HIV・i n t e g r a s e i n h i b i t o r y s u b s t a n c e s from C o l e u s p a r v i f o l i u s . P h y t o t h e r . R e s . ,
17:232・239,2 0 0 3 . F o r t h e p u r p o s e o f d i s c o v e r i n g a n t i ‑ H I V ‑ I
agents 企omn a t u r a l s o u r c e s , w a t e r a n d ｷ E t O H e x t r a c t s o f 5 0 T h a i p l a n t s w e r e ｷ s c r e e n e d f o r t h e i r i n h i b i t o r y a c t i v i t y a g a i n s t HIV‑I i p . t e g r a s e ( I N ) , a n
en勾rmee s s e n t i a l . f o r v i r a l r e p l i c a t i o n . Of t h e s e p l a n t s , a n EtOH e x t r a c t o f C o l e u s p a r v i f o l i u s B e n t h . ( a e r i a l p a r t s ) showed p o t e n t a c t i v i t y a g a i n s t HIV‑1 IN w i t h a n I C s o v a l u e o f 9 . 2 ｵ g / m L . From t h i s e x t r a c t , 1 1 compounds w e r e i s o l a t e d a n d i d e n t i f i e d a s l u t e o l i n
5-0・P・n ‑ g l u c o p y r a n o s i d e ( i ) , l u t e o l i n ( 2 ) , l u t e o l i n 7 ‑ m e t h y l e t h e r ( 3 ) , l u t e o l i n
5・0・f3-n-glucuronide( 4 ) , 5 ‑ 0 ‑ f 3 ‑ D ｭ
glucopyranosyl・luteolin 7・methyl
e t h e r ( 5 ) , r o s m a r i n i c a c i d ( 6 ) , r o s m a r i n i c a c i d m e t h y l e s t e r ( 7 ) , d a u c o s t e r o l ( 8 ) , a m i x t u r e
ofα， andf 3 ‑ a m y r i n s ( 9 , 1 0 ) a n d p h y t o l ( 1 1 ) . Of t h e s e c o m p o u n d s , r o s m a r i n i c a c i d m e t h y l e s t e r ( 7 ) , r o s m a r i n i c a c i d ( 6 ) , l u t e o l i n ( 2 ) a n d l u t e o l i n
7・methyle t h e r ( 3 ) e x h i b i t e d i n h i b i t o r y a c t i v i t i e s a g a i n s t HIV‑1 IN w i t h I C s o ‑ v a l u e s o f 3 . 1 , 5 . 0 , 1 1 . 0 a n d 1 1 . 0 ｵM, r e s p e c t i v e l y . Among r o s m a r i n i c a c i d d e r i v a t i v e s , t h e
HIV・ 1IN i n h i b i t o r y
~ctivity
i n c r e a s e d
in 旬mf o r a d i m e r ( I C s o
=5 . 0 ｵ M ) , a t r i m e r ( I C s o
=1 . 4 ｵM), a n d a t e t r a m e r ( I C s o
=1 . 0 ｵ M ) . 6) Xie L . , Akao T . , Hamasaki K . , Deyama T . , and Hattori M.: BiotransformatiOn of p i n o r e s i n o l
d i g l u c o s i d e t o mammalian l i g n a n s by human i n t e s t i n a l m i c r o f l o r a , and i s o l a t i o n of E n t e r o c o c c u s f a e c a l i s s t r a i n
PDG圃1r e s p o n s i b l e f o r t h e transformation of ( + ) ‑ p i n o r e s i n o l t o
（＋）ーlariciresinol.
Chem. Pharm. B u l l . , 5 1 :
508・515,2 0 0 3 .
By a n a e r o b i c i n c u b a t i o n o f p i n o r e s i n o l d i g l u c o s i d e ( 1 ) f r o m t h e b a r k o f Eucommia u l m o i d e s w i t h a f e c a l s u s p e n s i o n o f h u m a n s , e l e v e n m e t a b o l i t e s w e r e f o r m e d , a n d t h e i r s t r u c t u r e s w e r e i d e n t i f i e d a s ( + ) ‑ p i n o r e s i n o l ( 2 ) , (
＋）ーl a r i c i r e s i n o l ( 3 ) , 3 ’- demethyl-( + ) ‑ l a r i c i r e s i n o l ( 4 ) ,
（ー）~secoisolariciresinol( 5 ) ,
（ー）－3”（3”， 4”－dihydroxybenzyl)-2・（4’－h y d r o x y ‑ 3 ' ‑ m e t h o x y b e n z y l ) b u t a n e ‑ 1 , 4 ‑ d i o l ( 6 ) ,
2-(3’，4’－dihydroxyben勾rl)-3_-(3’’( 7 ) ,
3 －（~”－hydroxybenzyl)-2-( 4’－hydroxy回3’－methoxyben可l)butane-1,4・diol( 8 ) ,
.2・（3' , 4
'-dihydroxyben勾rl）・3-(3”－h y d r o x y b e n z y l ) b u t a n e ‑ 1 , 4 ‑ d i o l ( 9 ) ,
（ー）－enterodiol( 1 0 ) ,
（ー）ー（2R,3R)-3.:(3”，4"-dihydroxybenzyl)-2・（4’－hydroxy-3 ＇幽m e t h o x y b e n z y l } ‑ b u t y r o l a c t o n e ( 1 1 ) ,
（ー）ー（2R,3R)-f,田（3 ’，4'-dihydroxyben勾rl)-3-(3 ”，4＂四dihydroxybenzyl ) b u t y r o l a c t o n e
( 1 2 ) ,
（－）ー（2R,3R)-3・（3”－hydroxyben勾rl)-2・（4’－hydroxy-3にmethoxyben勾rl)butyrolactone( 1 3 ) ,
2・（3’，4'-dihydroxybenzyl）ー3・（3”－hydroxybenzyl)butyrolactone
( 1 4 ) , 2-(3 ’- hydroxybenzyl)-3-(3 ’’, 4"-dihydroxybenzyl)butyrolactone ( 1 5 ) a n d ( ‑ ) ( 2 R , 3 R ) ‑ e n t e r o l a c t 0 n e ( 1 6 ) by v a r i o u s s p e c t r o s c o p i c m e a n s , i n c l u d i n g two d i m e n s i o n a l (2D)‑NMR, mass
spec仕ome甘y
a n d . c i r c u l a r d i c h r o i s m . A p o s s i b l e m e t a b o l i c p a t h w a y was p r o p o s e d o n t h e b a s i s o f t h e i r
s甘UC旬resa n d t i m e
coぽse
e x p e r i m e n t s m o n i t o r e d by t h i n ‑ l a y e r c h r o m a t o g r a p h y . F u r t h e r m o r e , a b a c t e r i a l s t r a i n r e s p o n s i b l e f o r t h e t r a n s f o r m a t i o n o f ( + ) ‑ p i . n o r e s i n o l t o ( + ) ‑ l a r i c i r e s i n o l was i s o l a t e d f r o m a human f e c a l s u s p e n s i o n a n d i d e n t i f i e d a s E n t e r o c o c c u s f a e c a l i s s t r a i n
PDG・ 1.~·：））~~ ~
J50G~~lc 0 . J 5 0 H
2H凧；。。；H MeO.心不~~·MeM.~OX)工 : : J 0 1 : 5
eO.‑ Hか
PDG 1
F i g . 2
(+)‑Pinoresinol 2
9
例Larici;"sino~ロ 6 〆 Il10H
：：ぺ： t~~ ) : p " : 1 :
ｷ‑oH/ 7
~｛・｝・Enterodiol
10 ｛・）－Enterolactone
16 '15 14
7) Tan.aka N . , Sekiya N . , Hattori M., Goto H . , Shibahara N . , Shimada
y。， andTerasawa K . : Measurement of
plasm~procyanidin
B圃2a n ｫ : f procyanidin
B・3l e v e l s a f t l e i r o r a l a d m i n i s t r a t i o n i n r a t . P h y t o m e d i c i n e , 1 0 : 1 2 2 ‑ 1 2 6 , 2 0 0 3 .
