化学応用部門

(1)

化学応用部門

教授門田重利（薬学博士）

助教授畑中保丸（薬学博士）助手手塚康弘（薬学博士）技官長岡武馬（薬学修士）

。研究目的

本部門では，化学的手法を応用する和漢薬の基礎研究として，天然薬物を中心とする生理活性分子の医薬化学的及び生物有機化学的研究を行っている。即ち，天然、薬物の成分単離，構造解析，

合成等の，和漢薬成分に関する化学的研究を行う。さらに，その過程で構造が明らかとなる天然薬物成分について，その構造・活性相関，構造・機能相関の化学的解明に取り組んでいるo本年度の主な研究課題は下記の通りである。

。研究概要

I.天然薬物成分の単離，構造解析，合成，作用

1

）人参，丹参，草豆蓮，蒙蓋等の和漢生薬

2

）インドネシア，ベトナム，ミャンマー，ネパル等の薬用植物

3

）爵香から単離した新規成分ムスクライド類の合成及び誘導体化

4

）肝臓病や骨粗懸症に有効な天然薬物成分の開発研究

II.薬物・生体高分子相互作用系の生物有機化学

1）構造・機能相関解析に有用な独自の化学的手法の開発

2

）機能性生体高分子の構造生物学

上記の研究課題によって得られた本年度の成果（原著及び学会報告）は下記の通りである。

(2)

。原著

1 ) Fan W., Tezuka Y . , Komatsu K . , Namba T . , and Kadota S . : P r o l y l Endopeptidase I n h i b i t o r s from Underground Part o f Rhodiola s α e r αS. H. F u . B i o l . Pharm. B u l l . , 2 2 : 1 5 7 ‑ 1 6 1 , 1 9 9 9 .

Summary: P r o l y l e n d o p e p t i d a s e (PEP, EC 3 . 4 . 2 1 . 2 6 ) i s an enzyme which p l a y s a r o l e i n t h e metabolism o f p r a l i n e ‑ c o n t a i n i n g n e u r o p e p t i d e s , e . g . , v a s o p r e s s i n , s u b s t a n c e P and t h y r o t r o p i n ‑ r e l e a s i n g hormone (TRH), which have b e e n s u g g e s t e d t o b e i n v o l v e d i n l e a r n i n g and memory p r o c e s s e s . I n our s y s t e m a t i c s c r e e n i n g f o r PEP i n h i b i t o r s from t r a d i t i o n a l C h i n e s e m e d i c i n e s , we found t h a t MeOH e x t r a c t from t h e underground p a r t o f R h o d i o l a s a c r αS. H. Fu shows s i g n i f i c a n t 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 PEP from Fl α v o bαc t e r i u m m e n i n g o s e p t i c u m . Examination o f t h e c o n s t i t u e n t s o f t h e e x t r a c t r e s u l t e d i n t h e i s o l a t i o n o f n i n e t e e n known compounds, i d e n t i f i e d a s hydroquinone ( 1 ) , 4 hydroxybenzoic a c i d ( 2 ) , c a f f e i c a c i d ( 3 ) , 4‑hydroxycinnamic a c i d ( 4 ) , s u b e r i c a c i d ( 5 ) , p r o t o c a t e c h u i c a c i d ( 6 ) , g a l l i c a c i d ( 7 ) , ( ) ‑ e p i g a l l o c a t e c h i n 3‑0

^宇

g a l l a t e( 8 ) , 2 ‑ p h e n y l e t h y l / 3 D g l u c o p y r a n o s i d e ( 9 ) , 3 ‑ 0 ‑ g a l l o y l e p i g a l l o c a t e c h i n ( 4 β

→

8 ) ‑ e p i g a l l o c a t e c h i n 3 ‑ 0 ‑ g a l l a t e ( 1 0 ) , 2 ‑ p h e n y l e t h y l α

ーし

a r a b i nopyranosy 1 ‑( 1

→

6 ) ‑ / 3 ‑ D g l u c o p y r a n o s i d e ( 1 1 ) , s a c r a n o s i d e A ( 1 2 ) , / 3 D ‑ g l u c o p y r a n o s y l 4 ‑ hydroxybenzoate ( 1 3 ) , r h o d i o c y a n o s i d e A ( 1 4 ) , r h o d i o o c t a n o s i d e ( 1 5 ) , sarmentosin ( 1 6 ) , h e t e r o d e n d r i n ( 1 7 ) , a r b u t i n ( 1 8 ) and 4‑0

（−

β D ‑ g l u c o p y r a n o s y l ) ‑ g a l l i c a c i d ( 1 9 ) . Among t h e s e , 1 , 2 , 5 , 8 1 0 , 1 3 , 1 6 , 1 8 and 1 9 have been i s o l a t e d f o r t h e f i r s t t i m e from R . s α e r α

，

among which 5 , 9 , 1 0 , 1 3 , 1 6 , 1 8 and 1 9 have been i s o l a t e d from R h o d i o l a p l a n t s f o r t h e f i r s t t i m e . On t h e PEP i n h i b i t i o n , s e v e n compounds ( 6 8 , 1 0 , 1 2 , 1 8 , 1 9 ) showed i n h i b i t i o n with an I C 5 : o f 2 7 . 8 , 4 8 7 , 1 . 4 7 , 0 . 4 3 7 , 3 4 8 , 3 9 1 and 2 1 5 μ M, r e s p e c t i v e l y . The k i n e t i c study o f t h e s e i n h i b i t o r s i n d i c a t e d t h a t they a r e n o n c o m p e t i t i v e i n h i b i t o r s , e x c e p t f o r 6 which i s a c o m p e t i ‑ t i v e i n h i b i t o r .

2 ) Tezuka Y . , Terazono M . , Kusumoto

I.

T . , Kawashima Y . , Hatanaka Y . , Kadota S . , Hattori M . , Namba T . , Kikuchi T . , Tanaka K . , and Supriyatna S . : H e l i s t e r c u l i n s A and B , Two New ( 7 . 5

，

8 . 2

ト

Neolignans, and H e l i s o r i n , t h e F i r s t ( 6 . 4

，

7 . 5

，

8 . 2

）自

Neolignan, from Indonesian Medicinal Plant H e l i c t e r e s i s o r α . H e l v . Chim. A c t a , 8 2 : 4 0 8 ‑ 4 1 7 , 1 9 9 9 .

Summary : During a c h e m i c a l s t u d y o f I n d o n e s i a n m e d i c i n a l p l a n t s , we examined t h e c o n

s t i t u e n t s o f f r u i t s o f H e l i c t e r

^噌

e si s o r αL. ( S t e r c u l i a c e a e ) , one o f t h e famous Jamu m e d i c i n e s .

From a water e x t r a c t o f t h e f r u i t s , we i s o l a t e d t h r e e new n e o l i g n a n s , h e l i s t e r c u l i n s A ( 1 )

and B ( 2 ) and h e l i s o r i n ( 3 ) , and e l u c i d a t e d t h e i r s t r u c t u r e s by s p e c t r a l a n a l y s e s .

H e l i s t e r c u l i n s A ( 1 ) and B ( 2 ) a r e ( 7 . 5

，

8 . 2

）−

n e o l i g n a n s with a b i c y c l o [ 2 . 2 . 2 ] o c t e n e C

framework, w h i l e h e l i s o r i n ( 3 ) i s a ( 6 . 4 ' , 7 . 5 ' , 8 . 2

）−

n e o l i g n a n with a v e r y r a r e 4 , 4 a , 9 , 9 a ‑

t e t r a h y d r o ‑ 3 , 9 ‑ m e t h a n o ‑ 3 H ‑ f l u o r e n e C‑framework. The n a t u r a l p r o d u c t with t h e l a t t e r C

framework has no l i t e r a t u r e p r e c e d e n t . The n e o l i g n a n s 1 3 showed weak 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 r e v e r s e t r a n s c r i p t a s e from a v i a n m y e l o b r a s t o s i s v i r u s .

(3)

3 ) Prasain J . K . , Tezuka Y . , L i J . ‑ X . , Tanaka K . , Basnet P . , Dong H . , Namba T . , and Kadota S . : New Diarylheptanoid from t h e Seeds o f A l p i n i α b l e p h α r o e α l y x . Planta Medica, 6 5 : 1 9 6 , 1 9 9 9 .

Abstract: A l p i n i α b l e p h α r o e α l y x

K.

