7,3''-O- 二甲基橙皮素抗氣喘的作用
Anti-asthmatic action of hesperetin-7,3''-O -dimethylether
中文摘要
橙皮素 (hesperetin) 是一種選擇性 phosphodiesterase-4 (PDE4) 的抑制劑，為了提高其 抗氣喘效果，降低副作用，我們合成hesperetin 的甲基化衍生物 hesperetin-7-O-
methylether (7-HME) 及 hesperetin-7,3''-O-dimethylether (HDME)，7- HME 對 PDE4 幾乎沒有抑制作用，但 HDME 有很強的抑制作用，其 IC50 為 3.0 µM，HDME 抑制PDE4 的 IC50 比 hesperetin-5,7,3''-O-trimethylether (HTME) 還要小，
所以抗氣喘的效果有希望比HTME 更好。
將雌性小白鼠 (BALB/c) 腹腔內注射卵蛋白 (ovalbumin, OVA)，使其敏感化，再以氣化的卵 蛋白二次激釁 (secondary challenge) 後，利用整體體積描述器來分析因 methacholine (MCh) 所引起的氣道過度反應 (airway hyperresponsiveness； AHR)。本文實驗結果顯示，
HDME (3~30 µmol/kg, p.o.) 可以劑量依存性地、有意義地減低由 MCh (6.25~50 mg/ml) 所增加的enhanced pause (Penh) 值，也可以有意義地減少肺泡灌洗液 (BALF) 之總發炎細
胞、巨噬細胞、淋巴球、嗜中性白血球、及嗜伊紅性白血球的數量及第二型輔助T 細胞 (Th2
cells) 釋放的細胞介素 (cytokines)，如 interleukin (IL)-4、IL-5、tumor necrosis factor (TNF)-α 的含量，雖也會減少第一型輔助 T 細胞 (Th1 cells)釋放 IL-2。但是 interferon (INF)- γ 的含量不會減少，甚至於高劑量 (30 µmol/kg, p.o.) 時，反而會有意義地增加。HDME (3~30 µmol/kg, p.o.) 可以有意義地減少血清及肺泡灌洗液中 total 及 OVA-specific IgE 的 含量，相反的會使血清中IgG2a 劑量依存性地增加。此外，HDME 30 μM 會有意義地鬆弛敏 感化離體天竺鼠氣管的基本張力，HDME (3~30 μM) 也會有意義地抑制累加 OVA (10~100 μg/ml) 引起的敏感化離體天竺鼠氣管的收縮。
根據Lineweaver-Burk 的分析，HDME 對 PDE1、PDE3、及 PDE4 呈現競爭性的抑制，其 Ki 值分別為46.1、24.1 及 7.7 μM，雖然 HDME 對 PDE3 及 PDE4 的 Ki 值之間沒有意義差，但 PDE4 與 PDE1 的 Ki 值之間有意義差，暗示 HDME 對 PDE4 的親和力較好，HDME 抑制 PDE1、PDE3、及 PDE4 活性的 IC50 分別為 22.1、24.6 及 3.0 μM，也顯示對 PDE4 的抑制作 用較強，亦即與PDE4 的 low affinity rolipram binding sites (LARBS)的結合能力較強，而 且HDME 取代[3H]-rolipram 結合在敏感化天竺鼠全腦顆粒之 high affinity rolipram binding sites (HARBS)的 EC50 為 106.6 μM，所以 HDME 的 PDE4H/PDE4L 的比值為 106.6/3 (約 35.5)，比 HTME 的 PDE4H/PDE4L 的比值 (18.2) 還高。由於 HDME 同時抑制 PDE3，所以會加強其抗氣喘的作用。PDE4H/PDE4L 的比值雖為目前公認用於評估 PDE4 選擇 性抑制劑抗氣喘療效的方法，但畢竟是離體試驗的結果，本文獨創活體試驗的評估法，以減少 25% xylazine/ketamine 麻醉時間所需的劑量 (ADD25) ，除以抑制 75% Penh 値所需的劑 量 (ID75)，所得 rolipram，Ro 20-1724 及 luteolin 的比値非常接近它們 PDE4H/PDE4L 的 比值，如表二所列，値得信賴，而HDME (10~100 μmol/kg, s.c.) 並不會有意義地縮短 xylazine/ketamine 引起的麻醉時間，所以推測其噁心及嘔吐之副作用頗低，顯示 HDME 極 具抗氣喘的潛力。
結論，HDME 競爭性且選擇性抑制 PDE4 及 PDE3，而有支氣管擴張及抗發炎作用，但很少有 不良反應，它的PDE4H/PDE4L 比值是 35.5，是當今全世界最大者，顯示它具有治療氣喘的 潛力。
英文摘要
To improve antiasthmatic and reduce adverse effects of hesperetin, a
phosphodieasterase(PDE)-4 inhibitor, we synthesized hesperetin-7-O-methylether (7-HME) and hesperetin-7,3''-O-dimethylether (HDME). In the present results, HDME was highly effective in the inhibition of PDE4 activity with a IC50 value of 3.23 µM, but 7-HME almost had no effect on the PDE4 inhibition. The IC50 value of HDME is obviously smaller than that of hesperetin-5,7,3''-O- trimethylether (HTME) in the previouse report. Therefore, the anti-asthmatic effects of HDME may be better than those of HTME.
