細 胞 資 源 工 学
雄 子 継 夫 征 隆 博 憲 部 津 代 村 服 横 宮 中
授 授 手 官
教
教 助 助 技
本部門では，和漢薬資源、の恒久的維持，育成を図るため，薬用生物に関する細抱工学的研究を行なうと 同時に，動植物細胞の持つ遺伝 情報を解析して，その薬用資源開発への応用，あるいは微生物および動物 細胞を用いて生理活性物質の探索，和漢薬の薬効発現機構の解明を行なうことを目指している。本年度の 主な研究テーマと成果は下記の通りである。
I .腸内嫌気性菌によるバイオトランスフォーメーション
腸内細菌によるサイコサボニンの変換反応を検討し，Eubacteriumsp. A 44, Bijidobacterium sp. Saiko 1, Saiko‑2などの菌株がサイコサボニンをプロサイコゲニンに変換することを見いだした。Eubacteriumsp. A 44からサイコサポニン加水分解酵素（β−D−グルコシダーゼの一種），プロサイコゲニン加水分解酵素
（β−Dーフコシダーゼの一種）を精製し，その基質特異性，至適pHなどを調べた。
II.腸内細菌による薬物の代謝活性化に関与する遺伝子
Sennosideの加水分解に関与する Bijidobacteriumsp.由来の酵素遺伝子の塩基配列を決定した。
III .抗ウイルス薬の開発
霊芝およびその胞子から種々のラノスタン型のトリテルペンを単離し，それらの抗HIV作用， HIVプ ロテアーセ、阻害作用を見いだした。また，韓国産，スーダン産の植物の抗HIV作用，日IV−プロテアーゼ 阻害作用を検索した。
N.腎疾患における病態の解明と治療薬の開発
活性酸素，フリーラジカルが直接的，間接的に腎疾患に関わっていることから，丹参から単離したmag‑
nesium lithospermate B，緑茶，大黄牡丹皮湯，温牌湯を中心に腎不全モデル，虚血一再濯流による腎障害 のinvitro評価系， cisplatin,cephaloridineによる細胞障害系で検討し，その有効性を明らかにした。ま た漢方方剤，生薬，フラボノイドの抗酸化活性についても検討した。
く 〉 著 書
1 ）服部征雄（分担）：『漢方薬理学』高木敬次郎監修，
木村正康編集，南山堂，東京， 1997.
2 ）横淳隆子，大浦彦吉，西岡五夫：腎摘ラットで増 加する活性酸素をmagnesiumlithospermate B が軽減した。「腎とフリーラジカル 第3集」加 藤克己，長瀬光昌監修，山田晴生，中村一路編，
93‑96，東京医学社，東京， 1997.
3）横津隆子，藤塚直樹，大浦彦吉，柏木寛．腎不 全ラットにおけるフリーラジカルの産生と消去能 について。「腎とフリーラジカルー第3集一」加
藤克巳，長瀬光昌監修，山田晴生，中村一路編，
114 117，東京医学社，東京， 1997.
く 〉 原 著
1) Lim Y. A., Ma C., Kusumoto I. T., Miyashir。
H., Hattori M., Gupta M. P. and Correa M. : HIV 1 Reverse Transcriptase Inhibitory Principles of Chαmαesyce hyssopifoliα．
Phytother. Res. 11, 22‑27, 1997.
In the course of our studies on the development of anti acquired immunodeficiency syndrome (AIDS) agents, we isolated corilagin, quercetin 3‑0−β D
glucopyranoside and 1,3,4,6‑tetra ‑0‑galloyl一β−D‑
glucopyranose from C加mae.りcehyssoρifolia, as the main inhibitory sbstances against human im‑ munodeficiency virus (HIV) reverse transcriptase, an enzyme essential for the proliferation of HIV.
The IC50 of these substances were 20, 50 and 86 μM, respectively, their modes of inhibition being non ‑ competitive with respect to the substrate.
2) Helal A. M., Nakamura N., Meselhy R. M., Eト Fishawy A. M., Hattori, M. and Mahran G. H. : Guaianolides from Centαureα scopαriα．
Phytochemistry, 45, 551‑554, 1997.