Schum. ( Z i n g i b e r a c e a e ) i s used i n t r a d i t i o n a l C h i n e s e m e d i c i n e f o r t h e treatment o f stomach d i s o r d e r ( 1 ) . We have r e c e n t l y r e p o r t e d f i f t e e n n o v e l d i a r y l h e p t a n o i d s t o g e t h e r with s i x known p h e n o l i c compounds ( 2 5 ) . Further i n v e s t i g a t i o n on t h e e t h e r s o l u b l e f r a c t i o n o f t h e s e e d s o f A . b l e p h α r o e α l y x has l e d t o t h e i s o l a t i o n o f a new d i a r y l h e p t a n o i d , 5 , 6 ‑ d e h y d r o ‑ 4

−d

e ‑ 0 ‑ m e t h y l c e n t r o l o b i n ( 1 ) .

4 ) L i H . , Miyahara T . , Tezuka Y . , Namba T . , Suzuki T . , Dowaki R . , Watanabe M., Nemoto N . , Tonami S . , Seto H . , and Kadota S . : The E f f e c t of Kampo Formulae on Bone Resorption i n V i t r o and i n V i v o . I I . D e t a i l e d Study of B e r b e r i n e . B i o l . Pharm. B u l l . , 2 2 : 3 9 1 ‑ 3 9 6 , 1 9 9 9 .

Summary: We p r e v i o u s l y i s o l a t e d b e r b e r i n e from aqueous e x t r a c t s o f t s u ‑ k a n ‑ g a n , a Kampo formula used f o r t h e treatment o f o s t e o p o r o s i s . B e r b e r i n e c a u s e d an i n h i b i t o r y e f f e c t on p a r a t h y r o i d hormone (PTH)‑stimulated bone r e s o r p t i o n i n n e o n a t a l mouse b o n e . I n t h i s r e p o r t we d e s c r i b e t h e i n h i b i t o r y e f f e c t o f b e r b e r i n e on t h e formation o f o s t e o c l a s t ‑ l i k e m u l t i n u c l e a t e d c e l l s (OCLs) i n t h e c o ‑ c u l t u r e o f mouse o s t e o b l a s t i c c e l l s and bone marrow c e l l s i n t h e p r e s e n c e o f 1 α

，

25‑dihydroxyvitamin D o [ 1 α

，

2 5 ( 0 H ) 2 D 3 ] , PTH and i n t e r l e u k i n ‑ 1α(IL 1 α

）

• B e r b e r i n e d o s e ‑ d e p e n d e n t l y i n h i b i t e d t h e formation o f t a r t r a t e ‑ r e s i s t a n t a c i d phosphatase (TRAP)‑positive OCLs i n d u c e d by l α

，

25(0H)2D3, PTH and IL‑1α. We p r e ‑ pared OCLs i n t h e c o ‑ c u l t u r e o f o s t e o b l a s t i c c e l l s and bone marrow c e l l s . The e f f e c t o f b e r b e r i n e on p i t formation by OCLs was examined using d e n t i n s l i c e s . As OCLs a r e t e r m i ‑ n a l l y d i f f e r e n t i a t e d m u l t i n u c l e a t e d c e l l s , t h e s u r v i v a l o f OCLs a f f e c t s t h e b o n e ‑ r e s o r b i n g a c ‑ t i v i t y o f OCLs. This prompted us t o count t h e number o f TRAP‑positive OCLs on t h e s l i c e s . B e r b e r i n e d o s e ‑ d e p e n d e n t l y i n h i b i t e d p i t formation and c a u s e d a d e c r e a s e i n t h e number o f TRAP‑positive OCLs. C a l c i t o n i n (CT) i n h i b i t e d p i t formation without a f f e c t i n g t h e number o f OCLs. B e r b e r i n e a c c e l e r a t e d t h e c e l l death i n OCLs c u l t i v a t e d on a c u l t u r e p l a t e , but CT d i d not a f f e c t t h e c e l l d e a t h o f OCLs. T h i s s u g g e s t s t h a t t h e d e c r e a s e i n t h e number o f OCLs on d e n t i n s l i c e s may b e due t o a p o p t o t i c c e l l death i n OCLs. I n f a c t , Hoechst 3 3 2 5 8 s t a i n i n g r e v e a l e d t h a t t h e treatment o f OCLs with b e r b e r i n e r e s u l t e d i n condensed n u c l e i and a d e ‑ c r e a s e i n c e l l s i z e . Oral a d m i n i s t r a t i o n o f t h e b e r b e r i n e ( 3 0 and 5 0 mg/kg/d) t o o v a r i e c t o m i z e d r a t s p r e v e n t e d a d e c r e a s e i n bone mineral d e n s i t y (BMD) o f t h e lumbar v e r ‑ t e b r a without a f f e c t i n g t h e weight o f t h e u t e r u s and plasma c o n c e n t r a t i o n o f e s t r a r l i o l . These r e s u l t s s u g g e s t e d t h a t b e r b e r i n e p r e v e n t e d a d e c r e a s e i n BMD i n v i v o by i n h i b i t i n g o s t e o c l a s t i c bone r e s o r p t i o n .

5 ) Stampoulis P . , Tezuka Y . , Banskota A. H . , Tran K. Q . , S a i k i I . , and Kadota S . :

Staminol A, a Novel Diterpene from Orthosiphon s t a m i n e u s . Tetrahedron L e t t . , 4 0 :

(4)

4 2 3 9 ‑ 4 2 4 2 , 1 9 9 9 .

Abstract: From t h e a e r i a l p a r t o f a Vietnamese m e d i c i n a l p l a n t , O r t h o s i p h o n s t a m i n e u s BENTH. ( L a m i a c e a e ) , staminol A ( 1 ) , a d i t e r p e n e with a n o v e l carbon framework, was i s o ‑ l a t e d t o g e t h e r with f o u r new i s o p i m a r a n e ‑ t y p e d i t e r p e n e s , o r t h o s i p h o l s F‑I ( 2 5 ) . Their s t r u c t u r e s were e l u c i d a t e d by t h e s p e c t r o s c o p i c a n a l y s e s .

6 ) Xiong Q . , Hase K . , Tezuka Y . , Namba T . , and Kadota S . : Acteoside I n h i b i t s Apoptosis i n D

^闇

Galactosamineand Lipopolysaccharide‑lnduced L i v e r I n j u r y . L i f e S c i e n c e s , 6 5 : 4 2 1 ‑ 4 3 0 , 1 9 9 9 .

Summary: We a s s e s s e d t h e e f f e c t o f a c t e o s i d e , a n a t u r a l l y o c c u r r i n g a n t i o x i d a t i v e p h e n y l e t h a n o i d , on h e p a t i c a p o p t o s i s and t h e subsequent l i v e r f a i l u r e i n d u c e d by D ‑ galactosamine (D‑GalN) and l i p o p o l y s a c c h a r i d e (LPS). A c o ‑ a d m i n i s t r a t i o n o f D‑GalN ( 7 0 0 mg/kg) and LPS ( 3 5 mg/kg) t o mice evoked t y p i c a l h e p a t i c a p o p t o s i s c h a r a c t e r i z e d by DNA fragmentation and a p o p t o t i c body f o r m a t i o n , r e s u l t i n g i n fulminant h e p a t i t i s and l e t h a l i t y o f m i c e . P r e ‑ a d m i n i s t r a t i o n o f a c t e o s i d e a t 1 0 o r 5 0 mg/kg subcutaneously a t 1 2 and 1 h p r i o r t o D GalN/LPS i n t o x i c a t i o n s i g n i f i c a n t l y i n h i b i t e d h e p a t i c a p o p t o s i s , h e p a t i t i s and l e ‑ t h a l i t y . Tumor n e c r o s i s f a c t o r

−

α

（

TNF

−

α

）

s e c r e t e d from LPS‑stimulated macrophages i s an important mediator o f a p o p t o s i s i n t h i s m o d e l . A c t e o s i d e showed no apparent e f f e c t on t h e marked e l e v a t i o n o f serum TNF

−

α

，

but i t p a r t i a l l y p r e v e n t e d i n v i t r o TNF

−

α(100 ng /ml) ‑ i n d u c e d c e l l death i n D‑GalN ( 0 . 5 μ M)‑sensi t i z e d hep a t o c y t e s a t t h e c o n c e n t r a t i o n s o f 5 0 , 1 0 0 and 2 0 0

μ

M. These r e s u l t s i n d i c a t e d t h a t D‑GalN/LPS‑induced h e p a t i c a p o p t o s i s can b e b l o c k e d by an exogenous a n t i o x i d a n t , suggesting t h e involvement o f r e a c t i v e oxygen i n t e r m e d i a t e s (ROis) i n TNF

−

α

−

dependent h e p a t i c a p o p t o s i s .

7 ) Fan W., Tezuka Y . , Xiong Q . , Hattori M., Namba T . , and Kadota S . : Apocynins A D: New Phenylpropanoid‑substituted F l a v a n ‑ 3 ‑ o l s I s o l a t e d from Leaves o f Apocynum venetum (Luobuma‑Ye). Chem. Pharm. B u l l . , 4 7 : 1 0 4 9 ‑ 1 0 5 0 , 1 9 9 9 .