U s i n g a h i g h ‑ p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c method a n d m a s s s p e c t r o m e t r y a n a l y s i s , we s u c c e s s f u l l y m e a s ｭ u r e d t h e a b s o r p t i o n o f o r a l l y a d m i n i s t e r e d p r o c y a i l i d i n
B・2a n d p r o c y a n i d i n B ‑ 3 i s o l a t e d
仕omC i n n a m o n o i m i C o r t e x ( t h e b a r k o f Cinnamomum c a s s i a Blume) i n t h e r a t p l a s m a . T h i s method u s e d a TSK‑GEL
ODS・SOTSc o l u m n , t w o s o l v e n t s ( A : 0 . 0 1 % a c e t i c a c i d ; B : m e t h a n o l w i t h ‑ 0 . 0 1 % a c e t i c a c i d ) i n a l i n e a r g r a d i e n t a t a f l o w ‑ r a t e o f 1 . 0 m l / m i n , a n d f l u o r e s c e n c e d e t e c t i o n a t e x c i t a t i o n a n d e m i s s i o n w a v e l e n g t h s o f 2 2 0 a n d 3 2 7 n m .
8) Min B . , Lee S . , Kim J . , Lee J . , Kim T . , Kim D . , Kim Y . , Joung H . , Lee H . , Nakamura N . , , Miyashiro H . ; and Hattori
M~ ：Anti‑complement a c t i v i t y o f c o n s t i t u e n t ｧ from t h e stem‑bark of Jug/ans m a n d s h u r i c a . B i o l . P h a n n , . B u l l . , 26:1042‑1044, 2 0 0 3 .
F o u r known f l a v o n o i d s a n d two g a l l o y l g l u c o s e s
Isolated 企omt h e s t e m ‑ b a r k o f J u g / a n s m a n d s h u r i c a
(Juglandacea吋， namely
t a x i f o l i , n ( 1 ) , a f z e l i n ( 2 ) , q u e r c i t r i n ( 3 ) , m y r i c i t r i n ( 4 ) ,
1,2,6・trigalloylglucose( 5 ) , a n d
1,2ム6・tetragalloylglucose
( 6 ) , w e r e e v a l u a t e d f o r t h e i r a n t i ‑ c o m p l e m e n t a c t i v i t y a g a i n s t a c o m p l e m e n t s y s t e m .
A f z e l i n ( 2 ) a n d q u e r c i t r i n ( 3 ) showed i n h i b i t o r y a c t i v i t y a g a i n s t t h e c o m p l e m e n t s y s t e m w i t h 50% i n h i b i t o r y c o n c e n ｭ
t r a t i o n ( I C s o ) v a l u e s o f 2 5 8 a n d 4 4 0 ｵM.
1,2,6・Trigalloylglucose( 5 ) a n d 1 , 2 ; 3 , 6 ‑ t e t r a g a l l o y l g l u c o s e ( 6 ) e x h i b i t e d
a n t i ‑ c o m p l e m e n t a c t i v i t y w i t h I C s o ｷ v a l u e s o f 1 3 6 a n d 3 4 ｵM. I n t e r m s o f t h e e v a l u a t i o n o f t h e
struc旬re-activityr e l a t i o n s h i p o f
3 ム7・trihydroxyflavone,compounds 2 , 3 , a n d 4 w e r e h y d r o l y z e d w j t h n a r i n g i n a s e t o g i v e k a e m p f e r o l ( 2 a ) , q u e r c e t i n ( 3 a ) , a n d m y r i c e t i n ( 4 a ) a s t h e i r a g l y c o n e s , a n d t l i e s e w e r e a l s o . t e s t e d f o r t h e i r a n t i ‑ c o m p l e m e n t
ac・t i v i t y . Of t h e t h r e e a g l y c o n e s , k a e m p f e r o l ( 2 a ) e x h i b i t e d weak a n t i ‑ c o m p l e m e n t a c t i v i t y w i t h a n I C s o v a l u e o f 7 3 0 ｵM, w h i l e q u e r c e t i n ( 3 a ) a n d m y r i c e t i n ( 4 a ) w e r e i n a c t i v e i n t h i s a s s a y s y s t e m . Among t h e compounds t e s t e d ,
1 ム3,6・tetragalloylglucose
( 6 ) showed t h e m o s t p o t e n t a n t i c o m p l e m e n t a c t i v i t y ( I C s o , 3 4 ｵM).
9) Tazawa T . , Zhao H . , Li Y . , Meselhy M. R . , Nakamura N . , Akao T . , and Hattori M.: A new enzyme immunoassay f o r a c o n i t i n e and i t s a p p l i c a t f o n t o q u a n t i t a t i v e determination of a c o n i t i n e l e v e l s i n plasma. B i o l . P h f ! ‑ r m . B u l l . ,
26:1289・1294,2 0 0 3 .
A r e l i a b l e enzyme i m m u n o a s s a y ( E I A ) method was d e v e l o p e d f o r q u a n t i t a t i v e d e t e r m i n a t i o n o f a c o n i t i n e w i t h h i g h s e n s i t i v i t y a n d s p e c i f i c i t y . The b . o v i n e s e r u m a l b u m i n ( B S A ) ‑ a n d p ‑ g a l a c t o s i d a s e ( p ‑ G a l ) c o n u g a t e s a s immunogens a n d e n z y m e ‑ l a b e l e d a n t i g e n s w e r e p r e p a r e d by c o u p l i n g o f t h e i r p r o t e i n s w i t h s u c c i n i c a c i d ( s h o r t c h a i n l e n g t h ; n = 2 , w h e r e n r e p r e s e n t s t h e number o f m e t h y l e n e u n i t s ) a n d h e x a d e c a n e d i o i c a c i d ( l o n g c h a i n l e n g t h ; n = 1 4 ) h e m i e s t e r s o f b e n z o y l a c o n i n e t h r o u g h t h e r e s p e c t i v e N ‑ h y d r o x y s u c c i n i m i d e e s t e r s a s i n t e r m e d i a t e s . Two t y p e s o f t h e B S A ‑ c o n j u a g a t e s w i t h s h o r t a n d l o n g c h a i n s w e r e r e p e a t e d l y i n j e c t e d i n t o
rabbit~t o o b t a i n a n t i ‑, a c o n i t i n e a n t i s e r a ( A s l a n d A s 2 , r e s p e c t i v e l y ) . A l l c o m b i n a t i o n s o f P ‑ G a l ‑ l a b e l e d a n t i g e n s LAgl ( n = 2 ) a n d LAg2 ( n = l 4 ) w i t h a n t i s e r a Asl ( n = 2 ) a n d As2 ( n = 1 4 ) showed h i g h s e n s i t i v i t y t o a c o n i t i n e i n a r a n g e o f
0. ト 1.0n g . A l t h o u g h t h e c o m b i n a t i o n o f LAg2 ( n = l 4 ) w i t h a n t i s e r u m Asl ( n = 2 ) showed h i g h s p e c i f i c i t y t o a c o n i t i n e , t h e comｭ b i n a t i o n o f LAg2 ( n = 1 4 ) a n d As2 ( n = l 4 ) was h i g h l y s p e c i f i c t o b o t h a c o n i t i n e and m e s a c o n i t i n e . When a c o n i t i n e was i n t r a v e n o u s l y a d m i n i s t e r e d t o r a t s , t h e a c o n i t i n e c o n c e n t r a t i o n i n t h e i r p l a s m a r e m a r k a b l y d e c r e a s e d w i t h i n t h e f i r s t 6 0 m i n , a n d t h e n g r a d u a l l y d e c l i n e d , s u g g e s t i n g a t w o ‑ c o m p a r t m e n t p h a r m a c o k i n e t i c model i n (Ve 0 . 4 1 ｱ 0 . 0 9 1 / k g ,
Vdss1 . 7 ｱ 0 . 4 1 / k g , C L t o t
10 土 2 ml/min ・ kg,A U C o ‑ 4 s o o 2055ｱ294.3
ng ・ min/ml).F o l l o w i n g o r a l a d m i n i s t r a ‑ t i o n o f a c o n i t i n e t o r a t s a t two d o s e s o f 0 . 1 a n d 1 . 0 mg/kg b . w . , t h e maximum p l a s m a
concen仕ations( C m a x ) w e r e
0.73 土 0.08
a n d
3.3 土 0·.6n g / m l a t t i m e s o f 4 5 ｱ 9 a n d
150 土 52m i n , r e s p e c t i v e l y , a n d t h e
AUCo・1440v a l u e s w e r e 130ｱ4and,1600ｱ270
ng ・ min/ml.The b i o a v a i l a v i l i t y
（丹 ofa c o n i t i n e was d e t e r m i n e d t o b e 0 . 0 1 3 , w h e r e o n l y 1 . 3 % o f t h e a c o n i t i n e a d m i n i s t e r e d o r a l l y was a b s o r b e d i n t o t h e bo4y f l u i d .