In the sensitized and OVA-secondarily challenged BALB/c mice, an asthmatic animal model, the airway hyperresponsiveness (AHR) was measured in unrestrained animals by barometric plethysmography using a whole-body plethysmograph after exposure of methacholine (MCh, 6.25~50 mg/ml) and enhanced pause (Penh) values were determined. In the present results, HDME (3~30 µmol/kg, p.o.) dose-dependently and significantly suppressed the
enhancement of MCh (25~50 mg/ml)-induced Penh values. In the bronchoaveolar lavage fluid (BALF) of mice, it also significantly reduced the increase of total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils and significantly reduced the release of helper T cell subtype 2 (Th2) cytokines, such as interleukin(IL)-4, IL-5, and tumor necrosis fator(TNF)-α. In contrast, HDME at 30 µmol/kg (p.o.) significantly increased the release of interferon(INF)-γ, a Th1
cytokine, although it at dose ranged from 3 to 30 µmol/kg (p.o.) suppressed the release of IL-2, another Th1 cytokine. HDME (3~30 µmol/kg, p.o.) also significantly (P<0.05) reduced total and OVA-specific IgE in serum and BALF, but dose-
dependently and significantly increased IgG2a in serum. In addition, HDME (30 µM) significantly relaxed the baseline tension, and in concentration ranged from 3 to 30 µM it also suppressed OVA (10~100 µg/ml)-induced contractions in isolated sensitized guinea pig trachealis.
According to the Lineweaver-Burk analysis, HDME competitively inhibited PDE1, PDE3, and PDE4 activities, with a Ki value of 46.1, 24.1, and 7.7 μM, respectively.
Although the latter two values did not significantly differ from each other, the last one was significantly lower than the first one. It suggests that HDME has a higher affinity for PDE4. HDME concentration-dependently inhibited activities of PDE1, PDE3, and PDE4 with an IC50 value of 22.1, 24.6, and 3.0 μM, respectively. It also
revealed to have the most potency in PDE4 inhibition, suggesting that its binding ability on low affinity rolipram binding sites (LARBS) of PDE4 is the greatest. HDME displaced [3H]-rolipram from high affinity rolipram binding sites (HARBS) of
particulates of whole brains isolated from sensitized guinea pigs, with an EC50 value of 106.6 μM. Therefore, the PDE4H/PDE4L ratio of HDME was 106.6/3 (aproximately 35.3), which is greater than that of HTME (18.2). It is possibly due to its concomitant inhibition on PDE3, and potentiates its antiasthmatic effects. To evaluate the antiasthmatic effects of selective PDE4 inhibitor, the PDE4H/PDE4L ratio is recognized as the best method today. However, it eventually is a result from in vitro studies. In the present results, we first time reported an in vivo evaluating method, dose (ADD25) for reducing 25% of duration of anesthesia induced by xylazine/ketamine is divided by dose (ID75) for inhibiting 75% of enhanced pause value. The ADD25/ID75 ratios of rolipram, Ro 20-1724, and luteolin are similar to PDE4H/PDE4L ratios of these three compounds, as shown in Table 2. In the present results, HDME (10~100 μmol/kg, s.c.) did not significantly shorten duration of anathesia induced by xylazine/ketamine, therefore HDME may have little adverse effects, such as nausea and vomiting, although ID75 value of HDME was not determined.
In conclusion, HDME competitively and selectively inhibits PDE4/PDE3 and have bronchodilatory and anti-inflammatory effects with little adverse effect. The PDE4H/PDE4L ratio of HDME was 35.5, in the present result, and is the greastest nowadays in the world, suggesting HDME may have the potential for treating asthma.
