台灣產黃藤之化學與生物活性研究

黃藤?(Calamus quiquesetinervius) 為分佈於台灣山區之棕櫚科省藤屬有刺藤本植物，民間常用於高血壓與肝炎等疾病的治療。本研究已發現黃藤與果實部之乙醇萃取物具有清除自由基、抗發炎作用、抗血小板凝集及心臟與週邊血管系統舒張作用。針對黃藤活性成分的持續研究，利用化學性層析分離技術，首次由莖部之乙酸乙酯活性層及正己烷層中分離純化並鑑定出二十九個新穎之酚類衍生物，包括十五個tricin-type flavonolignans (1?{15)，六個8-O-4′ neolignans (16?{21)，兩個dihydrobenzofuran neolignans (22?{23) 及六個phenylpropanoid glycosides (24?{29)，分別命名為calquiquelignans A?{O (1–23) 與calquiquesides A?{F (24?{29)；另亦分離出十八個已知化合物 (30-48)。這些新穎木質素類化合物主要利用一維 (1H- 及 13C-) 與二維核磁共振技術 (COSY、HMQC、HMBC 及 NOESY)，並輔以其他光譜數據進行結構解析。利用逆向高壓液相層析系統，建立乙酸乙酯層 (KCQSE) 之化學性指紋圖譜，並比對出十三個化合物 (1-3、6、11、13 及 27、31-33、35-37) 的主要吸收訊號。生物活性試驗結果顯示，於離體試驗中，比較黃藤醇溶萃取物之其他管柱分離層，KCQSE具有最佳的抑制，由 AA或 PAF 所誘發之抗血小板凝集作用、大白鼠胸主動脈血管舒張作用、清除氧屬自由基與抑制LPS 刺激 RAW 264.7 巨噬細胞生成 NO 發炎反應。此外，以 TPA 誘發小鼠耳朵發炎反應試驗，證實 KCQSE 及其管柱分離層SE2 具有抑制急性發炎期TNF-α、IL-1及IL-6的表現；在慢性發炎的實驗結果顯示，SE2 除可調控pro-inflammatory cytokine的活性表現外，亦可藉由抑制 adhesion molecules 與 monocyte chemoattractant protein 之作用以及調節 arachidonic acid 異常代謝反應來降低慢性發炎症狀。在安全性評估方面，經由單一口服急毒性試驗結果顯示KCQSE 對 ICR 雄鼠與雌鼠之口服急毒性LD50 值均大於5g/kg體重。相較於 aspirin、calquiquelignan J、salcolins A-B 顯示對於 AA 與 PAF 引發之血小板凝集具有顯著地抑制作用 (p?? 0.05)；Calquiquelignan R 對於 collagen 所誘發血小板凝集活性則為 aspirin 的三倍。Calquiquelignan A、dihydrotricin 及 (2S)-naringenin 在反應濃度為 100 ?嵱時能使以 phenylephrine 誘發收縮的離體大白鼠胸主動脈血管環產生 60.3%、80.3% 及 60.9% 的舒張作用。Calquiquelignans A-B、dihydrotricin、tricin、calquiquelignan Q 與 calquiquesides A-C 及 E-F (IC50 ?? 10 ?嵱) 則具有較 Trolox?? 為佳的清除氫氧自由基 (OH?{) 能力。在抗發炎活性的表現上， calquiquelignan R 和 calquiqueside E-F 具有顯著抑制 LPS 刺激 RAW 264.7 巨噬細胞生成 NO 之能力，其抑制活性為 quercetin 的三至四倍。