1 0 ) Zhao J . , Sun X . , Nakamura N . , Zuo F . , Yang X . ; , Akao T . , and H a t t o r i M.: Development o f an enzyme immunoassay system f o r mesaconitine and i t s a p p l i c a t i o n t o t h e d i s p o s i t i o n study on m e s a c o n i t i n e . J . T r a d . M e d . ,
20:201・207,2 0 0 3 .
F o r t h e p u r p o s e o f q u a n t i t a t i v e d e t e r m i n a t i o n of m e s a c o n i t i n e , a n e x t r e m e l y t o x i c a n d major
‘alkaloid i n a c o n i t e t u b e r , a h i g h l y s e n s i t i v e enzyme i m m u n o a s s a y ( E I A ) was d e v e l o p e d a s f o l l o w s ; F i r s t l y , a h a p t e n [ N ｭ d e s e t h y l a c o n i t i n e N ‑ g l u t a r a t e (DEAG)J was s y n t h e s i z e d by i n t r o d u c t i o n o f a g l u t a r y l g r o u p t o t h e n i t r o g e n a t o m o f a c o n i t i n e a f t e r . e l i m i n a t i o n o f a n N ‑ e t h y l g r o u p . S u b s e q u e n t l y i t was c o u p l e d w i t h b o v i n e s e r u m a l b u m i n (BSA) a n d p ‑ g a l a c t o s i d a s e ( p ‑ G a l ) t o g i v e a n immunogen (DEAG‑BSA) a n d a l a b e l e d a n t i g e n
(DEAG・P・Gal),r e s p e c t i v e l y . A f t e r i m m u n i z a t i o n Of f e m a l e a l b i n o r a b b i t s w i t h DEAG‑BSA f o r s i x m o n t h s t o e l i c i t a p o l y c l o n a l a n t i s e r : u m ( A s ｭ DEAG), t h e e x p e r i m e n t a l c o n d i t i o n s w e r e o p t i m i z e d f o r a h i g h l y s e n s i t i v e E I A . At 1 0 3 ‑ f o l d d i l u t i o n o f DEAG‑P‑Gal a n d 2 . 5 ﾗ 1 0 4 ‑ f o l d d i l u t i o n
・ofAs‑DEAG, t h e a s s a y e x h i b i t e d a l i n e a r r a n g e o f 0 . 0 0 5 ‑ 5 n g / t u b e f o r m e s a c o n i t i n e . Under t h e same c o n d i t i o n s , t h e a n t i s e r u m h a d a 2 4 . 4 % c r o s s ‑ r e a c t i o n w i t h a c o n i t i n e a n d v i r t u a l l y n o r e a c t i o n w i t h b e n z o y l m e s a c o n i n e .
N e x t , t h e EIA method was a p p l i e d t o t h e d i s p o s i t i o n s t u d y on m e s a c o n : i t i n e t o m o n i t o r t h e p l a s m a c o n c e n t r a t i o n
a f t e r o r a l a d m i n i s t r a t i o n o f m e s a c o n i t i n e a t 1 mg/kg t o r a t s , i t s low b i o a v a i l a b i l i t y b e i n g
suggested 企omC m a x a n d
A UC v a l u e s o b t a i n e d .
1 1 ) Ahn E . , Nakamura N . , Akao T . , Komatsu K . , Qui M., and Hattmri M.: J P ' r e n y l a t e d f l a v o n o i d s from Moghania p h i l i p p i n e n s i s . P h y t o c h e m i s t r y ,
64:1389・1394,2 0 0 3 .
F i v e p r e n y l a t e d f l a v o n o i d s ,
8・（ 1,1 ・dimethylallyl)genistein( 1 ) ,
5,7,3’，4’－tetrahydroxy♂’，5'-di(3-methylbut-2・enyl)‑ i s o f l a v o n e ( 2 ) ,
5,7,3'-trihydroxy-2'-(3・methylbut-2・enyl）・4',5'-(3,3-dimethylpyrano) i s o f l a v o n e ( 3 ) ,
(2R）・5,2',4’－trihydroxy- 8,5’－di(3-methylbut-2-enyl)・6,7・（3,3・dimethylpyrano)flavanone( 4 a ) a n d
(2S)-:5,2',4’ー仕ihydroxy-"8，ターdi(3-methylbut- 2・enyl)-6,7-(3,3-dimethylpyrano)f l a v a n o n e ( 4 b ) , w e r e i s o l a t e d f r o m t h e r o o t s o f Moghania
phi!伊']Jinensis.The
S仕uctures
o f t h e s e compounds w e r e d e t e r m i n e d on t h e b a s i s o f s p e c t r o s c o p i c a n d c h e m i c a l m e a n s .
1 2 ) Takako K . , Jyo M., Nakamura N . , Komatsu K . , H a t t o r i M., S h D . m o t o l n ( [ ] ) n o K . , Shimotohono K . , and Kakiuchi N . : I n h i b i t o r y e f f e c t of Tibetan medicinal
pla四tson v i r a l polymerases. J . T r a d . Med.,
20:243・250,2 0 0 3 .
F o r t h e
puゅoseo f d e v e l o p m e n t o f n o v e l a n t i ‑ v i r u s a g e n t s f r o m e t h n i c a l d r u g s , we e x a m i n e d 7 6 t r a d i t i o n a l T i b e t a n m e d i c i n e s f o r i n h i b i t o r y e f f e c t on two v i r a l e n z y m e s , r e v e r s e t r a n s c r i p t a s e ( R T a s e ) o f HIV a n d RNA d e p e n d e n t RNA p o l y m e r a s e ( R d R p ) o f HCV. A l t h o u g h 2 8 m e t h a n o l
ex仕actsi n h i b i t e d RTase more t h a n 70% a t a c o n c e n t r a ｭ t i o n o f 1 0 0 ｵ g / m l , o n l y 3 s a m p l e s ,
T42（蔵青果， Terminaliac h e b u l a R E T Z . ) , T46
（横榔， Arecac a t e c h u L . ) a n d T 6 1
（紅横榔），
w e r e f o u n d s t i l l i n h i b i t o r y a f t e r e l i m i n a t i n g t h e e f f e c t o f t a n n i n s by a d d i t i o n o f BSA i n t h e
en勾rmer e a c ｭ t i o n
mixtur・e.I n t h e c a s e o f t h e RdRp, 7 e x t r a c t s ( I C s o v a l u e s o f l e s s t h a n 1 0 ｵ g / m l ) c o n t a i n e d l e s s t h a n 20% t a n n i n s . The e x t r a c t o f R h o d i o l a s a c r a , whose I C s o f o r RTase was 2 5 . 9 ｵ g / m l , was s u b j e c t t o p h y t o c h e m i c a l i n v e s t i g a t i o n . Out of 8 compounds i s o l a t e d f r o m t h e e x t r a c t , d a u c o s t e r o l was f o u n d e f f e c t i v e f o r R T a s e .