3β，8 α 0 Di (4 hydroxytigloyl) 1αH, 5αH, 6βH, 7αH‑guai 4 (15), 10 (14), 11 (13) triene 6,12 olide; a new guaianolide, has been isolated from the aerial parts of Centaurea scoρaria together with nine known ones.
3 ) Heikal 0. A., Miyashiro H., Akao T. and Hattori M. : 'Quantitative Determination of Paeoniflorin and Its Major Metabolites, Paeonimetabolin I, in the Rat Plasma by Enzyme Immunoassay. J. Trad. Med., 14, 15‑ 19, 1997.
After oral administration of paeoniflorin (PF) at a dose of 20 mg/kg in rats, the plasma concentra‑ tions of PF and its major metabolite paeonimetabolin I (PM I) were determined by using the enzyme immunoassay method. The plasma concentration of PF rapidly reached a Cmax
(95 ng/ml) at 30 min, then decreased to 0.2 ng/ml at 240 min after administration of PF. On the other hand, the plasma concentration of Pお1‑I reached a
Cmax (400 ng/ml) at 140土24.7min, then decreased to 0.5 ng/ml at 480 min. The AUCs were 7700±900 and 52400±17500 ng・min・ml 1, for PF and PM I, respectively, indicating that the latter was a major compound present in the plasma. These findings suggest that orally administered PF is scarcely absorbed from the gastrointestinal tract (GIT), while the unabsorbed one is transformed to PM I by intestinal bacteria, which is subsequently absorbed from GIT. This is the first report showing the presence of PM I in the plasma after oral admini‑ stration of PF.
4) Lim Y. A., Kida H., Miyaji M., Kusumoto I. T., Miyashiro H., Hattori M., Shimotohno K.,
Gupta M. P. and Correa M.: Inhibitory Effects of Some Panamanian Plants on Human Immunodeficiency Viral Reverse Transcriptase and Protease. J. Trad. Med., 14, 54‑58, 1997.
In the course of our studies on the development of anti‑HIV agents, we have screened thirty nine Panamanian plants for their inhibitory activity against HIV 1 reverse transcriptase (RT) and protease (PR), essential enzymes for proliferation of HIV. Water extracts of Chamaesyce hyssoρi命 的
(whole plant), Cordia宅pinescens (leaves), and Hyptis lantanifolia (aerial parts), and the methanol extracts of Tetri砂防ris macrocarpα（aerial parts) and Xylop￡αj均 的cens(leaves) appreciably inhib ited the activity of HIV 1 RT, with IC50 of 8, 6, 7, 8, and 11 μg/ml, respectively. Furthermore, the meth anol extracts of Erythroxylum citrifolium (trunk), Seγjαηぬmexicαηα （wholeplant), Wiαltheγiα in dieα
(branches), and a water extract of Lindackeria lauriηα(leaves) showed appreciable inhibition against HIV‑protease, with IC50 of 58, 48, 46, and 54 μg/ml, respectively.
5) Kida H., Nakamura N, Meselhy R. M., Akao T. and Hattori M.: Isolation and Identifica‑ tion of Human Intestinal Bacteria Capable of Hydrolyzing Saikosaponins. J. Trad. Med., 14, 34‑40, 1997.
For studying biotransformation of saikosaponins by human intestinal bacteria, thin layer chromato‑ graphy‑densitometric analysis was performed on the metabolites obtained by anaerobic incubation of saikosaponins with a human fecal suspension. It revealed that saikosaponins a, b1, bz and d were converted to the corresponding prosaikogenins and saikogenins in order. In the case of saikosaponin c, saikogenin E was obtained as a sole product. Of 31 defined human intestinal bacterial strains, only Eubαcterium sp. A 44. could metabolize sai‑ kosaponins a, and b1 to the corresponding prosai‑ kogenins and saikogenins. Saikosaponins d and b2 were hydrolyzed to the respective prosaikogenins, but no saikosaponin c by this strain.
After screening bacterial colonies from fresh human feces for metabolizing activity of sai‑ kosaponins, two of 60 isolates showed appreciable
