橙皮素在活體及離體抗氣喘的作用機轉
part I
橙皮素 (hesperein) 是一種選擇性 PDE4 的抑制劑，然其是否有治療氣喘的作用，目前還未有研究，因此是本篇研究 的目的。本實驗將 BALB/c 小鼠腹腔內注射卵蛋白 (ovalbumin; OVA) ，使其敏感化，再以卵蛋白 (OVA) 氣化噴霧刺激 (s econdary challenge) ，利用整體體積描述器來分析因 methacholine (MCh) 引起的氣道過度反應 (airway hyperresponsi veness response; AHR) ，結果顯示 hesperetin 能抑制卵蛋白 (OVA) 引起的氣道過度反應 (AHR) ， hesperetin (10~3 0 mol/kg, i.p.) 亦能依劑量相關性且有意義地減少 MCh (25~50 mg/ml) 引起的 Penh 值增加。投與 hesperetin (3~30
mol/kg, i.p.)
的小白鼠與暴露於 OVA 氣化噴霧但未敏感化 (non-sensitized) 之小白鼠無異。觀察肺泡灌流液 (BA LF) 的細胞沉澱物，發現 hesperetin (10~30 mol/kg, i.p.) 具有統計意義地 (P < 0.05) 減少總發炎細胞數、嗜中性血 球、嗜伊紅白血球、淋巴球及巨噬細胞。 Hesperetin (3~30 mol/kg, ip) 有統計意義地 (P < 0.05) 降低 IL-2, IL-4, IF N- 和 TNF- 的釋放，當劑量高達 30 mol/kg 時更可顯著地抑制 IL-5 的釋放 (P < 0.05) 。 Hesperetin (30 M) 有意義地抑制累加之 OVA (10~100 g/ml) 所造成敏感化離體天竺鼠氣管的收縮。由 Lineweaver-Burk 分析發現 he speretin (10~100 M) 對 PDE4 呈競爭性的抑制作用。結論， hesperetin 具選擇性且競爭性地抑制 PDE4 ，在所用劑 量 (10~30 mol/kg, i.p.) 具抗發炎及支氣管擴張的作用，因此推測 hesperetin 極具治療氣喘的潛力。
Part II
西方各國均認為選擇性的 PDE4 抑制劑是治療氣喘及慢性阻塞性肺病的希望，只可惜會結合到腦的 rolipram 高親和力 的結合點 (HARBS) ，而造成嘔吐及胃酸分泌的副作用。有些類黃鹼素也對 PDE4 具選擇性的抑制作用，因此有必要 了解它們在腦部顆粒 HARBS 結合的情形，以篩選出治療氣喘有用的藥物。
敏感化與未敏感化的天竺鼠全腦細胞顆粒 HARBS ，以 Scatchard plots 分析，結果顯示兩者之間之 Bmax 在統計上並 無差別。本文結果顯示 luteolin 及 genistein 會依濃度相關性地取代結合在敏感化天竺鼠全腦細胞顆粒 HARBS 之 [3H]-rolipram
，其 EC50 值分別為 11.2 及 47.7 ?M ，其他所測定的類黃鹼素其 EC50 值均超過 300 ?M 。因 diosmetin 之 EC50 值大 於 luteolin ，顯示 C-4? 之 OH 基被甲基化之後就比較不會與 HARBS 結合。而 luteolin-7-glucoside 之 EC50 值大於 lut eolin ，推測 C-7 之 OH 基被醣化後形成的龐大體積，使其不易與腦部的 HARBS 結合。 Biochanin A 或 prunetin 之 EC 50 值均大於 genistein ，顯示 C-4? 或 C-7 之 OH 基甲基化後，較難與 HARBS 結合。
Biochanin A 的 PDE4H/PDE4L 比大於 35 ，不過 biochanin A 對 PDE1 及 PDE2 也有抑制的作用，對 PDE4 的選擇性相 對較弱。 Hesperetin 和 hesperetin triacetate 的 PDE4H/PDE4L 比分別為 11 和 12 ，其中 hesperetin 可選擇性的抑制 PD E4 ，它們或許能成為治療氣喘及慢性阻塞性肺病的藥物。
Part I
Hesperetin selectively inhibits PDE4. To evaluate whether it possesses anti-asthmatic effect is the aim of this investigation. Female BALB/c mice were sensitized by an intraperitoneal injection of ovalbumin (OVA), then challenged two times via the airway by ultrasonic nebulization of 1% OVA.