1 3 ) Lipipun V . , Kurokawa M., S u t t i s r i R . , Taweehotipatr P . , Pramyothinn P . , Hattori M., and S h i r a k i K . : E f f i c a c y of Thai medicinal p l a n t e x t r a c t s a g a i n s t herpes s i m r n ] p l e x v i r u s type 1
in圃f e c t i o n i n v i t r o and i n v i v o . A n t i v i r a l
Research~ 60:175・180,2 0 0 3 .
Twenty T h a i m e d i c i n a l p l a n t e x t r a c t s w e r e e v a l u a t e d f o r a n t i ‑ h e r p e s s i m p l e x v i r u s t y p e 1 ( H S V ‑ 1 ) a c t i v i t y . E l e v e n o f them i n h i b i t e d . P l a q u e f o r m a t i o n o f HSV‑1 more t h a n 50% a t 1 0 0 ｵ g / m l i n a p l a q u e r e d u c t i o n a s s a y .
Aglaiαo d o r a t a , Moringa o l e i f e r a , a n d V e n t i l a g o d e n t i c u l a t e among t h e 1 1 w e r e a l s o e f f e c t i v e a g a i n s t t h y m i d i n e k i n a s e ｭ d e f i c i e n t HSV‑1 a n d
phosphonoacet~te-resistant HSV」 strains. . T h e s e t h e r a p e u t i c e f f i c a c i e s w e r e c h a r a c t e r i z e d u s i n g a c u t a n e o u s
HSV・ 1i n f e c t i o n i n m i c e . The e x t r a c t o f M o l e i f e r a a t a d o s e o f 7 5 0 mg/kg p e r d a y s i g n i f i c a n t l y d e l a y e d t h e d e v e l o p m e n t o f s k i n l e s i o n s , p r o l o n g e d t h e mean s u r v i v a l t i m e s a n d r e d u c e d t h e m o r t a l i t y o f HSV‑1
irトf e c t e d m i c e a s c o m p a r e d w i t h 2% DMSO i n d i s t m e d w a t e r (P < 0 . 0 5 ) , The e x t r a c t s o f A . o d o r a t a a n d V d e n t i c u l a t e w e r e a l s o s i g n i f i c a n t l y e f f e c t i v e i n l i m i t i n g t h e d e v e l o p m e n t o f s k i n l e s i o n s (P < 0 . 0 5 ) . T h e r e w e r e no s i g n i f i c a n t d i f f e r e n c e b e t w e e n a c y c l o v i r a n d t h e s e t h r e e p l a n t
ex仕actsi n t h e d e l a y o f t h e d e v e l o p m e n t o f s k i n l e s i o n s a n d no s i g n i f i c a n t d i f f e r e n c e b e t w e e n a c y c l o v i r a n d M o l e i f e r a i n mean s u r v i v a l t i m e s . T o x i c i t y ｷ o ( t h e s e p l a n t e x t r a c t s w e r e n o t o b s e r v e d i n t r e a t e d m i c e . T h u s , t h e s e t h r e e p l a n t e x t r a c t s may b e p o s s i b l e c a n d i d a t e s o f
anti-HSV・1a g e n t s .
1 4 ) Park J . C . , Miyashiro
H~，and H a t t o r i M.: I n h i b i t o r y e f f e c t s of m d l l u a n o l e x t r a c t s from Korean medicinal p l a n t s a g a i n s t HIV‑1 p r o t e a s e a c t i v i t y . Korean J . M r e d . Crop S c i . , 1 1 : 2 6 4 ‑ 2 6 7 , 2 0 0 3 .
K o r e a n m e d i c i n a l p l a n t s w e r e s c r e e n e d f o r t h e i r i n h i b i t o r y a c t i v i t y a g a i n s t HIV‑ l p r o t e a s e . The i n h i b i t o r y a c t i v i t y o f p r o t e a s e was d e t e r m i n e d by i n c u b a t i n g t h e e x t r a c t s i n r e a c t i o n m i x t u r e s c o n t a i n i n g p r o t e a s e a n d s u b s t r a t e H i s ｭ
Lys-Ala-Arg-Val・Leu-(p-N02-Phe）四Glu-Ala-Nle-Ser-NH2
t o p e r f o r m p r o t e o l y t i c c l e a v a g e r e a c t i o n s . I n t h i s s t u d y t h e
t w e n t y s i x e x t r a c t s f r o m m e d i c i n a l p l a n t s w e r e i n v e s t i g a t e d . Of t h e e x t r a c t s t e s t e d , t h e e x t r a c t s f r o m t h e s t e m o f
Morus a l b a e x h i b i t e d t h e s t r o n g e s t a c t i v i t y w i t h i n h i b i t i o n o f 8 1 % a t a c o n c e n t r a t i o n o f 1 0 0 ｵ g / m l . The e x t r a c t s o f
t h e f l o w e r o f
Si似折。ga stolonifer，α， ands t e m s o f Euonymus j a p o n i c a a n d C a s t a m e a c r e n a t a showed a p p r e c i a b l e i n ｭ
h i b i t o r y a c t i v i t y (>50%) a g a i n s t HIV‑1 p r o t e a s e a t t h e same c o n . c e n t r a t i o n .