Summary: Four new p h e n y l p r o p a n o i d ‑ s u b s t i t u t e d f l a v a n ‑ 3 o l s c a l l e d apocynins A ‑D Cl‑

4 ) have been i s o l a t e d from t h e l e a v e s o f Apocynum venetum ( A p o c y n a c e a e ) , t o g e t h e r with two known p h e n y l p r o p a n o i d ‑ s u b s t i t u t e d f l a v a n ‑ 3 ‑ o l s , c a t e c h i n ‑ [ 8 , 7 ‑ e ] 4 α

ベ

3 , 4dihydroxyph‑

e n y l ) dihydro‑2(3H)‑pyranone ( 5 ) and c i n c h o n a i n I a ( 6 ) , and f o u r known f l a v a n ‑ 3 ‑ o l s ,

（一）−

e p i c a t e c h i n , (

+

) ‑ c a t e c h i n , ( ) ‑ e p i g a l l o c a t e c h i n , and (

+

) ‑ g a l l o c a t e c h i n . Their s t r u c t u r e s were e l u c i d a t e d on t h e b a s i s o f s p e c t r a l a n a l y s i s , i n c l u d i n g 2D NMR and CD s p e c t r a . They showed h e p a t o 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 D ‑ g a l a c t o s a m i n e (D‑GalN) /tumor n e c r o s i s f a c t o r α (TNF

^守

α

−）

i n d u c e dc e l l death i n primary c u l t u r e d mouse h e p a t o c y t e s .

8 ) Tezuka Y . , Fan W., Kasimu R . , and Kadota S . : Screening of Crude Drug Extracts

f o r P r o l y l Endopeptidase I n h i b i t o r y A c t i v i t y . Phytomedicine, 6 : 1 9 7 2 0 3 , 1 9 9 9 .

Summary: P r o l y l e n d o p e p t i d a s e (PEP, EC 3 . 4 . 2 1 . 2 6 ) i s an enzyme t o p l a y a r o l e i n

(5)

metabolism o f p r a l i n e ‑ c o n t a i n i n g n e u r o p e p t i d e s , such a s v a s o p r e s s i n , s u b s t a n c e P and t h y r o t r o p i n ‑ r e l e a s i n g hormone (TRH), which were s u g g e s t e d t o b e i n v o l v e d w i t h l e a r n i n g and memory p r o c e s s e s . T h e n , s p e c i f i c i n h i b i t o r o f PEP i s e x p e c t e d t o have a n t i a m n e s i c e f ‑ f e c t s , and t h u s we s c r e e n e d f o r t y ‑ s i x w a t e r ‑and m e t h a n o l ‑ e x t r a c t s from c r u d e drugs s e ‑ l e c t e d on t h e b a s i s o f t r a d i t i o n a l C h i n e s e m e d i c i n e t h e o r y , f o r F l α u o bαc t e r i u m p r o l y l e n d o p e p t i d a s e i n h i b i t i o n . Among t h e m , t h e water

^四

e x t r a c t so f Rhodia

！

α s αe r α C I C 5 o , 0 . 7 7 μg /ml) and t h e m e t h a n o l ‑ e x t r a c t s o f Lycopodium c l α u αtum ( I C 5 0 , 1 . 3 μ g / m l ) , Pαe o n iα αc J t i f l o r α v a r . t r i c h o c α r p α ( I C 5 0 , 5 . 7 μ g / m l ) , Pαe o n iαu e i t c h i i ( I C 5 o , 2 . 4 μ g/ml) and R h o d i o l a

s αe r α ( I C 5 0 , 0 . 6 7 μ g/ml) showed s t r o n g i n h i b i t o r y a c t i v i t y . I n a d d i t i o n , we a l s o examined t h e PEP i n h i b i t o r y a c t i v i t y o f e l e v e n compounds from S αl u i α d e s e r tα

，

and found t h a t i n ad d i t i o n t o a c a t e c h o l groupα

−

hydroxy para q u i n o n e group may b e r e l a t e d t o t h e PEP i n h i b i ‑ t i o n .

9 ) Ohsugi M . , Fan W . , Hase K . , Xiong Q . , Tezuka Y . , Komatsu K . , Namba T . , Saitoh T . , Tazawa K . , and Kadota S . : Active‑oxygen Scavenging A c t i v i t y o f T r a d i t i o n a l N o u r i s h i n g ‑ t o n i c Herbal Medicines and A c t i v e C o n s t i t u e n t s o f Rhodiola s α e r α . J . Ethnopharmacol., 6 7 : 1 1 1 ‑ 1 1 9 , 1 9 9 9 .

A b s t r a c t : A c t i v e ‑ o x y g e n s c a v e n g i n g a c t i v i t y o f s e v e n t y 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 s used i n China and Japan a s n o u r i s h i n g ‑ t o n i c s were e v a l u a t e d by e l e c t r o n s p i n r e s o n a n c e (ESR) t e c h ‑ n i q u e , i n o r d e r t o e v a l u a t e t h e i r e f f e c t i v e n e s s f o r a n t i ‑ a g i n g and t o s e a r c h f o r new a c t i v e ‑ oxygen s c a v e n g e r s from n a t u r a l r e s o u r c e s . Most o f t h e s e v e n t y h e r b a l m e d i c i n e s showed s c a v e n g i n g a c t i v i t y with v a r i o u s i n t e n s i t i e s . E s p e c i a l l y Arec α c αt e c h u (methanol e x t r a c t ) , Dendrobium p l i c α t i l e ( m e t h a n o l ‑ e x t r a c t ) , Jug

！

α n s r e g iα

（

w a t e r ‑ e x t r a c t ) , Pαe o n iα J αc t i f l o r α

( m e t h a n o l ‑ e x t r a c t ) , P s y c h o t r i α s e r p e n s ( w a t e r and methanol e x t r a c t s ) , R h o d i o l αsαe r α ( w a t e r and m e t h a n o l ‑ e x t r a c t s ) and Unc α r iαr h y n c h o p h y l l α

（

w a t e r ‑ e x t r a c t ) showed strong s c a v e n g i n g a c t i v i t y a g a i n s t s u p e r o x i d e a n i o n r a d i c a l

・（

0 2

） , 一

w h i l e Jug

！

α n s r e g iα

（

w a t e r ‑and m e t h a n o l ‑ e x t r a c t s ) , Morusαl bα

（

water e x t r a c t ) and S c h i sαndr αc h i n e n s i s ( w a t e r e x t r a c t ) r e v e a l e d s t r o n g s c a v e n g i n g a c t i v i t y a g a i n s t hydroxyl r a d i c a l (HO

）・

• I

n

a d d i t i o n , t h e a c t i v e ‑ oxygen s c a v e n g i n g a c t i v i t i e s o f n i n e t e e n compounds i s o l a t e d from R . s α e r a was a l s o exam i n e d , and hydroquinone ( 1 ) , c a f f e i c a c i d ( 3 ) , p r o t o c a t e c h u i c a c i d ( 6 ) , g a l l i c a c i d ( 7 ) ,

（一）−

e p i g a l l o c a t e c h i n 3 ‑ 0 ‑ g a l l a t e ( 8 ) , 3 ‑ 0 ‑ g a l l o y l e p i g a l l o c a t e c h i n ‑ ( 4 β

→

8 ) ‑ e p i g a l l o c a t e c h i n 3 ‑ 0 ‑ g a l l a t e ( 1 0 ) , h e t e r o d e n d r i n ( 1 7 ) and g a l l i c a c i d 4 0 ‑ / 3

心−

g l u c o p y r a n o s i d e( 1 9 ) were found t o show m i l d o r s t r o n g 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 s u p e r o x i d e a n i o n r a d i c a l

・（

0 2) , w h i l e 4 ‑ hydroxybenzoic a c i d ( 2 ) , 3 , 4‑hydroxycinnamic a c i d ( 4 ) , 6 8and19 i n h i b i t e d hydroxyl r a d i ‑ c a l (OH

）・

• These a c t i v e ‑ o x y g e n s c a v e n g e r s may c o n t r i b u t e , with d i f f e r e n t e x t e n t , t o t h e i r a n t i ‑ a g i n g a c t i o n .

1 0 ) Stampoulis P . , Tezuka Y . , Banskota A. H . , Tran K . Q . , S a i k i I . , and Kadota S . : Staminolactone A, B and Norstaminol A: Three Highly Oxygenated Staminane‑

type D i t e r p e n e s from O r t h o s i p h o n s t a m i n e u s . Org. L e t t . , 1 : 1 3 6 7 ‑ 1 3 7 0 , 1 9 9 9 .