關鍵字

黃藤

並列摘要

Calamus quiquesetinervius Burret, the most abundant species of Calamus (Arecaceae) endemic to Taiwan, is a clustered prickly rattan palm that grows at low and medium elevations. It has also been used as a treatment for several diseases, such as hypertension, stroke and hepatitis in folk herbal medicine. As part of our discovery of bioactive constituents from the titled plant, twenty-nine rivatives, including fifteen tricin-type flavonolignans (1–15), six 8-O-4′ neolignans (16–21), two dihydrobenzofuran neolignans (22, 23), and six phenylpropanoid glycosides (24–29), and eighteen known phenolic compounds (30–48), were isolated from the ethanol extract of the stems C. quiquesetinervius. These new lignoids are named as calquiquelignans A–O (1–15) and calquiquesides A–F (24–29). Structural elucidation of the new isolates was accomplished on the basis of their spectroscopic data, including 1D and 2D NMR techniques (COSY, HMQC, HMBC and NOESY) and mass spectrometry. Biological evaluations of the fractionated layers resulted from the ethanol extracts of the title plant were also carried out in vitro. The EtOAc fraction (KCQSE) showed the most potency for anti-platelet aggregation, vasodilatory activities, free-radical scavenging activities, and anti-inflammatory activities employing the inhibition of nitric oxide (NO) production. Furthermore, KCQSE and its subfraction, SE2, possessed strong inhibitory effects on TNF-α, IL-1 and IL-6 in the acute inflammatory stage on 12-O-tetradecanoylphorbol-13-acetate (TPA)- induced mouse ear inflammatory model. In the chronic inflammation, the chronic inflammatory symptom can reduce by SE2 via the regulation of up-expression of pro-inflammatory cytokine, the inhibition of adhesion molecules and monocyte chemoattractant protein, and the modulation of arachidonic acid metabolism. A single dose 5 g/kg body weight of KCQSE did not show any adverse effect on clinical sign for two weeks, implying that KCQSE extracts do not cause any acute toxicity. This result indicated the LD50 value of KCQSE could be >5 g/kg body weight/day. Calquiquelignan J (10), salcolin A, and (3, 34) showed potent platelet aggregation inhibition induced by AA and PAF compared with aspirin; calquiquelignan R (18) had three folds collagen-antagonistic activity compared with aspirin, but was inactive against thrombin-induced platelet aggregation. Calquiquelignan A (1), dihydrotricin (31), and (2S)-naringenin (36) exhibited significant vasodilatory potencies at 100 ?嵱, as indicated by 60.3%, 80.3% and 60.9% relaxations, respectively. Calquiquelignan A, B (1, 2), calquiquelignan Q (17), calquiqueside A–C (24–26), E, F (28, 29), dihydrotricin (31), and tricin (32) also exhibited more potent hydroxyl radical (.OH) scavenging activities (IC50 ??10 ?嵱) than Trolox?? as characterized by the ultraweak chemiluminescence assay. The anti-inflammatory potencies of calquiquelignan R (18) and calquiqueside E, F (28, 29) were three to four folds higher compared with quercetin employing the inhibition of nitric oxide (NO) production. Biological evaluations of the fractionated layers resulted from the ethanol extracts of the title plant were also carried out in vitro. The EtOAc fraction (KCQSE) showed the most potency for anti-platelet aggregation,vasodilatory activities, free-radical scavenging activities, and anti-inflammatory activities employing the inhibition of nitric oxide (NO) production. Furthermore, KCQSE and its subfraction, SE2, possessed strong inhibitory effects on TNF-α, IL-1 and IL-6 in the acute inflammatory stage on 12-O-tetradecanoylphorbol-13-acetate (TPA)- induced mouse ear inflammatory model. In the chronic inflammation, the chronic inflammatory symptom can reduce by SE2 via the regulation of up-expression of pro-inflammatory cytokine, the inhibition of adhesion molecules and monocyte chemoattractant protein, and the modulation of arachidonic acid metabolism. A single dose 5 g/kg body weight of KCQSE did not show any adverse effect on clinical sign for two weeks, implying that KCQSE extracts do not cause any acute toxicity. This result indicated the LD50 value of KCQSE could be >5 g/kg body weight/day. Calquiquelignan J (10), salcolin A, and (3, 34) showed potent platelet aggregation inhibition induced by AA and PAF compared with aspirin; calquiquelignan R (18) had three folds collagen-antagonistic activity compared with aspirin, but was inactive against thrombin-induced platelet aggregation. Calquiquelignan A (1), dihydrotricin (31), and (2S)-naringenin (36) exhibited significant vasodilatory potencies at 100 ?嵱, as indicated by 60.3%, 80.3% and 60.9% relaxations, respectively. Calquiquelignan A, B (1,2), calquiquelignan Q (17), calquiqueside A–C (24–26), E, F (28, 29), dihydrotricin (31), and tricin (32) also exhibited more potent hydroxyl radical (.OH) scavenging activities (IC50 ??10 ?嵱) than Trolox?? as characterized by the ultraweak chemiluminescence assay. The anti-inflammatory potencies of calquiquelignan R (18) and calquiqueside E, F (28, 29) were three to four folds higher compared with quercetin employing the inhibition of nitric oxide (NO) production.