1 5 ) Yokozawa T . , Rhyu D . Y . , Cho E . J . , Aoyagi K . : P r o t e c t i v e A c t i v i t y of
（ー）－Epicatecbin 3・0圃g a l l a t e a g a i n s t
Peroxynitrite圃mediatedRenal Damage. F r e e
Radi，ι Res., 37:561・571,2 0 0 3 . The p r o t e c t i v e e f f e c t o f
（ー）－epicatechin 3-0・gallate( E C g ) a g a i n s t p e r o x y n i t r i t e
(ONOO・）－mediateddamage was e x ‑ ‑ a m i n e d ｷ u s i n g a n a n i m a l r r i o d e l a n d a c e l l c u l t u r e s y s t e m . I n r a t s s u b j e c t e d t o l i p o p o l y s a c c h a r i d e ( L P S ) a d m i n i s t r a ‑ t i o n p l u s i s c h e m i a ‑ r e p e r f u s i o n , t h e p l a s m a
3-ni仕otyrosi_nel e v e l , a n i n d i c a t o r o f ONOO‑p r o d u c t i o n i n
viνo,was e l e v a t e d , w h e r e a s i t d e c l i n e d s i g n i f i c a n t l y a n d d o s e ‑ d e p e n d e n t l y a f t e r t h e o r a l a d m i n i s t r a t i o n o f ECg a t d o s e s o f 1 0 a n d 20 mmoles/kg body w e i g h t / d a y f o r 2 0 d a y s p r i o r t o t h e p r o c e s s . M o r e o v e r , o r a l a d m i n i s t r a t i o n o f ECg s i g n i f i ｭ c a n t l y e n h a n c e d t h e a c t i v i t i e s ｷ o f t h e a n t i o x i d a n t e n z y m e s , s u p e r o x i d e d i s m u t a s e , c a t a l a s e a n d g l u t a t h i o n e p e r o x i d a s e , a n d t h e a n t i o x i d a n t g f o t a t h i o n e , s h o w i n g e n h a n c e m e n t o f t h e b i o l o g i c a l d e f e n s e s y s t e m a g a i n s t t h e damage i n d u c e d by ONOO‑. I n a d d i t i o n , t h e s i g n i f i c a n t i n c r e a s e i n t h e r e n a l m i t o c h o n d r i a l t h i o b a r b i t u r i c a c i d ‑ r e a c t i v e s u b s t a n c e l e v e l o f LPS a n d ischemic
”reperfused c o n t r o l r a t s was a t t e n u a t e d i n r a t s g i v e n ECg. F u r t h e r m o r e , t h e
elevatio~si n t h e p l a s m a u r e a n i t r o g e n a n d c r e a t i n i n e ( C r ) l e v e l s a n d t h e u r i n a r y m e t h y l g u a n i d i n e / C r r a t i o i n d u c e d by t h e
proce・d u r e w e r e a t t e n u a t e d m a r k e d l y a f t e r o r a l a d m i n i s t r a t i o n o f ECg, i m p l y i n g a m e l i o r a t i o n o f r e n a l i m p a i r m e n t . The a d ｭ d i t i o n o f ECg ( 2 5 o r 1 2 5 mM) p r i o r t o 3 ‑ m o r p h o l i n o s y d n o n i m i n e ( S I N ‑ 1 , 8 0 0 mM) e x p o s u r e r e d u c e d ONooｭ f o r m a t i o n a n d . i n c r e a s e d t h e v i a b i l i t y o f c u l t u r e d r e n a l e p i t h e l i a l ‑ ( L L C ‑ P K 1 ) c e l l s i n a d o s e ‑ d e p e n d e n t m a n n e r . I n p a r t i c u l a r , ECg i n h i b i t e d ONoo‑‑ m e d i a t e d a p o p t o t i c c e l l d e a t h , w h i c h was c o n f i r m e d by d e c r e a s e s ｷ i n t h e DNA f r a g m e n t a t i o n r a t e a n d t h e p r e s e n c e o f a p o p t o t i c m o r p h o l o g i c a l c h a n g e s , i . e . , s m a l l n u c l e i a n d n u c l e a r f r a g m e n t a t i o n . F u r t h e r m o r e , a d d i n g ECg b e f o r e S I N ‑ I t r e a t m e n t r e g u l a t e d t h e c e l l ‑c y c l e by e n h a n c i n g G2/M p h a s e
a町est.T h i s s t u d y p r o v i d e s e v i d e n c e t h a t ECg h a s p r o t e c t i v e a c t i v i t y a g a i n s t t h e r e n a l damage i n d u c e d ‑by e x c e s s i v e ONOO‑i n c e l l u l a r a n d i n v i v o s y s t e m s .
1 6 ) Yokozawa T . , I s h i d a A . , Cho E . J . , Nakagawa T . ｷ : The e f f e c t s of C o p t i d i s Rhizoma e x t r a c t on
I
A h y p e r c h o l e s t e r o l e m i c animal model. P h y t o m e d i c i n e , 1 0 : 1 7 ‑ 2 2 , 2 0 0 3 .
The s e r u m c h o l e s t e r o l ( t o t a l , f r e e , e s t e r i f i e d , low d e n s i t y l i p o p r o t e i n (LDL) a n d o x i d i z e d LDL) l e v e l s o f r a t s f e d a d i e t c o n t a i n i n g , by w e i g h t , 1% c h o l e s t e r o l a n d 0.5% c h o l i c a c i d i n c r e a s e d c o m p a r e d w i t h t h o s e o . f r a t s f e d a n o r m a l d i e t . H o w e v e r , t h e l e v e l s , e s p e c i a l l y , o f t o t a l c h o l e s t e r o l , LDL a n d o x i d i z e d LDL, w e r e s i g n i f i c a n t l y r e d u c e d , i n a d o s e ‑ d e p e n d e n t m a n n e r , i n r a t s g i v e n C o p t i d i s Rhizoma e x t r a c t o r a l l y a t d o s e s ̲ o f 5 0 a n d 1 0 0 mg/kg body
weight/day仁f o r 3 0 d a y s . T h i s r e s u l t i n d i c a t e s t h a t C o p t i d i s Rhizoma e x t r a c t i s e f f e c t i v e i n r e d u c i n g t h e p a t h o l o g i c a l damage c a u s e d by h y p e r c h o l e s t e r o l e m i a t h r o u g h l o w e r i n g s e r u m c h o l e s t e r o l l e v e l s . I n a d d i t i o n , C o p t i d i s Rhizoma e x t r a c t r e d u c e d t h e l e v e l o f l i v ｷ e r c h o l e s t e r o l , w h e r e a s i t d i d n o t r e d u c e t h a t o f f e c a l c h o l e s t e r o l , s u g g e s t i n g t h a t t h e c h o l e s ｭ t e r o l l e v e l ‑ l o w e r i n g e f f e c t r e s u l t e d f r o m t h e r e d u c t i o n o f c h o l e s t e r o l s y n t h e s i s , n o t t h e e n h a n c e m e n t o f i t s
excr~tion.F u r t h e r m o r e , t h e s e r u m t h i o b a r b i t u r i c a c i d ‑ r e a c t i v e s u b s t a n c e l e v e l d e c r e a s e d a f t e r o r a l a d m i n i s t r a t i o n o f C o p t i d i s R h i : z o m a e x t r a c t , i n d i c a t i n g
’that C o p t i d i s Rhizoma c o u l d p r e v e n t h y p e r c h o l e s t e r o l e m i c d i s e a s e t h r o u g h r e d u c i n g l i p i d p e r o x i d a t i o n . T h i s s t u d y d e m o n s t r a t e s t h a t C o p t i d i s Rhizoma may b e a u s e f u l t h e r a p y f o r h y p e r c h o l e s t e r o l e m i a by r e d u c i n g o x i d a t i v e s t r e s s a n d c h o l e s t e r o l l e v e l s .
1 7 ) Yokozawa T . , Cho E . J . , Nakagawa T . : Influence of green t e a polyphenol i n r a t s with a r g i n i n e ‑ induced r e n a l f a i l u r e . . 1 . ｷ A g r i c . Food C h e m . ,
51:2421圃2425,2 0 0 3 .
To d e t e r m i n e w h e t h e r g r e e n t e a p o l y p h e n o l a m e l i o r a t e s t h e p a t h o l o g i c a l c o n d i t i o n s i n d u c e d by e x c e s s i v e d i e t a r y a r g i n i n e , g r e e n t e a p o l y p h e n o l was a d m i n i s t e r e d t o r a t s a t a d a i l y d o s e o f 5 0 o r 1 0 0 mg/kg body w e i g h t f o r 3 0 d a y s w i t h a 2% w/w a r g i n i n e d i e t . I n a r g i n i n e ‑ f e d c o n t r o l r a t s , u r i n a r y a n d / o r s e r u m l e v e l s o f g u a n i d i n o c o m p o u n d s , n i t r i c o x i d e ( N O ) , u r e a , p r o t e i n a n d g l u c o s e i n c r e a s e d s i g n i f i c a n t l y , w h i l e t h e r e n a l a c t i v i t i e s o f t h e o x y g e n s p e c i e s ｭ s c a v e n g i n g enzymes s u p e r o x i d e d i s m u t a s e (SOD) a n d c a t a l a s e d e c r e a s e d , c o m p a r e d w i t h c a s e i n ‑ f e d r a t s . H o w e v e r ,
r剖s
g i v e n g r e e n t e a p o l y p h e n o l showed s i g n i f i c a n t a n d d o s e ‑ d e p e n d e n t d e c r e a s e s i n s e r u m
lev~lso f c r e a t i n i n e ( C r )
and 町ea
n i t r o g e n a n d u r i n a r y e x c r e t i o n
o~C r , a n d t h e y e x e r t e d a s l i g h t r e d u c t i o n o f n i t r i t e p l u s n i t r a t e , i n d i c a t i n g
t h a t g r e e n t e a p o l y p h e n o l r e d u c e d t h e p r o d u c t i o n o f u r e m i c t o x i n s a n d NO. : I n a d d i t i o n , i n a r g i n i n e ‑ f e d r a t s t h e u r i ｭ n a r y u r e a , p r o t e i n a n d . g l u c o s e l e v e l i n c r e a s e s w e r e r e v e r s e d by t h e
adminis仕ationo f g r e e n t e a p o l y p h e n o l . M o r e o v e r , i n r a t s g i v e n g r e e n t e a p o l y p h e n o l t h e SODｷ a n d c a t a l a s e a c t i v i t i e s s u p p r e s s e d by e x c e s s i v e a r g i n i n e a d ｭ m i n i s t r a t i o n i n c r e a s e d d o s e ‑ d e p e n d e n t l y , i m p l y i n g t h e b i o l o g i c a l d e f e n s e s y s t e m was a u g m e n t e d a s a r e s u l t
of 仕切r a d i c a l s c a v e n g i n g . T h e s e r e s u l t s s u g g e s t t h a t g r e e n t e a p o l y p h e n o l would a m e l i o r a t e r e n a l f a i l u r e i n d u c e d by e x c e s ｭ s i v e
dietaηra r g i n i n e by d e c r e a s i n g u r e m i c t o x i n , a n d NO p r o d u c t i o n a n d i n c r e a s i n g r a d i c a l ‑ s c a v e n g i n g e r i z y m e a c ｭ t i v i t y .