After secondary challenge, the airway hyperresponsiveness was measured in unrestrained animals nebulized methacholine (MCh, 6.25~50 mg/ml), by barometric plethysmography using a whole-body plethysmograph. Hesperetin (10~30 μmol/kg, i.p.) revealed it dose-dependently and significantly atte nuated the enhanced pause (Penh) value induced by MCh (25~50 mg/ml). The Penh values of mice administered hesperetin (3~30 μmol/kg, i.p.) did n ot significantly differ from those challenged by OVA without sensitization mice (non-sensitized). Hesperetin (10~30 μmol/kg, i.p.) also significantly i nhibited total inflammatory cells, macrophages, lymphocytes, neutrophils, eosinophils,in bronchoalveolar lavage fluid (BALF). Hesperetin (10~30 μm ol/kg, i.p.) also significantly attenuated the release of IL-2, IL-4, IFN-γ and TNF-α. It at a dose of 30 μmol/kg further significantly inhibited the release of IL-5. Hesperetin (30 μM) significantly inhibited cumulative OVA (10~100 μg/ml)-induced contractions of isolated sensitized guinea pig trachea.
By Lineweaver-Burk analysis, hesperetin (10~100 μM) competitively inhibited PDE4 activities.
In conclusion, hesperetin selectively and competitively inhibited PDE4 activities. At doses of 10~30 μmol/kg (i.p.), it possessed anti-inflammatory and bronchodilating effects. Therefore it may have potential in the treatment of asthma.
Part II
In western countries , it is realized a hope that selective PDE4 inhibitors may be used in the treatment of asthma and chronic obstructive pulmonary dis ease. However , they may bind to brain high affinity rolipram binding sites (HARBS) from which some side effects, such as vomiting and gastric hype rsecretion, are developed. Some flavonoids also selectively inhibit PDE4, therefore it is nessary to understand their binding on the HARBS of brain par ticulate and to screen out useful drugs.
By Scatchard polt analysis, it revealed that Bmax values of HARBS of sensitized and non-sensitized guinea pig brain particulate did not significantly d iffer from each other.
In the present result, luteolin and genistein concentration-dependently displaced [3H]-rolipram bound on the HARBS of the sensitized guinea pig whol e brain particulate. Their EC50 values were 11.24 and 47.75 ?M, respectively. Those of other flavonoids used were beyond 300 ?M. It revealed that th e methylation on C-4?OH group of flavones reduced the binding on the HARBS, because the EC50 values of diosmetin was greater than that of luteoli n. It also showed glycosylation on C-7 group of flavones may reduced the binding on the HARBS, because the EC50 value of luteolin-7-glucoside was greater than that of luteolin. It also revealed that the methylation on C-4?or C-7 OH group of isoflavones may reduced the binding on the HARBS, bec ause both EC50 values of biochanin A and prunetin were greater than that genistein.
The PDE4H/PDE4L ratio of biochanin A was greater than 35, but it also inhibited PDE1 and PDE2. In other words, its selectivity on PDE4 inhibition was not so high. However, those of hesperetin and hesperetin triacetate were 11 and 12, respectively. They selectively inhibited PDE4, therefore they may become therapeutic drugs for the treatment of asthma and chronic obstructive pulmonary disease
Machanisms of anti-asthmatic action of hesperetin in vivo and in vitro
Machanisms of anti-asthmatic action of hesperetin in vivo and in vitro
