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Flavone類衍生物之氣管鬆弛作用及其結構-活性的關係

Flavone類衍生物之氣管鬆弛作用及其結構-活性的關係. 我們分析十二種 flavone 類衍生物，包括 6-hydroxyflavone, 7-hydroxyflavone, chrysin, baicalein, luteolin, 5-methoxyflavone, 6-methoxyflavone, diosmetin, diosmin, acacetin, tangeretin 及 luteolin-7-glucoside 對 histamine (30 μ M) 預縮的離體天竺鼠氣管，產生劑量依存性的鬆弛作用，由其 IC50 得

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Flavone類衍生物之氣管鬆弛作用及其結構-活性的關係

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  1. Flavone類衍生物之氣管鬆弛作用及其結構-活性的關係Flavone類衍生物之氣管鬆弛作用及其結構-活性的關係 • 我們分析十二種 flavone 類衍生物，包括 6-hydroxyflavone, • 7-hydroxyflavone, chrysin, baicalein, luteolin, • 5-methoxyflavone, 6-methoxyflavone, diosmetin, diosmin, • acacetin, tangeretin 及 luteolin-7-glucoside 對 histamine (30 μ • M) 預縮的離體天竺鼠氣管，產生劑量依存性的鬆弛作用，由其 IC50 得 • 知其活性大致依序為6-hydroxyflavone, 7-hydroxyflavone, luteolin, • tangeretin > chrysin, 5-methoxyflavone, > baicalein, acacetin, • luteolin-7-glucoside > 6-methoxyflavone, diosmetin, diosmin.， • 其 structure-activity relationship (SAR) 如下: (a) 第 7 位之 OH • 接上 sugar,如 luteolin 變成 luteolin-7-glucoside﹔及diosmetin 變 • 成 diosmin, 則活性顯著下降; (b) 第 6 或 3' 及位之 H 被 OH 取代, • 如 flavone 變成 6-hydroxyflavone 或 apigenin 變成 luteolin 活性 • 皆明顯增加，但第 7 位之 H 被 OH 取代，如 flavone 變成 • 7-hydroxyflavone 則活性不變，若第 5 及第 7 位之 H 同時被 OH 取代 • ，如 flavone 變成 chrysin 亦然，反觀第5, 6 及 7 位之 H 被 OH 取 • 代，如 flavone 變成 baicalein, 或原先已在第 5 或 7 位有 OH 只單 • 獨在第 6 位以 OH 取代 H 如 chrysin 變成 baicalein 則活性會下降； • (c) 第 5 位之 H 被 OCH3 取代﹐如 flavone 變成 5-methoxyflavone • 或進一步多位 H 被 OCH3 取代如 5-methoxyflavone 變成 tangeretin • 活性不變，但然而第 6 位之 H 被 OCH3 取代，如 flavone 變成 • 6-methoxyflavone 則活性明顯下降﹔(d) 第 6 位之 OH 被 OCH3 取代如 • 6-hydroxyflavone 變成 6-methoxyflavone；或第 4' 位之 OH 被 OCH3 • 取代，如 luteolin 及 apigenin 分別變成 diosmetin 及 acacetin﹐ • 活性會明顯下降。上述十二種flavone類衍生物中較強的六種，包括 • 6-hydroxyflavone, 7-hydroxyflavone, chrysin, luteolin, • 5-methoxyflavone 及 tangeretin 預處理均能非競爭性地對抗累加 • histamine, carbachol 或 KCl 引起的收縮，它們的 pD2'值大致上均有 • 意義地小於它們的 -logIC50 值， 顯示他們抑制內鈣釋放的能力小於抑 • 制外鈣內流的能力。在高鉀 (60 mM)無鈣溶液中，它們也能非競爭性地抑 • 制累加外鈣引起的收縮，也對 histamine (30 μM)預縮而nifedipine • (10 μM) 引起的最大鬆弛產生更進一步的鬆弛，表示除了能抑制 • voltage (VOC) 及/或 receptor operated calcium channels (ROC) 外 • ，尚有其他的鬆弛機轉。然而除了下列三點外，這六種flavone 類衍生物 • 的鬆弛反應並不因上皮細胞去除或 propranolol (1 μM)、 • glibenclamide (10 μM)、methylene blue (25 μM) 及 • 2',5'-dideoxyadenosine (10 (M) 存在的影響，(一) 6-hydroxyflavone