(6)

Abstract : S t a m i n o l a c t o n e s A ( 1 ) and B ( 2

）

ιnd n o r s t a m i n o l A ( 3 ) , t h r e e h i g h l y oxygen‑

a t e d s t a m i n a n e ‑ t y p e d i t e r p e n e s having m i l d c y t o t o x i c a c t i v i t i e s a g a i n s t h i g h l y l i v e r m e t a s t a t i c c o l o n 2 6 ‑ 1 5 carcinoma c e l l s , were i s o l a t e d from t h e a e r i a l p a r t o f Vietnamese m e d i c i n a l p l a n t O r t h o s i p h o n s tαmineus ( L a m i a c e a e ) . T h e i r s t r u c t u r e s were e l u c i d a t e d b a s e d on t h e e x t e n s i v e s p e c t r a l a n a l y s e s .

1 1 ) Gewali M. B . , Tezuka Y . , Banskota A.H., A l i M. S . , S a i k i I . , Dong H . , and Kadota S . : Epicalyxin F and Calyxin I ; Two Novel A n t i p r o l i f e r a t i v e D i a r y l h e p t a n o i d s from t h e Seeds o f A l p i n i αb l e p h α r o e α l y x . Org. L e t t . , 1 : 1 7 3 3 ‑ 1 7 3 6 , 1 9 9 9 .

A b s t r a c t : E p i c a l y x i n F ( 1 ) and c a l y x i n I ( 2 ) , two n o v e l d i a r y l h e p t a n o i d s were i s o l a t e d from a r e s i d u a l f r a c t i o n o f an EtOH e x t r a c t o f A l p i n iαb l e p hαr o e α l y x . Calyxin I ( 2 ) r e p r e s e n t e d a new carbon s k e l e t o n and e p i c a l y x i n F ( 1 ) p o s s e s s e d p o t e n t a n t i p r o l i f e r a t i v e a c t i v i t y t o ‑ wards HT‑1080 f i b r o s a r c o m a and c o l o n 2 6 ‑ 1 5 carcinoma w i t h E D 5 1 v a l u e s 1 . 7 1 and 0 . 8 9 μM, r e s p e c t i v e l y .

1 2 ) Tezuka Y . , Zhao W., I s h i i E . , and Kadota S . : A n t i ‑ H e l i c o b α c t e r p y l o r i A c t i v i t y o f S t e r o i d a l A l k a l o i d s Obtained from Three Ver α trum P l a n t s . J . Trad. Med., 1 6 : 1 9 6 ‑ 2 0 0 , 1 9 9 9 .

Abstract : Anti H e l i c o bαc t e r p y l o r i (HP) a c t i v i t i e s were e x a m i n e d , by d i s c method, on t h r e e t o t a l a l k a l o i d f r a c t i o n s and f o u r t e e n s t e r o i d a l a l k a l o i d s o b t a i n e d from t h r e e Veratrum p l a n t s ( V . mα αc k i i , V . nigrum v a r . u s s u r i e n s e and V . p a t u l u m ) , which a r e u s e d a s a name o f L i ‑ l u

（葉麗）

t ot r e a t a p h a s i a a r i s i n g from a p o p l e x y , wind t y p e d y s e n t e r y , j a u n d i c e , h e a d a c h e , s c a b i e s , c h r o n i c m a l a r i a , e t c . Among them, v e r a p a t u l i n ( 1 2 ) and v e r a t r a m i n e ( 1 3 ) r e v e a l e d anti‑HP a c t i v i t i e s , and t h e disc‑minimum i n h i b i t o r y c o n c e n t r a t i o n ( d i s k ‑ M I C ) v a l u e ( 1 0

μ

g/ml) o f 1 2 a g a i n s t two s t a n d a r d HP s t r a i n s , NCTC11637 and NCTC11916, was h i g h e r than t h a t o f a c l i n i c a l l y u s e d a n t i b i o t i c , erythromycin (

<

0 . 0 1 3 μ g / m l ) , but was com p a r a b l e t o t h o s e o f p e n i c i l l i n G ( 3 . 1 μ g/ml and 1 . 6 μ g / m l , r e s p e c t i v e l y ) .

1 3 ) L i H . , Miyahara T . , Tezuka Y . , Watanabe M . , Nemoto N . , S e t o H . , and Kadota S . : The e f f e c t o f low molecular weight Chitosan on bone r e s o r p t i o n i n v i t r o and i n v i v o . Phytomedicine, 6 : 3 0 5 ‑ 3 1 0 , 1 9 9 9 .

Summary: We s t u d i e d t h e e f f e c t o f low m o l e c u l a r weight c h i t o s a n (LMWC) on t h e forma‑

t i o n o f o s t e o c l a s t ‑ l i k e m u l t i n u c l e a t e d c e l l s (OCLs) i n t h e c o ‑ c u l t u r e o f mouse o s t e o b l a s t i c

c e l l s and bone marrow c e l l s i n t h e p r e s e n c e o f l α

，

2 5

^四

dihydroxyvitaminD 3 [ 1 α

，

2 5 ( 0 H ) 2 D 3 ] .

LMWC a t 4 4 0 μ g/ml i n h i b i t e d t h e formation o f t a r t r a t e r e s i s t a n t a c i d p h o s p h a t a s e

( T R A P ) ‑ p o s i t i v e OCLs i n d u c e d by l α

，

25(0H)2D3

・

Wep r e p a r e d OCLs i n t h e c o ‑ c u l t u r e o f

o s t e o b l a s t i c c e l l s and bone marrow c e l l s . The e f f e c t o f LMWC on p i t f o r m a t i o n by OCLs was

examined u s i n g d e n t i n s l i c e s , a r i d LMWC i n h i b i t e d p i t fo r mat i on a t 4 4 0 μ g / m l . O r a l a d ‑

m i n i s t r a t i o n o f t h e LMWC t o o v a r i e c t o m i z e d r a t s p r e v e n t e d a d e c r e a s e i n bone m i n e r a l den

s i t y (BMD) o f t h e lumbar v e r t e b r a without a f f e c t i n g t h e body and u t e r u s w e i g h t s . These

(7)

r e s u l t s s u g g e s t e d t h a t LMWC p r e v e n t e d a d e c r e a s e i n BMD i n v i v o by i n h i b i t i n g o s t e o c l a s t i c bone r e s o r p t i o n .

1 4 ) Hase K . , Xiong Q . , Basnet P . , Namba T . , and Kadota S . : I n h i b i t o r y E f f e c t o f Tetrahydroswertianolin on Tumor N e c r o s i s Factor

−

α

−

Dependent Hepatic Apoptosis i n M i c e . Biochem. P h a r m a c o l . , 5 7 : 1 4 3 1 ‑ 1 4 3 7 , 1 9 9 9 .

Abstract : We i n v e s t i g a t e d t h e e f f e c t o f t e t r a h y d r o s w e r t i a n o l i n (THS), a h e p a t o p r o t e c t i v e a g e n t from S w e r t i α J α p a n i c α

，

on tumor n e c r o s i s f a c t o r

−

α

（

TNF

−

α

）−

d e p e n d e n t hep a t i c a p o p t o s i s i n d u c e d by D ‑ g a l a c t o s a m i n e ( D ‑ G a l N ) ( 7 0 0 mg/kg, i . p . ) and l i p o p o l y s a c c h a r i d e (LPS) ( 1 0 , u g/kg, i . p . ) i n m i c e . A p o p t o t i c symptoms were o b s e r v e d a t t h e i n i t i a l s t a g e o f l i v e r damage. By 5 hr a f t e r i n t o x i c a t i o n , h e p a t i c DNA f r a g m e n t a t i o n had r i s e n t o 2 1 2 3 % , w i t h t h e v a l u e i n u n t r e a t e d mice s e t a t 1 0 0 % , without a s i g n i f i c a n t e l e v a t i o n o f serum a l a n i n e t r a n s a m i n a s e (ALT) a c t i v i t y . There was a p a r a l l e l i n c r e a s e i n hep a t o c y t e s undergoing c h r o ‑ matin c o n d e n s a t i o n and a p o p t o t i c body f o r m a t i o n . By 8 hr a f t e r i n t o x i c a t i o n , serum ALT a c t i v i t y had r i s e n t o 3 7 0 7 U / L . P r e t r e a t m e n t w i t h THS ( 5 0 mg/kg, p