1 8 ) Nakagawa T . , Yokozawa T . , Terasawa K . , Nakanishi K . : Therapeutic
~sefulnessof
Keishi・bukuryo・gan
f o r d i a b e t i c nephropathy. J . Pharm. P h a r m a c o l . ,
55:219~Z27,2 0 0 3 .
K e i s h i ‑ b u k u r y o ‑ g a n i s a t r a d i t i o n a l h e r b a l . m e d i c i n e , w h i c 4 i s u s e d c l i n i c a l l y a s a v a s c u l a r s y s t e m
disorder~e l i m i n a t i n g d r u g . I n t h i s
s旬dy,i t s e f f e c t s on t h e p r o g r e s s i o n o f
diabeti~n e p h r o p a t h y i n e x p e r i m e n t a l r a t s w e r e i n v e s ‑ t i g a t e d . The d i a b e t i c n e p h r o p a t h y m o d e l ｷ u s e d i n t h i s s t u d y s h o w s f u n c t i o n a l a n d
·mo中hologicalc h a n g e s o f t h e k i d n e y r e s e m b l i n g t h o s e s e e n i n p a t i e n t s w i t h d i a b e t i c n e p h r o p a t h y . I n c r e a s e d p r o t e i n u r i a a n d s e r u m u r e a n i t r o g e n a n d c r e a t i n i n e l e v e l s a n d d e c r e a s e d c r e a t i n i n e c l e a r a n c e , w h i c h a r e i m p o r t a n t p a r a m e t e r s o f r e n a l f u n c t i o n , w e r e o b ｭ s e r v e d i n r a t s w i t h d i a b e t i c n e p h r o p a t h y . P a t h o l o g i c a l e x a m i n a t i o n . o f t h e k i d n e y r e v e a l e d d i f f u s e , n o d u l a r a n d e x u d a t i v e l e s i o n s a n d a r t e r i o l a r h y a l i n ( ) s i s . The d e t e r i o r a t i o n o f r e n a l f u n c t i o n was a m e l i o r a t e d i n r a t s t r e a t e d w i t h K e i s h i ‑ b u k u r y o ‑ g a n f o r 1 5 weeks a n d t h e s e r e s u l t s a g r e e d w i t h t h e r e n a l h i s t o l o g i c a l f i n d i n g s . I n a d d i t i o n , m e t a b o l i c a b n o r m a l i t i e s m e d i a t e d by p e r s i s t e n t h y p e r g l y c e m i a ( t h e g l y c a t i o n r e a c t i o n , e x c e s s i v e p o l y o l p a t h w a y a c t i v i t y , o x i ｭ d a t i v e s t r e s s a n d l i p i d m e t a b o l i c a b n o r m a l i t i e s ) w e r e a l s o o b s e r v e d . H o w e v e r ,
Keishi°'bukuryo・ganr e d u c e d a c c u m u ｭ l a t i o n o f a d v a n c e d g l y c a t i o n e n d ‑ p r o d u c t s , d e t e r m i n e d by m e a s u r i n g f l u o r e s c e n c e , a n d s e r u m l i p i d p e r o x i d a t i o n ,
仕iglyceride
a n d t o t a l c h o l e s t e r o l l e v e l s d o s e ‑ 4 e p e n d e n t l y . T h u s , t h i s s t u d y i n d i c a t e s t h e p o t e n t i a l t h e r a p e u t i c u s e f u l ｭ n e s s o f
Keishi・bukuryo・gan-for. r e t a r d i n g t h e p r o g r e s s i o n o f r e n a l ｷ d a m a g e . a n d s u g g e s t s t h a t i t s b e n e f i c i a l e f f e c t s w e r e d u e t o i t s a b i l i t y t o i m p r o v e m e t a b o l i c a b n o r m a l i t i e s a s s o c i a t e d w i t h d i a b e t e s .
1 9 ) Yokozawa T . , Rhyu D . Y . , Chen C . P . : . P r o t e c t i v e e f f e c t s of
Acant~（］）]panaxRadix e x t ̲ r a c t a g a i n s t endotoxemia induced by l i p o p o l y s a c c h a r i d e .
Ph~totheれ Res., 17:353・357,2 0 0 3 . E n d o t o x e m i a c a u s e s a n e n h a n c e d p r o d u c t i o n o f r e a c t i v e o x y g e n r a d i c a l s , w h i c h c o n t r i b u t e t o m u l t i p l e o r g a n d y s ｭ f u n c t i o n . When r a t s w e r e g i v e n i n t r a v e n o u s l i p o p o l y s a c c h a r i d e a n d t e s t e d 6 h l a t e r we f o u n d t h a t t h e a c t i v i t i e s o f c a t a l a s e a n d
glutathion~p e r o x i d a s e (GSH‑Px) i n k i d n e y , w e r e a c u t e l y s u p p r e s s e d w h i l e i n s e r u m t h e l e v e l s of n i t r i c o x i d e ( N O ) , l i p i d p e r o x i d a t i o n , u r e a n i t r o g e n a n d c r e a t i n i n e w e r e
signi白candyi n c r e a s e d , i n d i c a t i n g t h e e x c e s s i v e p r o d u c t i o n o f r e a c t i v e . o x y g e n r a d i c a l s a n d t h e p r e s e n c e o f r e n a l i n j u r y . P r e t r e a t m e n t o f r a t s w i t h A c a n t h o p a n a x R a d i x e x t r a c t a d m i n i s t e r e d o r a l l y f o r 3 0 d a y s r e d u c e d t h e NO a n d l i p i d p e r o x i d a t i o n l e v e l s , i n c r e a s e d t h e a c t i v i t i e s o f c a t a l a s e a n d GSH‑Px, a n d a t t e n u a t e d t h e r e n a l d y s f u n c t i o n . t h e s e r e s u l t s s u g g e s t e d t h a t s c a v e n g i n g o f r e a c t i v e o x y g e n r a d i c a l s i s p a r t o f t h e mechanism by w h f o h A c a n t h o p a n a x R a d i x e x t r a c t w o r k s a s a n e f f e c t i v e a g e n t i n p r e ｭ v e n t i n g m u l t i p l e o r g a n d y s f u n c t i o n .
2 0 ) Kim H Y . . . , Yokozawa T . , Cho
Eム CheighH . S . , C h o i .
