3-O-Methylquercetin 在活體及離體抗氣喘的作用機轉
Mechanisms of anti-asthmatic action of 3-O-methylquercetin in vivo and in vitro
中文摘要
第一部份
中原鼠李Rhamnus Nakaharai (Hayata) Hayata (Rhamnaceae，鼠李科)，在台灣作為民 間藥，用於治療便秘、發炎、腫瘤及氣喘等疾病。3-O- Methylquercetin (3-MQ) 為此植物主要 有效成分，已被報告在低濃度下就會抑制天竺鼠氣管cAMP-phosphodiesterase (PDE)及 cGMP-PDE 的活性。所以我們有興趣研究 3-MQ 在活體及離體抑制卵蛋白 (OVA)¾ 引起的氣道 過度反應。3-MQ (3-30 mmol/kg, i.p.) 有意義地抑制敏感化小白鼠第二次卵蛋白刺激後，因 吸入氣化methacholine (50 mg/ml) 而引起的 Penh 值增加，也有意義地抑制總發炎細胞、
巨噬細胞、嗜中性血球、嗜伊紅白血球，但非淋巴球，當與它們的賦形劑(vehicle)比較，3-MQ (1-10 mM) 及 Ro 20-1724 (3-30 mM) 有意義地降低 OVA (10 mg/ml)¾ 引起的收縮。3- MQ (30 mM)與 PDE3 選擇性抑制劑 milrinone 有意義地增強離體天竺鼠左、右心房的基本收 縮力，然而，3-MQ 及 milrinone 不影響右心房的基本心拍數，也不移動 isoprenaline 引起的 收縮及心跳反應。3-MQ (3-30 mmol/kg, i.p.)對清醒小白鼠的收縮壓、及被動逃避的步入猶疑 時間 (step-through latency)不影響，也不影響活性 (naive behavior)、正向反射、牽引 (traction)、及滾桿 (rotarod)試驗。結論，3-MQ 有抗發炎及支氣管擴張作用，在沒有明顯副 作用的劑量下，具有治療氣喘的潛力。
第二部份
中原鼠李Rhamnus Nakaharai (Hayata)Hayata (Rhamnaceae，鼠李科)，在台灣作為民 間藥，用於治療便秘、發炎、腫瘤及氣喘等疾病。3-O-Methylquercetin (3-MQ) 為此植物主要 有效成分，已被證實具有抑制天竺鼠氣管cAMP-phosphodiesterase (PDE)及 cGMP-PDE 的 作用。所以我們有興趣研究及探討3-MQ 對於來自天竺鼠肺臟及心臟的 PDE 各種亞型酵素 (isozyme)的抑制效果。
天竺鼠的肺臟及心臟經研磨及離心步驟，取其上清液使通入Q-Sepharose 陽離子交換樹脂，
藉著改變NaCl 的濃度，便可依序由肺臟分離得到 PDE1、PDE5、PDE2 及 PDE4，而由心臟得 到PDE3。3-MQ 對於 PDE1~5 的百分之五十抑制濃度 (IC50)分別為 31.9 mM、18.6 mM 、1.6 mM、28.5 mM 及 86.9 mM。
3-MQ 濃度為 10~100 mM 時，對於 PDE2 為非競爭性抑制，但卻會競爭性抑制 PDE4，若濃 度介於1~10 mM 時，對於 PDE3 也是呈現競爭性抑制。3-MQ (10~30 mM)雖然有點傾向於 競爭性抑制PDE1，但若再提高 3-MQ 的濃度，則非競爭性抑制 PDE1，所以仍認為 3-MQ (10~100 mM)對於 PDE1 及 PDE5 屬於非競爭性抑制。目前的實驗數據顯示出，3-MQ 作用於 PDE3 及 PDE4 之 Ki 值分別與 milrinone 及 Ro 20-1724 相似。
雖然3-MQ 抑制 PDE4 的效果並非很強，但結論仍認為 3-MQ 呈現有選擇性、競爭性的抑制 PDE3 及 PDE4。因此 3-MQ 除了對抗病毒外，亦可能具有相當潛力的氣喘治療效果。
英文摘要
Part I
Rhamnus Nakaharai Hayata (Rhamnaceae), has been used as a flok medicine in Taiwan for treating constipation, inflammation, tumors and asthma. 3-O-
Methylquercetin (3-MQ), a main constituent of the plant, has been reported to inhibit total cAMP- and cGMP-phosphodiesterase (PDE) of guinea-pig trachealis at low concentration. Therefore we are interested in investigating its suppressive effects on ovalbumin (OVA)-induced airway hyperresponsiveness in vivo and in vitro. 3-MQ (1-10 mg/kg, i.p.) significantly suppressed the enhanced pause (Penh) value induced by aerosol methacholine (50 mg/ml) in sensitized mice after
secondary allergen challenge. 3-MQ (3-30 mmol/kg, i.p.) also significantly
suppressed total inflammatory cells, marcrophages, neutrophils and eosinophils, but not lymphocytes. 3-MQ (1-10 mM) as well as Ro 20-1724 (3-30 mM), a selective PDE4 inhibitor, significantly attenuated OVA (10 mg/ml)-induced
contractions when compared to their vehicle. 3-MQ (30 mM) as well as milrinone (1-10 mM), a selective PDE3 inhibitor, significantly enhanced baseline
contractions in isolated guinea pig left and right atria. However, 3-MQ and milrinone did not significantly affect baseline beating rate in right atria, and also did not significantly shift log concentration-response curves of isoprenaline in both inotropic and chronotropic effects. 3-MQ (3-30 mmol/kg, i.p.) did not significantly affect systolic pressure and step-through latency of passive avoidance behavior in conscious mice. 3-MQ (3-30 mmol/kg, i.p.) also did not affect naive behavior, right reflex, traction and rotarod tests. In conclusion, 3-MQ has both anti-inflammatory and bronchodilatory effects, and has a potential in the treatment of asthma at a dose without obvious side effects.
Part II
Rhamnus Nakaharai (Hayata)Hayata (Rhamnaceae), has been used as a flok medicine in Taiwan for treating constipation, inflammation, tumors and asthma. 3- O-Methylquercetin (3-MQ), a main constituent of the plant, has been reported to inhibit total cAMP- and cGMP-phosphodiesterase (PDE) of guinea-pig trachealis.
Therefore we are interested in investigating the inhibitory effect of 3-MQ on various PDE isozymes from guinea-pig lungs and hearts.
Isolated guinea-pig lungs and hearts were homogenized and centrifuged. The supernatant was chromatographed over a column of Q-sepharose, and eluted with various concentrations of NaCl. In the following order, PDE subtype 1, 5, 2, 4 from lungs, and 3 from hearts were separated. The IC50 values of 3-MQ on these isozymes were 31.9, 86.9, 18.6, 28.5 and 1.6 mM, respectively.
3-MQ (10-100 mM) non-competitively inhibited PDE2, but competitively inhibited PDE4. 3-MQ (1-10 mM) also competitively inhibited PDE3. However, 3-MQ (10-100
mM) did not competitively inhibit PDE1 and 5, though it had a tendency to competitively inhibit PDE1 at concentrations of 10-30 mM. The present results showed that Ki values of 3-MQ was similar to that of milrinone in PDE3, and was not significantly different from that of Ro 20-1724 in PDE4, respectively.
In conclusion, 3-MQ revealed selective and competitive PDE3/PDE4 inhibitor, although its inhibitory effect on PDE4 was not potent. Therefore, 3-MQ may have a potential in the treatment of asthma beside its antiviral activity.