  2. 在 2&apos;,5&apos;-dideoxyadenosine (10 μM) 存在下，能使 6-hydroxyflavone • 的對數濃度-反應曲線向右平行移動，表示 6-hydroxyflavone 可能會活 • 化 adenylate cyclase ；(二) luteolin 在 methylene blue (25 μM) • 存在下，能使 luteolin 的對數濃度-反應曲線向右移動，表示 luteolin • 可能會活化 guanylate cyclase，(三) 5-methoxyflavone 在 • glibenclamide (10 μM) 存在下，能使 5-methoxyflavone 的對數濃度- • 反應曲線向左平行移動，原因不明。6-hydroxyflavone (20 μM) 及 • luteolin (20 μM) 能使 forskolin 的對數濃度-反應曲線向左平行移動 • ，而使 forskolin 的 pD2 值增加， 6-hydroxyflavone (10, 20 μM), • luteolin (20 μM) ,5-methoxyflavone (20 μM) 及 tangeretin (20 • μM)能使 nitroprusside 的對數濃度-反應曲線向左平行移動，而使 • nitroprusside 的 pD2 值增加。顯示6-hydroxyflavone, luteolin, • 5-methoxyflavone 及 tangeretin 可能有抑制 phosphodiesterase • (PDE) 的作用，由 PDE 的直接測定得知，luteolin 在 100 μM 及 300 • μM 會大大地抑制 cAMP-PDE 及 cGMP-PDE 的活性，而抑制 cGMP-PDE 之 • 程度有意義地大於抑制 cAMP-PDE；其他 flavone 如 6-hydroxyflavone, • 7-hydroxyflavone, chrysin, 5-methoxyflavone, 及tangeretin 雖能抑 • 制 cAMP-PDE 及 cGMP-PDE, 但並不強，即使 300 μM， 尚不完全 ( < • 70 % )； 然而 6-hydroxyflavone 100 μM 抑制 cAMP-PDE 之程度有意 • 義地大於抑制 cGMP-PDE 以及 tangeretin 300 μM 抑制 cGMP-PDE 之程 • 度有意義地大於抑制 cAMP-PDE。綜觀以上，luteolin 的鬆弛機轉大部份 • 來自對 PDE 的抑制，特別對 cGMP-PDE的抑制較具選擇性，6- • hydroxyflavone 可能來自活化 adenylate cyclase 及輕微的 PDE 抑制 • 作用，其他四種對 PDE 僅具輕微的抑制作用； 以上六種均能抑制外鈣流 • 入及內鈣釋放，除 leteolin 外，對外鈣流入的抑制均比對內鈣釋放的抑 • 制較具選擇性。

  3. Tracheal relaxant effects of flavone derivatives and their structure-activity relationships • The tracheal relaxant activities and action mechanisms offlavone derivatives, including 6-hydroxyflavone,7-hydroxyflavone, chrysin, baicalein, luteolin,5-methoxyflavone, 6-methoxyflavone, diosmetin, diosmin,acacetin, tangeretin and luteolin-7-glucoside were analyzed tounderstand their structure-activity relationships (SAR). Theabove tweleve flavones concentration-dependently relaxed thehistamine (30 μM)-, carbachol (0.2 μM)-, and KCl (30 mM)-induced precontractions of isolated guinea-pig trachea. Roughly,according to their IC50 values, the order of their relaxantpotency was 6-hydroxyflavone, 7-hydroxyflavone, luteolin,tangeretin > chrysin, 5-methoxyflavone, > baicalein, acacetin,luteolin-7-glucoside > 6-methoxyflavone, diosmetin, diosmin. TheSAR was concluded as follows: (a) The substitution of sugargroup at position 7, such as luteolin to luteolin-7-glucosideand diosmetin to diosmin, reduced their relaxant activities ;(b) The substitution of OH group at position 6 or 3&apos;, such asflavone to 6-hydroxyflavone or apigenin to luteolin,respectively, increased their relaxant activity, but at position7 , such as flavone to 7-hydroxyflavone, and even at bothpositions 5 and 7, such as flavone to chrysin did not change itsrelaxant activity. On the contrary, substitution of OH group atpositions 5, 6, and 7, such as flavone to baicalein, or atposition 6 of 5, 7-dihydroxyflavone compound, such as chrysin tobaicalein decreased their relaxant activities. (c) Thesubstitution of OCH3 group at position 5, such as flavone to5-methoxyflavone, or further substitution at many otherpositions, such as 5-methoxyflavone to tangeretin, did notchange its relaxant activity, whereas at position 6, such asflavone to 6-methoxyflavone attenuate its relaxant activity. (d)The substitution of OCH3 to OH group at position 6, such as6-hydroxyflavone to 6-methoxyflavone or at position 4&apos;, such asluteolin and apigenine to diosmetin and acacetin, respectively,markedly decreased their relaxant activity. The preincubation ofthe six more potent flavones, 6-hydroxyflavone,7-hydroxyflavone, chrysin, luteolin, 5-methoxyflavone ortangeretin among the above twelve compounds, non-competitivelyinhibited contraction induced by cumulatively adding histamine,carbachol or KCl in isolated guinea-pig trachea. In general,their pD2&apos; values were significantly less than their -logIC50values. Therefore, their abilities of inhibition on calciumrelease from intracellular calcium stores may be less potentthan those of suppression on calcium influx from extracellularfluid. They also non-competitively inhibited contractions of thetrachealis induced by cumulatively adding calcium into highpotassium (60 mM)-Ca2+ free medium in the trachealis. Aftermaximal relaxation on histamine (30 μM)-induced precontractionby nifedipine (10 μM), they caused further relaxation of thetrachealis. The result suggests that they may have otherrelaxing mechanisms in addition to inhibiting voltage (VOC) and/or receptor operated calcium channels (ROC) in the trachealis.