Jふ， ChungH.Y。： InV i t r o and i n V i v o
Antioxidant e f f e c t s of mustard l e a f ( B r a s s i c a J u n c e a ) . P h y t o t h e r . R e s . ,
li7:465・471,2 0 0 3 .
To i n v e s t i g a t e t h e a n t i o x i d a n t a c t i v i t y o f m u s t a r d l e a f ( B r a s s i c a j u n c e a ) , we p r e p a r e d
four 企actions( C H 2 C b , EtOAc,
BuOH
andH20 丘actions)a n d e x a m i n e d t h e i r r a d i c a l s c a v e n g i n g a c t i v i t i e s i n v i t r o a n d i n v i v o . B a s e d on t h e
in νitror e s u l t s o f s p i n t r a p p i n g a n d 1 ,
1 ・diphenyl-2・picrylhydrazylr a d i c a l , we
’·carried o u t a n i n v i v o s t u d y w i t h t h e BuOH
f r a c t i o n t o i n v e s t i g a t e i t s e f f e c t on o x i d a t i v e s t r e s s i n r a t s w i t h s t r e p t o z o t o c i n ‑ i n d u c e d d i a b e t e s . We f o u n d t h a t i n
c o m p a r i s o n w i t h u n t r e a t e d d i a b e t i c c o n t r o l r a t s , o r a l a d m i n i s t r a t i o n o f t h e BuOH f r a c t i o n ( 1 0 0 o r 2 0 0 mg/kg body
w e i g h t / d a y f o r 1 0 d a y s ) i n d u c e d a s i g n i f i c a n t d e c r e a s e i n s e r u m g l u c o s e a n d g l y c o s y l a t e d p r o t e i n , which i s
g l y c o s y l a t e d w i t h h e m o g l o b i n a s a n i n d i c a t o r o f o x i d a t i v e
s仕ess.M o r e o v e r , a d m i n i s t r a t i o n o f t h e
BuOH 合actiona l s o e f f e c t i v e l y r e d u c e d t h e s e r u m s u p e r o x i d e a n d n i t r i t e / n i t r a t e l e v e l s . F u r t h e r m o r e , t h e l e v e l s o f t h i o b a r b i t u r i c a c i d ｭ r e a c t i v e s u b s t a n c e s i n s e r u m a n d l i v e r w e r e a l s o s i g n i f i c a n t l y l o w e r t h a n i n t h e c o n t r o l g r o u p . T h e s e r e s u l t s i n d i c a t e t h a t t h e BuOH f r a c t i o n o f m u s t a r d l e a f c o n t r o l s g l u c o s e m e t a b o l i s m a n d r e d u c e s l i p i d p e r o x i d a t i o n a s w e l l a s t h e l e v e l o f o x y g e n r a d i c a l s , a m e l i o r a t i n g t h e damage c a u s e d by o x i d a t i v e s t r e s s i n d i a b e t e s .
2 1 ) Yokozawa T . , Kim H . Y . , Cho E . J . , Yamabe N . , Choi J . S . : P r o t e c t i v e e f f e c t s of mustard ‑ l e a f ( B r a s s i c a juncea) a g a i n s t d i a b e t i c o x i d a t i v e s t r e s s . J . N u t r . S c i . V i t a m i n o l . , 4 9 : 8 7 ‑ 9 3 , 2 0 0 3 . Of
four 企actions(CH2Ch, e t h y l a c e t a t e ( E t O A c ) , b u t a n o l (BuOH) a n d
H20）仕omm u s t a r d l e a f ( B r a s s i c a j u n c e a ) , t h e EtOAc f r a c t i o n showed t h e s t r o n g e s t i n h i b i t o r y e f f e c t s , w h i c h w e r e c o n c e n t r a t i o n ‑ d e p e n d e n t , on t h e f o r m a t i o n o f a d v a n c e d g l y c a t i o n e n d p r o d u c t s a n d
合eer a d i c a l ‑ m e d i a t e d p r o t e i n damage i n a n i n v i t r o s y s t e m , i n d i c a t i n g a p o ｭ t e n t i a l p r o t e c t i v e r o l e o f
this 企actiona g a i n s t d i a b e t e s a n d / o r i t s c o m p l i c a t i o n s . B a s e d on t h e s e r e s u l t s , we c a r r i e d o u t a n i n v i v o s t u d y t o d e t e r m i n e w h e t h e r t h e
EtOAc 丘actionp r o t e c t e d a g a i n s t d i a b e t i c 9 , X i d a t i v e s t r e s s i n d u c e d by s t r e p t o z o t o c i n . O r a l a d m i n i s t r a t i o n o f t h e EtOAc f r a c t i o n a t d o s e s o f 5 0 a n d 2 0 0 mg/kg body w e i g h t / d a y ‑ f o r 1 0 d a y s r e d u c e d s e r u m l e v e l s o f g l u c o s e a n d g l y c o s y l a t e d p r o t e i n , i m p l y i n g t h a t t h e i m p a i r e d g l u c o s e m e t a b o l i s m d u e t o d i a ｭ b e t e s h a d b e e n a m e l i o r a t e d . I n a d d i t i o n , t h e
EtOAc 丘actions i g n i f i c a n t l y r e d u c e d t h e t h i o b a r b i t u r i c a c i d ‑ r e a c t i v e s u b s t a n c e l e v e l s o f s e r u m a n d h e p a t i c a n d r e n a l m i t o c h o n d r i a . F u r t h e r m o r e , t h e e l e v a t e d l e v e l s o f s u p e r o x i d e a n d n i ｭ t r i t e / n i t r a t e w e r e r e d u c e d , i n a d o s e ‑ d e p e n d e n t m a n n e r , by o r a l a d m i n i s t r a t i o n of t h e
EtOAc 仕action.T h e s e f i n d i n g s s u g g e s t t h a t t h e
EtOAc 企action 合omm u s t a r d l e a f m i g h t b e b e n e f i c i a l i n a t f o n u a t i n g t h e damage c a u s e d by o x i d a t i v e s t r e s s i n v o l v e d i n d i a b e t e s a n d i t s c o m p l i c a t i o n s .
2 2 ) Cho E . J . ; Yokozawa T . , Rhyu D . Y . , Kim S . C . , Shibahara N . , Park J . C . : Study on t h e i n h i b i ｭ t o r y e f f e c t s of Korean medicinal p l a n t s and t h e i r main compounds on t h e
1,1・diphenyl-2・p i c r y l h y d r a z y l r a d i c a l . Phytomedinine,
10:544・551,2 0 0 3 .
A 1 ,
l-diphenyl-2・picrylhydrazyl(DPPH) r a d i c a l ‑ g e n e r a t i n g s y s t e m was u s e d t o e v a l u a t e t h e a n t i o x i d a n t p r o p e r t i e s o f K o r e a n m e d i c i n a l p l a n t s , w h i c h h a v e b e e n w i d e l y u s e d a s f o l k m e d i c i n e s f o r s e v e r a l d i s o r d e r s , a n d compounds i s o l a t e d f r o m t h e m . Among t h e . R o s a c e a e , Rosa r u g o s a a n d Rosa d a v u r i c a showed s t r o n g DPPH r a d i c a l ‑ s c a v e n g i n g a c t i v i t y . The m o s t e f f e c t i v e m e d i c i n a l p l a n t o f t h e f a m i l i e s o t h e r t h a n R o s a c e a e was C e d r e l a s i n e n s i s , f o l l o w e d i n o r d e r by Nelumbo n u c i f e r a , Eucommia u l m o i d e s , Z a n t h o x y l u m p i p e r i t u m , C u d r a n i a t r i c u s p i d a t a a n d H o u t t u y n i a
cordαta.