・

o . )a t 1 8 and 2 hr b e ‑ f o r e i n t o x i c a t i o n s i g n i f i c a n t l y r e d u c e d DNA f r a g m e n t a t i o n t o 821% o f t h a t i n u n t r e a t e d mice and p r e v e n t e d t h e emergence o f chromatin c o n d e n s a t i o n and a p o p t o t i c body f o r m a t i o n . A s i g n i f i c a n t and d o s e ‑ d e p e n d e n t r e d u c t i o n i n serum ALT a c t i v i t y a t 8 hr a l s o was o b s e r v e d w i t h THS p r e t r e a t m e n t . These e f f e c t s o f THS were d i f f e r e n t from t h o s e o b s e r v e d from p r e ‑ t r e a t m e n t with g l y c y r r h i z i n (GCR), which i s a c l i n i c a l l y u s e d h e p a t o p r o t e c t i v e agent with m e m b r a n e ‑ s t a b i l i z i n g a c t i v i t y . GCR p r e t r e a t m e n t ( 1 0 0 mg/kg, p

・

o . )d i d n o t i n h i b i t h e p a t i c DNA f r a g m e n t a t i o n ( 1 5 8 8 % o f u n t r e a t e d m i c e ) , a l t h o u g h t h i s compound s i g n i f i c a n t l y p r o ‑ t e c t e d a g a i n s t serum ALT e l e v a t i o n ( 1 4 6 3 U / L ) . These d a t a s u g g e s t t h a t an i n h i b i t o r y e f f e c t on t h e p r o g r e s s i o n o f h e p e t i c a p o p t o s i s p r i o r t o l i v e r i n j u r y may b e i n v o l v e d i n t h e h e p a t o p r o t e c t i v e mechanisms o f THS, whereas i t a p p e a r s t h a t GCR a f f e c t s t h e p r o c e s s e s a f t e r a p o p t o s i s . I n a s e p a r a t e e x p e r i m e n t , we found t h a t t h e c o n c e n t r a t i o n o f serum TNF αrose t o 2 0 1 6 pg/mL a t 1 hr a f t e r i n t o x i c a t i o n o f m i c e with D‑GalN and LPS, b u t t h i s i n ‑ c r e a s e was s u p p r e s s e d by THS p r e t r e a t m e n t ( 1 0 , 5 0 , o r 2 0 0 mg/kg, p . o . ) t o 7 1 6 , 4 5 4 , o r 4 0 6 pg/mL, r e s p e c t i v e l y . Further study w i t h a r e v e r s e t r a n s c r i p t a s e ‑ p o l y m e r a s e c h a i n r e a c t i o n method showed t h a t THS b l o c k e d TNF

−

αproduction a t t h e t r a n s c r i p t i o n a l l e v e l . B e c a u s e TNFαis a c r i t i c a l mediator t o e l i c i t a p o p t o s i s i n t h i s m o d e l , t h e p r o p e r t y o f s u p p r e s s i n g TNF

−

αproduction may b e o f prime i m p o r t a n c e f o r THS i n h i b i t i o n o f h e p a t i c a p o p t o s i s .

1 5 ) Kurokawa M . , Basnet P . , Ohsugi M . , Hozumi T . , Kadota S . , Namba T . , Kawana T . ,

and S h i r

叫

dK . : Anti‑Herpes Simplex Virus A c t i v i t y o f Moronic Acid P u r i f i e d from

Rhusj α u α n i c α I n V i t r o and I n V i v o . J . Pharmacol. Exp. T h e r . , 2 8 9 : 7 2 ‑ 7 8 , 1 9 9 9 .

Ab st ra ct : Rhus j α u α n i c α ， a m e d i c i n a l h e r b , has b e e n shown t o e x h i b i t o r a l t h e r p e u t i c a n t i ‑

h e r p e s s i m p l e x v i r u s (HSV) a c t i v i t y i n m i c e . We p u r i f i e d two major anti‑HSV compounds,

moronic a c i d and b e t u l o n i c a c i d , from t h e h e r b a l e x t r a c t by e x t r a c t i o n w i t h e t h y l a c e t a t e a t

pH 1 0 f o l l o w e d by chromatographic s e p a r a t i o n s and examined t h e i r anti‑HSV a c t i v i t y

仇

(8)

v i t r o and i n v iυ o . Moronic a c i d was q u a n t i t a t i v e l y a major anti‑HSV compound i n t h e e t h y l a c e t a t e ‑ s o l u b l e f r a c t i o n . The e f f e c t i v e c o n c e n t r a t i o n s f o r 50% p l a q u e r e d u c t i o n o f moronic a c i d and b e t u l o n i c a c i d f o r w i l d ‑ t y p e HSV t y p e 1 (HSV 1 ) were 3 . 9 and 2 . 6 , u g/ml, r e s p e c ‑ t i v e l y . The t h e r a p e u t i c i n d e x o f moronic a c i d ( 1 0 . 3 ‑ 1 6 . 3 ) was l a r g e r than t h a t o f b e t u l o n i c a c i d ( 6 . 2 ) . S u s c e p t i b i l i t y o f a c y c l o v i r

^四

p h o s p h o n o a c e t i c a c i d

^守

r e s i s t a n t HSV‑1, thymidine k i n a s e ‑ d e f i c i e n t HSV 1 , and w i l d ‑ t y p e HSV t y p e 2 t o moronic a c i d was s i m i l a r t o t h a t o f t h e w i l d t y p e HSV‑1. When t h i s compound was a d m i n i s t e r e d o r a l l y t o m i c e i n f e c t e d c u t a n e o u s l y with HSV‑1 t h r e e t i m e s d a i l y , i t s i g n i f i c a n t l y r e t a r d e d t h e development o f s k i n l e s i o n s and/‑

o r 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 o f i n f e c t e d m i c e without t o x i c i t y compared with t h e c o n t r o l . Moronic a c i d s u p p r e s s e d v i r u s y i e l d s i n t h e b r a i n more e f f i c i e n t l y than t h o s e i n t h e s k i n . This was c o n s i s t e n t with t h e p r o l o n g a t i o n o f mean s u r v i v a l t i m e s . Thus, moronic a c i d was p u r i f i e d a s a major anti‑HSV compound from t h e h e r b a l e x t r a c t o f Rhus j α u αn i c α . Mode o f t h e anti‑HSV a c t i v i t y was d i f f e r e n t from t h a t o f ACV. Moronic a c i d showed o r a l t h e r a ‑ p e u t i c e f f i c a c y i n H S V ‑ i n f e c t e d m i c e and p o s s e s s e d n o v e l anti‑HSV a c t i v i t y t h a t was c o n s i s ‑ t e n t with t h a t o f t h e e x t r a c t .

1 6 ) Watanabe C . , Satoh T . , Tahara E . , Murakami K . , Hayashi K . , Hase K . , Andoh T . , Kuraishi Y . , Kadota S . , Nagai H . , and S a i k i I . : I n h i b i t o r y Mechanisms o f Glycoprotein Fraction Derived from Misc α n t h u s s i n e n s i s f o r t h e Immediate Phase Response o f an lgE

^園

MediatedCutaneous R e a c t i o n . B i o l . Pharm. B u l l . , 2 2 : 2 6 ‑ 3 0 , 1 9 9 9 .

Summary: We i n v e s t i g a t e d t h e i n h i b i t o r y e f f e c t o f t h e g l y c o p r o t e i n f r a c t i o n ( f r a c t i o n 2 ) ex t r a c t e d from Misc α n t h u s s i n e n s i s ANDERSSON (M. s i n e n s i s ) on b i p h a s i c c u t a n e o u s r e a c t i o n s i n mice p a s s i v e l y s e n s i t i z e d with I g E . B i p h a s i c s k i n r e a c t i o n s with peak r e s p o n s e s a t 1 ( I P R , immediate p h a s e r e a c t i o n ) and 2 4 h (LPR, l a t e p h a s e r e a c t i o n ) were c a u s e d by p a s s i v e s e n s i ‑ t i z a t i o n with an a n t i d i n i t r o p h e n o l IgE monoclonal a n t i b o d y ( a n t i DNP IgE mAb) f o l l o w e d by an e p i c u t a n e o u s c h a l l e n g e o f 0 . 1 % d i n i t r o f l u o r o b e n z e n e (DNFB) i n 1 0 0 % e t h a n o l . I n t r a p e r i t o n e a l i n j e c t i o n o f f r a c t i o n 2 b e f o r e t h e DNFB c h a l l e n g e s i g n i f i c a n t l y i n h i b i t e d t h e b i p h a s i c e a r s w e l l i n g r e s p o n s e i n p a s s i v e l y s e n s i t i z e d mice i n a d o s e ‑ d e p e n d e n t manner (1‑

