台灣款冬 (Petasites formosanus Kitamura) 成分Petasins 在離體天竺鼠氣管的鬆弛作用

Relaxant effects of petasins, ingredients of Petasites formosanus Kitamura in isolated guinea-pig trachea

雷建邦(Lei Chien-Bang)

指導教授 ： 柯文昌

臺北醫學大學/藥學院/藥學研究所/碩士(1998年)

摘要

我們分析台灣款冬 (Petasites formosanus Kitamura) 所抽提的 petasins 類化合物， 包括 petasi、iso-petasin、S-petasin與 iso-S-petasin 對氣管的鬆弛活性，上述 pet asins 類化合物對 histamine (10 mM)、carbachol (0.2 mM)、KCl (30 mM) 及 leukotr iene D4 (10 nM)預縮的離體天竺鼠氣管，產生濃度依存性的鬆弛作用，S-petasin 雖對 四種收縮劑無特殊的選擇性，但 iso-S-petasin 對 carbachol 和 KCl 預縮的鬆弛作用 較具有選擇性，它們的 IC50 都在 10 mM 左右，就構造-活性之關係而言，含有硫原子的 petasins 對氣管的鬆弛活性強度 (potency) 比沒有硫原子的 petasins 要強。上述 pe tasins 類化合物中，除了 iso-S-petasin (50∼200 mM) 使 carbachol之對數濃度─反 應曲線平行向右移動，且不改變其最大收縮，顯示具抗毒蕈素 (antimuscarinic effect) 之競爭作用外，S-petasin (10∼200 mM) 或 iso-S-petasin (10∼200 mM)，均非競爭 性地抑制 histamine、carbachol 或 KCl累加引起之收縮，在無鈣環境中， S-petasin或 iso-S-petasin預處理對 histamine (100 mM) 、carbachol (10 mM) 或 KCl (60 mM) 去極化因累加外鈣引起的收縮能非競爭性地抑制﹔在完全無鈣 (含 0.02 mM EGTA)環境中 ，兩者預處理對 histamine 或 carbachol 累加引起之收縮亦能非競爭性地抑制，顯示兩 者對細胞外鈣離子流入或者細胞內鈣離子釋放都有抑制作用。對 carbachol (0.2 mM) 預 縮而 nifedipine (10 mM) 引起的最大鬆弛情況下，S-petasin 或 iso-S-petasin 也會 產生更進一步的鬆弛，表示不管有無抑制 voltage 及/或 receptor operated calcium c hannels，一定尚有其他的鬆弛機轉。然而其鬆弛反應不受 Nw-nitro-L-arginine (20 mM )、a-chymotrypsin (1 U/ml)、propranolol (1 mM)、glibenclamide (10 mM)、methyle ne blue (25 mM)及 2',5'-dideoxyadenosine (10 mM) 存在的影響，表示其鬆弛作用與 nitric oxide、vasoactive intestinal polypeptide、b-adrenoceptor 受體活化、ATP- 敏感的鉀通道開啟、adenylate cyclase 或 guanylate cyclase 活化無關。S-petasin ( 10∼20 mM) 或 iso-S-petasin (10∼20 mM) 不能使 forskolin 及 sodium nitroprussi de 的對數濃度─反應曲線濃度依存性地向左移動，亦不能增加 forskolin 及 sodium ni troprusside 的 pD2 值，由 phosphodiesterase (PDE) 活性的直接測定，得知 S-petas in (100∼300 mM)能有意義地抑制 cAMP-dependent PDE 的活性，但最高只能抑制 33.94 ±6.06 % (n=5)，顯示 S-petasin 只有輕微的抑制作用，而 iso-S-petasin (30∼300 m M) 不能有意義地抑制此酵素，S-petasin 及 iso-S-petasin (3∼300 mM) 亦不能有意義 地抑制 cGMP-dependent PDE 的活性。綜合以上結果，兩者對細胞外鈣離子流入或者細胞 內鈣離子釋放都有抑制作用，iso-S-petasin具有較強的抗毒蕈素作用，而 S-petasin 對 cAMP-dependent PDE 只有輕微的抑制作用。