  4. With exception of the following three flavones, their relaxantresponses were not affected by the removal of epithelial cellsor by the preincubation of propranolol (1 μM), glibenclamide(10 μM), methylene blue (25 μM) or 2&apos;,5&apos;-dideoxyadenosine (10μM), suggesting their relaxing effects may not be related toepithelium derived relaxing factor(s), activation of β-adrenoreceptor, opening of ATP-sensitive potassium channels, oractivation of guanylate cyclase or adenylate cyclase. First,2&apos;,5&apos;-dideoxyadenosine (10 μM) parallelly rightward shifted thelog concentration-response curve of 6-hydroxyflavone, suggestingthat 6-hydroxyflavone may activate adenylate cyclase. Secondary,methylene blue (25 μM) parallelly to the rightward shifted thelog concentration-response curve of luteolin, suggesting thatluteolin may activate guanylate cyclase. Third, glibenclamide(10 μM) parallelly leftward shifted the log concentration-response curve of 5-methoxyflavone with unknown mechanism.6-hydroxyflavone (20 μM) and luteolin (20 μM) parallellyleftward shifted the log concentration-response curve offorskolin, and enhance the pD2 value of forskolin.6-hydroxyflavone (10, 20 μM), luteolin (20 μM),5-methoxyflavone (20 μM) and tangeretin (20 μM) alsoparallelly leftward shifted the log concentration-response curveof nitroprusside and enhanced the pD2 value of nitroprusside. Itseems that 6-hydroxyflavone, luteolin, 5-methoxyflavone, andtangeretin may inhibit phosphodiesterase (PDE) activity. Fromdetermination of PDE activity, we found that luteolin (100 and300 μM) markedly inhibited cAMP-PDE and cGMP-PDE activity andthat the inhibition on cGMP-PDE activity was significantlylarger than on that of cAMP-PDE. Other flavone derivatives, suchas 6-hydroxyflavone, 7-hydroxyflavone, chrysin,5-methoxyflavone, and tangeretin partially inhibited cAMP-PDEand cGMP-PDE activities. Even at a high concentration such as300 μM the inhibition was less than 70%. However, theinhibition on cAMP-PDE activity by 6-hydroxyflavone (100 μM)was significantly larger than that on cGMP-PDE, but theinhibition on cGMP-PDE activity by tangeretin (300 μM) wassignificantly larger than that on cAMP-PDE. These resultssuggest that the relaxant mechanism of luteolin is mainly theinhibition on PDE activity, especially on cGMP-PDE activity.6-Hydroxyflavone may activate adenylate cyclase and slightlyinhibit PDE activity. 7-Hydroxyflavone, chrysin,5-methoxyflavone and tangeretin only slightly inhibit theactivity of PDE. The above six flavones inhibit both calciuminflux and calcium release from calcium store. In addition toluteolin, these six flavones may inhibit calcium influx moremarkedly than that on calcium release.

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