3 0 mg/kg). We a l s o found t h a t f r a c t i o n 2 was e f f e c t i v e a t i n h i b i t i n g t h e v a s c u l a r p e r m e a b i l ‑ i t y i n mouse e a r i n d u c e d by an i n j e c t i o n o f compound 4 8 / 8 0 , h i s t a m i n e o r s e r o t o n i n . I n a d d i t i o n , f r a c t i o n 2 i n h i b i t e d s c r a t c h i n g b e h a v i o r a s w e l l a s e a r edema o b s e r v e d w i t h i n 2 h a f t e r DNFB c h a l l e n g e . Marked i n h i b i t i o n was o b s e r v e d i n both p a s s i v e l y s e n s i t i z e d and non‑

s e n s i t i z e d m i c e . The locomotor a c t i v i t y o f mice was a l s o r e d u c e d by t h e a d m i n i s t r a t i o n o f

f r a c t i o n 2 a s w e l l a s by diphenhydramine. These r e s u l t s s u g g e s t t h a t t h e i n h i b i t o r y e f f e c t o f

g l y c o p r o t e i n f r a c t i o n 2 o f M. s i n e n s i s on an IgE‑mediated a l l e r g i c inflammatory r e a c t i o n i s

due t o t h e p r o t e c t i o n o f mediator

^司

i n d u c e dv a s c u l a r p e r m e a b i l i t y and t h a t i n a d d i t i o n t o t h e

i n h i b i t i o n o f an inflammatory r e a c t i o n , a s e d a t i v e a c t i o n i s r e s p o n s i b l e f o r t h e i n h i b i t i o n o f

a l l e r g y ‑ i n d u c e d s c r a t c h i n g r e s p o n s e s .

(9)

1 7 ) Hashimoto M. and Hatanaka Y . : I d e n t i f i c a t i o n o f P h o t o l a b e l e d P e p t i d e s f o r t h e Acceptor S u b s t r a t e Binding Domain o f β 1 , 4 ‑ G a l a c t o s y l t r a n s f e r a s e . Chem. Pharm.

B u l l . , 4 7 : 6 6 7 ‑ 6 7 1 , 1 9 9 9 .

A b s t r a c t : We s u c c e s s f u l l y a p p l i e d a c a r b e n e ‑ g e n e r a t i n g N ‑ a c e t y l g l u c o s a m i n e d e r i v a t i v e c a r r y i n g a b i o t i n y l group t o t h e r a d i o i s o t o p e ‑ f r e e i d e n t i f i c a t i o n o f p e p t i d e s w i t h i n b o v i n e U D P ‑ g a l a c t o s e : N ‑ a c e t y l g l u c o s a m i n e β 1 , 4 ‑ g a l a c t o s y l t r a n s f e r a s e ( G a l T , EC 2 . 4 . 1 . 3 8 ) c a t a ‑ l y t i c d o m a i n . Owing t o t h e low y i e l d o f c r o s s ‑ l i n k i n g , c o n v e n t i o n a l p h o t o a f f i n i t y l a b e l i n g e x ‑ p e r i m e n t s u s u a l l y e n c o u n t e r a thorny problem i n a t t e m p t i n g t o i s o l a t e l a b e l e d components from v e r y complex m i x t u r e s . A b i o t i n t a g i n t r o d u c e d w i t h our p h o t o a f f i n i t y p r o b e e n a b l e d u s t o s e p a r a t e t h e p h o t o l a b e l e d p r o t e i n from a l a r g e amount o f c o e x i s t i n g u n l a b e l e d G a l T . The i n t r o d u c t i o n o f b i o t i n was a l s o u s e f u l f o r t h e r a d i o i s o t o p e ‑ f r e e d e t e c t i o n o f a l a b e l e d p r o ‑ t e i n b a s e d on a h i g h l y s e n s i t i v e c h e m i l u m i n e s c e n t t e c h n i q u e . We d e v e l o p e d a n o v e l p o l y v i n y l i d e n e d i f l u o r i d e membrane f o r t h e i d e n t i f i c a t i o n o f l a b e l e d p e p t i d e s i n a s i m p l e d o t b l o t a s s a y . Using t h i s membrane, we s u c c e s s f u l l y i d e n t i f i e d b i o t i n y l p e p t i d e s among a num‑

b e r o f HPLC s e p a r a t e d fragments d e r i v e d from t h e p r o t e a s e d i g e s t i o n o f p h o t o l a b e l e d GalT p r o t e i n s . The s e q u e n c e a n a l y s i s r e v e a l e d t h a t t h e b i o t i n t a g was i n c o r p o r a t e d w i t h i n a t r y p t i c GalT fragment o f Y 1 9 7 ‑ R 2 0 8 . Our a p p r o a c h y i e l d s , f o r t h e f i r s t t i m e , i n f o r m a t i o n on t h e a c c e p t o r s u b s t r a t e b i n d i n g ‑ s i t e fragment i n t h i s e n z y m e , t h a t h a s b e e n d i f f i c u l t t o o b t a i n u s i n g o t h e r a p p r o a c h e s . T h e s e d a t a a r e c o n s i s t e n t w i t h p r e v i o u s s u g g e s t i o n s c o n c e r n i n g t h e GalT a c c e p t o r s i t e and c l e a r l y demonstrate t h e e f f e c t i v e n e s s o f our approach f o r r a p i d i d e n ‑ t i f i c a t i o n o f p h o t o l a b e l e d p e p t i d e s .

1 8 ) Kempin U . and Hatanaka Y . : A Novel Reagent f o r P r e p a r a t i o n o f P h o t o a f f i n i t y Probes from Unprotected Carbohydrates. Glycoconjugate J . , 1 6 : 8 5 4 , 1 9 9 9 .

A b s t r a c t : We h a v e d e v e l o p e d an e f f i c i e n t method f o r t h e p r e p a r a t i o n o f p h o t o a f f i n i t y c a r ‑ b o h y d r a t e p r o b e s . P h o t o r e a c t i v e c a r b o h y d r a t e s a r e p o w e r f u l c h e m i c a l t o o l s f o r t h e s t r u c ‑ t u r a l a n a l y s i s o f p r o t e i n s t h a t s p e c i f i c a l l y i n t e r a c t with g l y c o c o n j u g a t e s . C o n v e n t i o n a l method o f p r o b e s y n t h e s i s , h o w e v e r , u s u a l l y r e q u i r e s t h e m u l t i p l e s e q u e n c e o f r e a c t i o n s i n v o l v i n g p r o t e c t i o n , a c t i v a t i o n and d e p r o t e c t i o n s t e p s . Here we r e p o r t a n o v e l r e a g e n t f o r t h e o n e ‑ s t e p i n t r o d u c t i o n o f a c a r b e n e ‑ g e n e r a t i n g p h o t o r e a c t i v e group t o t h e r e d u c i n g t e r m i n u s o f u n p r o t e c t e d c a r b o h y d r a t e s . The s y n t h e s i s o f p r o b e s t a k e s a d v a n t a g e o f t h e oxime forma‑

t i o n r e a c t i o n between an aminooxy group o f t h e r e a g e n t and an a l d e h y d e a t t h e r e d u c i n g e n d . T h u s , t h e p h o t o r e a c t i v e d e r i v a t i v e o f

N二

a c e t y l l a c t o s a m i n e ,3

−

s i a l y l l a c t o s e , 3 ' ‑ s i a l y l ‑ N ‑ a c e t y l l a c t o s a m i n e , Lewis x t r i s a c c h a r i d e , o r Lewis x t e t r a s a c c h a r i d e was e a s i l y p r e p a r e d . Combination o f t h e p r e s e n t method w i t h r e c e n t l y d e v e l o p e d n o n ‑ r a d i o a c t i v e approach w i l l p r o v i d e a p o w e r f u l s t r a t e g y f o r p h o t o a f f i n i t y l a b e l i n g .

1 9 ) Kempin U . and Hatanaka Y . : P h o t o c r o s s l i n k i n g o f Carbohydrate‑Lectin I n t e r a c t i n g Systems. Photomedicine and P h o t o b i o l o g y , 2 1 : i n p r e s s , 1 9 9 9 .