關鍵字

Petasins ； 台灣款冬 ； 抗毒蕈素作用 ； 外鈣內流 ； 內鈣釋放 ； 磷酸雙酯酵素

並列摘要

Four petasins, including petasin, iso-petasin, S-petasin and iso-S-petasin, we re isolated from Petasites formosanus Kitamura. They concentration-dependently relaxed histamine (10 *M)-, carbachol (0.2 *M)-, KCl (30 mM)- or leukotriene D4 (10 nM)-induced precontractions of isolated guinea-pig trachealis. Iso-S-pe tasin selectively relaxed carbachol- and KCl-induced precontractions, although S-petasin non-selectively relaxed the precontractions induced by these contra ctile agents. Their IC50s were approximately about 10 mM. It seems that the re laxant effects of sulfur containing petasins, S-petasin and iso-S-petasin, wer e more potent than those of non-sulfur containg petasins, petasin and iso-peta sin.The preincubation of S-petasin or iso-S-petasin non-competitively inhibite d contractions induced by cumulatively adding histamine, carbachol and KCl in isolated guinea-pig trachealis, with an exception that the preincubation of is o-S-petasin (50~200 *M) competitively inhibited cumulative carbachol-induced c ontractions, suggesting that iso-S-petasin had an antimuscarinic effect. Both S-petasin and iso-S-petasin had a selectively inhibitory effect on cumulative KCl-induced contractions. In Ca2+-free medium, preincubation of S-petasin or i so-S-petasin non-competitively inhibited cumulative Ca2+-induced contractions in histamine (100 mM)-, carbachol (10 mM)- or KCl (60 mM)-depolarized tracheal is. In Ca2+-free medium containing 0.02 mM EGTA, the incubations of S-petasin and iso-S-petasin also non-competitively inhibited cumulative histamine- or ca rbachol-induced contractions. The above results suggest that S-petasin and iso -S-petasin may inhibit Ca2+-influx from extracellular space and Ca2+-release f rom intracellular Ca2+ stores. In normal Ca2+-medium, S-petasin was significan tly more potent than iso-S-petasin on the inhibition of Ca2+-influx from extra cellular space and Ca2+-release from intracellular Ca2+ stores induced by hist amine. However, iso-S-petasin was significantly more potent than S-petasin on the inhibition of Ca2+-influx from extracellular space via receptor (ROC) and/ or voltage operated calcium channels (VOC), which were opened by carbachol-dep olarization in Ca2+-free medium. After a maximal inhibition on carbachol (0.2 *M)-induced precontraction by nifedipine (10 *M), S-petasin or iso-S-petasin c aused a further relaxation of the trachealis. The result suggests S-petasin an d iso-S-petasin may have other relaxant mechanisms regardless of whether inhib iting VOC in the trachealis. However, their relaxant effects were not affected by the presence of propranolol (1 *M), 2*,5*-dideoxyadenosine (10 *M), methyl ene blue (25 *M), glibenclamide (10 *M), Nw-nitro-L-arginine (20 *M) or *-chym otrypsin (1 U/ml). It suggests their relaxing effect may be unrelated to activ ation of *-adrenoceptor, adenylate cyclase or guanylate cyclase, the opening o f ATP-sensitive potassium channels and the liberation of nitric oxide (NO) or vasoactive intestinal polypeptide (VIP).S-petasin and iso-S-petasin (10~20 *M) did not produce a parallel leftward shift of the log concentration-response c urves of forskolin and sodium nitroprusside. They did not affect pD2 values of forskolin and sodium nitroprusside. Neither cAMP- nor cGMP-dependent phosphod iesaterase (PDE) activity was inhibited by S-petasin and iso-S-petasin, except that S-petasin (100~300 *M) had a slightly inhibitory effect on cAMP-dependen t PDE activity. The maximal inhibition on the enzyme was only 33.94 * 6.06 % ( n=5). In conclusion, S-petasin and iso-S-petasin inhibited both Ca2+-influx fr om extracellular space and Ca2+-release from intracellular stores. In addition , iso-S-petasin had an antimuscarinic effect and S-petasin slightly inhibited cAMP-dependent PDE.

並列關鍵字

petasins ； Petasites formosanus Kitamura ； antimuscarinic activity ； calcium influx ； calcium release ； phosphodiesterase