T h e s e r e s u l t s s e r v e a s a good i n d e x o f
the 企eer a d i c a l ‑ s c a v e n g i n g a c t i v i t i e s o f K o r e a n m e d i c i n a l p l a n t s . F u r t h e r m o r e , t h e p o l y p h e n o l s i s o l a t e d
合omt h e s e
plants~p r o c y a n i d i n B ‑ 3 , ( + ) ‑ c a t e c h i n , g a l l i c a c i d , m e t h y l g a l l a t e , q u e r c e t i n , q u e r c e t i n
3・0-P-n-glucoside,q u e r c e t i n 3 ‑ 0 ‑ P ‑ n ‑ g a l a c t o s i d e , q u e r c e t i n 3 ‑ 0 ‑ r u t i n o s e a n d k a e m p f e r o l , e x ｭ t ; r t e d s t r o n g DPPH r a d i c a l ‑ s c a v e n g i n g a c t i v i t y . T h e s e r e s u l t s s u g g e s t t h a t t h e K o r e a n m e d i c i n a l p l a n t s a n d p o l y p h e n o l s i s o l a t e d f r o m them t h a t e x h i b i t e d e f f e c t i v e r a d i c a l ‑ s c a v e n g i n g a c t i v i t y may b e p r o m i s i n g a g e n t s f o r s c a v e n g i n g f r e e r a d i c a l s a n d t r e a t i n g d i s e a s e s a s s o c i a t e d w i t h
excess 企eer a d i c a l s .
2 3 ) Nakagawa T . , Y okozawa T . , Oowada S . , Goto
HィShibaharaN . , Shimada Y . , Terasawa K . : Amelioration of kidney damage i n spontaneously d i a b e t i c WBN/Kob r a t s a f t e r treatment with
̲ K e i s
hi占ukuryo・gan.J . T r a d . Med., 2 0 :
156・164,2 0 0 3 .
I n t h i s s t u d y , we i n v e s t i g a t e d w h e t h e r
Keishi・~bukuryo・ganc a n r e t a r d t h e
occu町encea n d p r o g r e s s i o n o f d i a b e t i c
n e p h r o p a t h y i n s p o n t a n e o u s l y d i a b e t i c WBN/Kob r a t s . A d m i n i s t r a t i o n o f
Keishi・bukuryo-gand i d n o t a f f e c t body
w e i g h t l o s s o r b l o o d g l u c o s e l e v e l s b u t e f f e c t i v e l y l o w e r e d u r i n a r y p r o t e i n e x c r e t i o n a n d s e r u m c r e a t i n i n e l e v e l s , a n d
a m e l i o r a t e d g l o m e r u l a r , v a s c u l a r a n d t u b u l o i n t e r s t i t i a l l e s i o n s . I n a d d i t i o n , t r e a t m e n t o f t h e d i a b e t i c r a t s w i t h K e i s h i ｭ
b u k u r y o ‑ g a n r e d u c e d r e n a l l e v e l s o f t h i o b a r b i t u r i c a c i d r e a c t i v e s u b s t a n c e s a n d a d v a n c e d g l y c a t i o n e n d p r o d u c t s s i g ｭ
n i f i c a n t l y a n d e l e v a t e d r e n a l s u p e r o x i d e d i s m u t a s e a c t i v i t y s i g n i f i c a n t l y . T h e s e r e s u l t s s u g g e s t t h a t K e i s h i ‑ b u k u r y o ‑
gan e x e r t s a n t i o x i d a n t e f f e c t s i n t h e k i d n e y s o f d i a b e t i c s . a n d may p r o v e t h a t t h e h e r b a l m e d i c i n e i s u s e f u l f o r i n h i b ｭ i t i n g t h e p r o g r e s s i o n o f d i a b e t i c k i d n e y d i s e a s e .
24) Yokozawa T . , Rhyu D . Y . , Cho. E . J . : Protection by the Chinese p r e ｧ ( C r i p t i o n We‑Pi‑Tang a g a i n s t r e n a l tubular LLC‑PK1 c e l l damage induced by
3・morpholinosydnonimine.J . Phann.
P h a r m a c o l . ,
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We i n v e s t i g a t e d t h e e f f e c t s o f Wen‑Pi‑Tang e x t r a c t on t h e p r o t e c t i v e mechanisms o f r e n a l t u b u l a r LLC‑PK1 c e l l s , a s
renal 旬bularc e l l s a r e t h e most v u l n e r a b l e r e n a l t i s s u e t o o x i d a t i v e
s仕ess.E x p o s u r e t o 800 ｵM 3 ‑ m o r p h o l i n o s y d n o n i m i n e ( S I N ‑ 1 ) r e s u l t e d i n a marked i n c r e a s e i n c e l l u l a r p e r o x y n i t r i t e (ONOO‑) t h a t c o n v e r t e d n o n f l u o r e s c e n t d i h y d r o r h o d a m i n e 1 2 3 t o f l u o r e s c e n t r h o d a m i n e 1 2 3 , a d e t e c t a b l e p r o b e f o r t h e l o n g ‑ l i v e d ONOO-.
>I n a d d i t i o n , i t r e s u l t e d i n a p o p t o t i c c e l l d e a t h , a s s e s s e d by a
DNA 丘agmentationa s s a y .
However，住eatmentw i t h Wen‑Pi‑Tang e x t r a c t , a t c o n c e n t r a t i o n s o f 5 0 and 1 0 0 ｵ g / m l , t o g e t h e r w i t h S I N ‑ 1 p r o t e c t e d r e n a l t u b u l a r c e l l s a g a i n s t
ONOO・ throughs c a v e n g i n g
ONOO・ andi n h i b i t i n g a p o p t o t i c c e l l d e a t h i n a d o s e ‑ d e p e n d e n t I l l : a n n e r . M o r e o v e r , t r e a t m e n t w i t h Wen-Pi
”Tang e x t r a c t b o t h b e f o r e and a f t e r e x p o s u r e t o S I N ‑ 1 was a l s o p r o t e c t i v e : i t
re・d u c e d c e l l u l a r ONOO‑l e v e l s , i n c r e a s e d c e l l v i a b i l i t y and d e c r e a s e d t h e
DNA 企agmentationr a t e . T h e s e r e s u l t s s u g ‑ g e s t t h a t Wen‑Pi‑Tang would b e e x p e c t e d t o h a v e p r o t e c t i v e a c t i v i t y a g a i n s t ONoo
- ”induced r e n a l t u b u l a r i n j u r y t h r o u g h t h e i n h i b i t i o n o f ONOO‑p r o d u c t i o n and a p o p t o t i c c e l l d e a t h by b o t h p r e v e n t i n g
and 仕eatingr e n a l
i吋町－F u r t h e r m o r e , m o r p h o l o g i c a l c h a r a c t e r i s t i c s o f a p o p t o s i s were o b s e r v e d i n S I N ‑ 1 t r e a t e d t u b u l a r c e l l s , w h i l e t h e
ad・d i t i o n o f
Wen-Pi・Tange x t r a c t w i t h S I N ‑ 1 a t t e n u a t e d t h e s e m o r p h o l o g i c a l c h a n g e s .
ONOO~g e n e r a t e d by S I N ‑ 1 a l s o d i s t u r b e d t h e c e l l c y c l e by d e c r e a s i n g t h e c e l l u l a r G2/M p h a s e r a t i o , w h i l e W
erト·Pi-Tange x t r a c t r e g u l a t e d t h e c e l l c y c l e by
G2／乱fp h a s e a r r e s t .
25) Soung D.Y., Rhee S . H . , Kim J . S . , Lee J . Y . , Yang H . S . , Choi J . S . , Y o k ( [ ] ) z a w a T . , Han Y . N . , Chung H . Y . : ｷ P e r o x y n i t r i t e scavenging a c t i v i t y of lithospermate B from S a l v i a
miltiorrhiza. よPharm. P h a r m a c o l . ,
55:1427・1432,2003.
Perox戸ii甘ite (ONOO・）