A b s t r a c t : We h a v e d e v e l o p e d an e f f i c i e n t method f o r t h e p r e p a r a t i o n o f p h o t o a f f i n i t y

(10)

carbohydrate p r o b e s . P h o t o r e a c t i v e c a r b o h y d r a t e s a r e p o w e r f u l c h e m i c a l t o o l s f o r t h e s t r u c t u r a l a n a l y s i s o f p r o t e i n s t h a t s p e c i f i c a l l y i n t e r a c t with g l y c o c o n j u g a t e s . C o n v e n t i o n a l method o f probe s y n t h e s i s , h o w e v e r , u s u a l l y r e q u i r e s t h e m u l t i p l e s e q u e n c e o f r e a c t i o n s i n ‑ v o l v i n g p r o t e c t i o n , a c t i v a t i o n and d e p r o t e c t i o n s t e p s . Here we r e p o r t a n o v e l r e a g e n t f o r t h e o n e ‑ s t e p i n t r o d u c t i o n o f a c a r b e n e g e n e r a t i n g p h o t o r e a c t i v e group t o t h e r e d u c i n g terminus o f u n p r o t e c t e d c a r b o h y d r a t e s . The s y n t h e s i s o f p r o b e s t a k e s advantage o f t h e oxime forma‑

t i o n r e a c t i o n between an aminooxy group o f t h e r e a g e n t and an a l d e h y d e a t t h e r e d u c i n g e n d . T h u s , t h e p h o t o r e a c t i v e d e r i v a t i v e s o f N ‑ a c e t y l l a c t o s a m i n e , Lewis x t r i s a c c h a r i d e , o r Lewis x t e t r a s a c c h a r i d e were e a s i l y p r e p a r e d . Combination o f t h e p r e s e n t method with r e ‑ c e n t l y d e v e l o p e d n o n ‑ r a d i o a c t i v e approach w i l l p r o v i d e a p o w e r f u l s t r a t e g y f o r p h o t o a f f i n i t y l a b e l i n g .

。学会報告

1 ) Arjun H. B a n s k o t a , Yasuhiro T e z u k a , Le Kim Phung, Kim Qui T r a n , Ikuo S a i k i , Y o s h i h i s a Miwa, Tooru Taga, and S h i g e t o s h i K a d o t a : Seven Novel C y c l o a r t a n e ‑ t y p e T r i t e r p e n e s from Combretum qu

α

dr

α

n g u l

α

r e and T h e i r C y t o t o x i c A c t i v i t i e s .

日本薬学会第

1 1 9

年会，

1 9 9 9 ,3 / 2 9 3 1

，徳島．

2

）李慧英，手塚康弘，難波恒雄，門田重平

j l

，宮原龍郎，渡辺誠，堂脇理沙，根本信雄：

漢方方剤による抗骨粗意活性成分の研究（II）ーベルペリンの骨吸収抑制機構について一．

日本薬学会第

1 1 9

年会，

1 9 9 9 , 3 / 2 9 3 1 ,

徳島．

3

）漬崎憲夫，石井営次，手塚康弘，長岡武馬，門田重利，富永和作，樋口和秀，荒川哲男，

黒木哲夫，矢野郁也：

H e l i c o b

α

c t e rp y l o r i

に対する漢方生薬呉莱英湯の抗菌活性とその成分追求について．第

8 5

回日本消化器病学会総会，

1 9 9 9 , 4 / 2 2 2 4

，長崎．

4) N . Hamasaki, T . Arakawa,

K. Tominaga, T

. Watanabe, Y . F u j i w a r a , K

.

H i g u c h i , T . K u r o k i , E . I s h i i , Y . Tezuka, T . Nagaoka, S . K a d o t a , I

.

Yano: Newly p u r i f i e d com‑

pounds o f two q u i n o l o n e a l k a l o i d s from ' G o s y u ‑ Y u ' have a s p e c i f i c a n t i b a c t e r i a l a c t i v ‑ i t y a g a i n s t H e l i c o bαc t e r p y l o r i . American G a s t r o e n t e r o l o g i c a l A s s o c i a t i o n : D i g e s t i v e D e s e a s e Week ( R ) : S c i e n t i f i c S e s s i o n s , 1 9 9 9 , 5 / 1 6 1 9 , O r l a n d o , F L , USA.

5

）熊泉波，長谷耕二，

I Ketu t Adnyana

，範文哲，相嬉，長岡武馬，手塚康弘，門田重平

j l

：天然抗酸化物質の

TNF

−

α

誘発初代培養肝細胞死に対する作用．日本組織培養学会第

7 2

回大会，

1 9 9 9 , 5 / 2 0 2 1

，富山．

6 ) Arjun H . Banskota

，手塚康弘，

KimQui Tran

，済木育夫，門田重利：

F i v eNovel C y c l o a r t a n e ‑ t y p e T r i t e r p e n e s from Combretum quαdr α n g u l a r e .

日本薬学会北陸支部第

1 0 0

回記念例会，

1 9 9 9 , 6 / 1 9 2 0

，金沢．

7 ) P a v l o s Stampoulis

，手塚康弘，

ArjunH . B a n s k o t a , Kim Qui Tran

，済木育夫，門田重利：

A Novel Carbon s k e l e t o n D i t e r p e n e from O r t h o s i p h o n s tαm i n e u s .

1 0 0

回記念例会，

1 9 9 9 , 6 / 1 9 ‑ 2 0

，金沢．

8 ) Mohan B . Gewali

，手塚康弘，

ArjunH . B a n s k o t a , Mohammad Shawkat A l i

，済木育夫，

門田重利：

NewD i a r y l h e p t a n o i d s from t h e S e e d s o f A l p i n i

α

b l e p h

α

r o e

α

l y x .

1 0 0

回記念例会，

1 9 9 9 , 6 / 1 9 2 0

，金沢．

(11)

9

）長阿武馬，金子卓嗣，手塚康弘，

ArjunH . Banskota

，入川志保，門田重手iJ：花械のマクロファジ

iNOSmRNA

発現抑制活性成分の研究．日本薬学会北陸支部第

1 0 0

回記念例会，

1 9 9 9 ,

6/19~20，金沢．

1 0

）活文哲，手塚康弘，門田重手jl：桃核承気湯のプロリルエンドペプチダゼ阻害活性成分に関する研究．第

1 6

回和漢医薬学会大会，

1 9 9 9 , 8 / 2 8 ‑ 2 9

，千葉．

1 1

）熊泉波，範文哲，手塚康弘，服部征雄，難波恒雄，門田重利：羅布麻（

Apocynum venetum L

.）葉の肝臓保護作用及びその活性成分に関する研究．第

1 6

回和漢医薬学会大会，

1 9 9 9 , 8 / 2 8 2 9

，千葉．

1 2 ) Arjun H . B a n s k o t a , K a t s u m i c h i M a t s u s h i g e , Yasuhiro T e z u k a , Takema Nagaoka, Quanbo X i o n g , I k u o S a i k i , and S h i g e t o s h i K a d o t a : P h a r m a c o l o g i c a l A c t i v i t i e s o f P r o p o l i s . XXXVIth Apimondia I n t e r n a t i o n a l A p i c u l t u r a l C o n g r e s s , 1 9 9 9 , 9 / 1 2 1 8 , V a n c o u v e r , C a n a d a .

1 3 ) I Ketut Adnyana

，手塚康弘，

ArjunH . Banskota

，熊泉波，門田重利，

KimQui Tran : F i v e New T r i t e r p e n e G l u c o s i d e s from S e e d s o f Combretum qu

α

dr

α

n g u l a r e .

日本生薬学会第

4 6

回年会，

1 9 9 9 , 9 / 1 7 1 8

，大阪．

1 4 ) Arjun H . Banskota

，手塚康弘，済木育夫，門田重利，

KimQui Tran : E i g h t New C y c l o a r t a n e t y p e T r i t e r p e n e s from L e a v e s o f Combretum qu

α

dr

α

n g u l a r e .

日本生薬学会第

4 6

回年会，

1 9 9 9 , 9 / 1 7 ‑ 1 8

，大阪．

1 5

）晴山亨，手塚康弘，李慧英，門田重利，宮原龍郎：漢薬牛膝の生理活性成分の研究．

1 0 1

回例会，

1 9 9 9 , 1 1 / 1 3

，富山．

1 6 ) P a v l o s S t a m p o u l i s , Yasuhiro T e z u k a , Arjun H . B a n s k o t a , Kim Qui T r a n , I k u o S a i k i , S h i g e t o s h i Kadota : T h r e e n o v e l s t a m i n a n e ‑ t y p e d i t e r p e n e s from O r t h o s i p h o n s t

α

m i n e u s .

1 0 1

回例会，

1 9 9 9 , 1 1 / 1 3

，富山．

1 7 ) Kempin U .

，畑中保丸：高速光アフィニティーラベリング：光反応性糖鎖プローブのー段階合成と応用．日本薬学会第

1 1 9

年会，

1 9 9 9 , 3 / 2 9 3 1

，徳島．

1 8

）畑中保丸，

KempinU .

：糖鎖ーレクチン相互作用系の光クロスリンキング．第

2 1

回日本光医学・光生物学会，

1 9 9 9 , 8 / 6 ‑ 7

，金沢．

1 9

）日

atanakaY . and Kempin U . : A N o v e l Reagent f o r P r e p a r a t i o n o f P h o t o a f f i n i t y P r o b e s from U n p r o t e c t e d C a r b o h y d r a t e s . XVth I n t e r n a t i o n a l Symposium on G l y c o c o n j u g a t e s , 1 9 9 9 , 8 / 2 2 2 7 , Tokyo, J a p a n .

2 0 ) Hatanaka Y . and Kempin U . : A S i m p l e Method f o r P r e p a r a t i o n o f P h o t o a f f i n i t y P r o b e s from U n p r o t e c t e d C a r b o h y d r a t e s . New F r o n t i e r o f G l y c o ‑and L i p i d ‑ B i o l o g y toward 2 1 s t C e n t u r y , 1 9 9 9 , 8 / 2 8 3 0 , Tokushima, J a p a n .

2 1 ) Hatanaka Y . , Kempin U . , and Kanaoka Y . : O n e ‑ S t e p P r e p a r a t i o n o f P h o t o a f f i n i t y C a r b o h y d r a t e P r o b e s . 1 4 t h F r e n c h ‑ J a p a n e s e Synposium on M e d i c i n a l and F i n e C h e m i n s t r y , 1 9 9 9 , 9 / 1 9 2 2 , La B a u l e , F r a n c e .

2 2

）田中啓友，日向須美子，畑中保丸，畑中研一，日向昌司，山形貞子，山形達也：

IV

型コラーゲンに対する細胞接着を制御するガングリオシド

GDl a .

第

7 2

回日本生化学会大会，

1 9 9 9 , 1 0 / 6 ‑ 9

，横浜．

2 3

）上回晴子，畑中保丸，小島京子，小

J I I

温子：自己会合性エンジュ樹皮レクチン

(12)

( S o p h o r a g i n

）と内在性リガンドとの複合体形成．第

7 2

回日本生化学会大会，

1 9 9 9 , 1 0 / 6 9

，横浜．

2 4

）畑中保丸，

Kempin U.

：糖鎖光アフィニティープロープの効率的合成と応用．第

2 5

回反応と合成の進歩シンポジウム，

1 9 9 9 , 1 1 / 4 ‑ 5

，富山．

2 5 ) Natanaka Y . : C h e m i c a l Approach f o r t h e A n a l y s i s o f Drug R e c e p t o r . The 3 r d I n t e r n a t i o n a l Symposium on R e c e n t Advances i n N a t u r a l P r o d u c t s R e s e a r c h and 1 9 9 9 Korea‑Japan J o i n t Symposium, 1 9 9 9 , 1 1 / 1 9 , S e o u l , K o r e a .

く〉その他

1

）門田重利：天然薬物の生理活性物質の探索.

1 9 9 9 , 6 / 8

，新潟薬科大学．

2

）畑中保丸：光アフィニティーラベル法による蛋白質の構造解析．サントリー生物有機化学研究所，

1 9 9 9 , 1 1 / 3 0

，大阪．

3

）手塚康弘：

NewD i a r y l h e p t a n o i d s o f A l p i n i a b l e p h a r o c a l y x (Yunnan‑Caodoukou).

1 9 9 9 , 8 / 5

，インドネシア・ノーススマトラ大学．

。海外調査

1

）門田重利：ミャンマ一保健省伝統薬品局に対する技術協力（

J I C A ) , 1 9 9 9 , 3 / 2 0 4 / 9 ,

ミャンマー．

2

）門田重利：マンダレー伝統医療専門学校に対する技術協力（

J I C A ) , 1 9 9 9 , 1 1 / 1 8 1 2 / 1 5 ,

ミャンマー．

。共同研究

1

）宮原龍郎：富山医科薬科大学薬学部，「骨組軽症に有効な漢方方剤の開発研究」，

1 9 9 8 , 4〜

2

）中村薫：京都大学化学研究所，「生体触媒を使ったキナ酸誘導体の合成研究」，

1 9 9 8 , 4

〜

3

）古賀大三：山口大学農学部，「植物生体防御におけるキチナーゼ誘導とキチン・キトサンの糖鎖組成との関連性

J , 1 9 9 8 , 4〜

4

）山形達也：日本皮革研究所，「

FJB

骨肉腫細胞の運動能・細胞一基質接着性・転移能を調節するガングリオシド

GDl a

，」

1 9 9 8 , 4〜

5

）小川温子：お茶の水女子大学大学院，「自己会合性エンジュ樹皮レクチン

S o p h o r a g i n

の

m a l t o t r i o s e

プロープによる光アフィニティーラベリング」，

1 9 9 9 , 4

〜

6

）中川昌子：千葉大学薬学部，「キニーネ・スフィンゴ脂質ハイブリッド構造を基本とする抗マラリヤ剤の開発」，

1 9 9 9 , 4

〜

7

）河野敬一，今中常雄：富山医科薬科大学薬学部，「

E p i r e g u l i n

の立体構造とレセプターとの相互作用解析による癌細胞の成長制御機構解明」，

1 9 9 9 , 4 〜

8

）挑新生：中国・藩陽薬科大学「和漢薬人参の化学的研究」，

1 9 9 8 , 4 〜

9

）陳英烹：中国・藩陽薬科大学「抗骨粗軽症に有効な薬物の開発研究」，

1 9 9 9 , 1 0 〜

(13)

。研究費取得状況

1

）文部省科学研究費，基盤研究

Al

（分担：門田重利）「新規高次神経変性疾患モデル動物，

細胞の開発，神経変性機序の解析と薬物評価法の確立」，

5 0

万

2

）文部省科学研究費，特定領域研究

A

（代表：畑中保丸）「スーパーバイオシステムの高次認識糖鎖分子による構築：光反応性高次認識糖鎖分子の設計と応用」，

1 3 0

万

3 )

（財）薬学研究奨励財団第

2 0

回研究助成金（代表：畑中保丸）「固相上に集積したプローブを用いる高速光アフィニティーラベル法の開発」，

1 0 0

万

4)

（財）内藤記念科学奨励金（代表：畑中保丸）「高速アフィニティーラベル法によるベロトキシンの糖鎖結合部位解析」，

1 3 0 万

。研究室在籍者

大学院前期2年：晴山亨，本田和之，

P a v l o sS t a m p o u l i s

，菅原弘之大学院後期

1

年：

TranLe Quan ( 1 9 9 9 , 4

〜），朴鍾集（

1 9 9 9 , 4

〜）大学院後期

2

年：

IKetut Adnyana

，活文哲，

MohammadShawkat A l i

大学院後期

3

年：

ArjunH . Banskota

，熊泉波

研究生：長谷耕二，

S u r e s hA w a l e

受託研究員：松繁克道，緑川淑

外国人客員研究員：；

MohanB . G e w a l i ( 1 9 9 8 , 5 , 3 0

〜

1 9 9 9 , 5 , 2 3 ) , Kyaw Myint Tun ( 1 9 9 9 , 3 , 1 0 〜 1 9 9 9 , 3 , 1 7 ) , T h e i n Han Oo ( 1 9 9 9 , 3 , 1 0 〜 1 9 9 9 , 3 , 1 7 ) , Warunee Thiramongkolchai ( 1 9 9 9 , 8 , 1 〜 1 9 9 9 , 9 , 2 9 ) , Nguyen Thanh Hong ( 1 9 9 9 , 9 , 1 〜 1 9 9 9 , 9 , 3 0

），議競康

( 1 9 9 9 , 9 , 1 5

〜

1 9 9 9 , 1 1 , 1 0

），越偉傑（

1 9 9 9 , 7 , 2 2

〜

2 0 0 0 , 1 , 1 1 ) , Daw Khin May Ni ( 1 9 9 9 , 3 , 3 〜 2 0 0 0 , 1 , 2 8 ) , L u c i a Yoshie Sumioka ( 1 9 9 9 , 1 0 , 1 2

〜

2 0 0 0 , 9 , 2 2 ) , Tran Le Quan ( 1 9 9 8 , 1 0

〜

1 9 9 9 , 3

），朴鍾集

( 1 9 9 8 , 1 0

〜

1 9 9 9 , 3 )

非常勤研究員：李慧英

。学位（修士・博士）取得者修士：

金子卓嗣：「花棋メタノールエキスのマウスマクロファージ誘導型

NO

合成酵素

mRNA

発現抑制活性成分の研究」

課程博士：

李慧英：「伝統薬物接骨木

c s α mbucuss i e b o l d i a n a

^BLUME

e x

GRAEBN.の茎）及び漢方方剤通関丸の抗骨粗緊症活性に関する研究」

論文博士：

長谷耕二：「肝障害抑制作用を有する新規和漢薬成分の検索及びその作用機序に関する研究」

超偉傑：「中国・東北産

Veratrum

属植物のアルカロイド成分に関する研究」

化 学 応 用 部 門