- 學位論文
山楂果多酚成份及木犀草素抗增生及發炎作用之研究
Studies on the anti-inflammation and anti-proliferation of Crataegus polyphenol and luteolin
摘要
PART I 山楂果（Crataegi fructus）屬薔薇科（Rosaceae）植物，其成熟果實去籽乾燥後可供藥用，過去傳統醫學上主要用於活血化瘀、腹瀉、消化不良、驅蟲解毒等症狀。經現代藥理研究後發現，山楂果對於心血管系統有多方面的藥理作用，能擴張冠狀動脈增加血流量，改善心臟活力、興奮中樞神經系統，具有降血脂、降膽固醇、降血壓、抗心律不整等作用，但對於抗發炎作用與抗癌活性目前並不清楚。在先前研究中，我們已得知山楂果（Crataegus pinnatifida）含有多酚（polyphenols）如類黃酮類（flavonoids）等成份，且具抗氧化活性及抑制低密度 脂蛋白（LDL）之氧化，並利用試管實驗與巨噬細胞體外實驗（in vitro）中証實山楂果多酚萃取物有抑制黃嘌呤氧化酶（xanthine oxidase）之活性，另其可抑制內毒素所誘發一氧化氮（nitric oxide; NO）及前列腺素E2（prostaglandin; PGE2） 的產生，由於山楂果中類黃酮成份佔多酚成份中之比例最高，因此，在本實驗中，我們基於以上基礎進行山楂果類黃酮成份進一步之抗發炎研究，我們以老鼠巨噬細胞（RAW 264.7）為細胞模式，結果發現，當以100 ng/ml 的內毒素（lipopolysaccharide; LPS）處理RAW 264.7 細胞12 小時，可以刺激與發炎有關之PGE2 和Nitric oxide 大量產生，當在處理內毒素時同時處理山楂果類黃酮萃取 物（CF-Fs, 0.1-0.75 mg/ml），則可降低有意義的降低PGE2 和Nitric oxide 之產生。為了更進一步確定CF-Fs 的抗發炎作用，我們利用大白鼠為動物模式來觀察，結果顯示當預先給予連續五天50~200 mg/kg 劑量之CF-Fs，可有意義的降低因LPS（5 mg/kg）造成的丙胺酸轉胺酶（ALT）與天門冬胺酸轉胺酶（AST）之上升。 在組織病理學的檢查方面，亦可發現預先給予連續五天CF-Fs（50-200 mg/kg）可降低LPS 造成老鼠肝臟之嗜中性白血球浸潤及壞死之現象。另外在發炎有關之環氧化酶-2（COX-2）與誘導型一氧化氮合成酶（iNOS）蛋白質表現方面，CF-Fs（50-200 mg/kg）同樣發現可有意義的降低因LPS 誘導之COX-2 與iNOS蛋白質表現。因此，我們認為山楂果中之類黃酮成分具有抗發炎之效果。 PART II 山楂果（Crataegus fructus）去籽乾燥後除可供藥用外，現在在台灣的飲料亦常添加來增加風味並希望山楂果可發揮去油解膩功效。先前研究中，我們發現山楂果水萃取物含許多多酚化合物（polyphenols）如procyanidins、flavonoids、rutin、 gallic acid、hyperoside 等，且具有抗氧化及抗發炎之功效，因發炎與癌化一直有密切之關連，因此我們於本研究中收集山楂果多酚成份進行體外細胞抗轉形及活體抗癌促進作用之探討。在細胞實驗方面，我們先以anchorage-independent transformation assay 為模式，用12-O-tertradecanoylphorbol-13-acetate（TPA）處 理JB6 P+細胞（老鼠promotion-sensitive 之上皮細胞），使其轉形（transformation）得以在soft agar 中生長，在實驗組加入山楂果多酚成份（CF-TP, 0.1-0.5 mg/ml）觀察其影響，結果顯示，在同時處理CF-TP 時可有效降低JB6 P+細胞轉型，同時我們發現CF-TP 亦可抑制osteopontin （OPN；一種transformational marker） 之蛋白表現，而此抑制JB6 P+細胞轉型與OPN 蛋白表現之功能可能與CF-TP 能抑制轉錄因子nuclear factor-κB（NF-κB）與 activator protein-1（AP-1）有關。為進一步研究CF-TP 是否具有化學預防及抗腫瘤促進活性，我們以ICR 小鼠為動物實驗模式進行觀察。在短期實驗方面，我們利用TPA 分別塗抹於背部及耳 朵造成背部皮膚產生大量H2O2、myeloperoxidase（MPO）活性上升、上皮層增厚、白血球浸潤及耳朵水腫等現象，而處理CF-TP 則可有效抑制這些因處理TPA引起之活性氧系（reactive oxygen species, ROS）產生及發炎現象。另一方面由於TPA 會誘導老鼠皮膚炎指標酵素ornithine decarboxylase （ODC）之活性與COX-2 與iNOS 蛋白表現，我們亦發現預先處理CF-TP 可有效的抑制ODC 活性與這些蛋白質的表現。最後我們以two-stage carcinogenesis 模式分別以benzo[a]pyrene當起始劑，TPA 當促進劑塗抹於老鼠背部造成老鼠腫瘤之產生，當在每次塗藥之前先以CF-TP 處理時，結果顯示老鼠產生腫瘤之機率降低，腫瘤產生之數目也降低。因此我們認為山楂果多酚成分具有抗癌促進作用。 PART III 長鏈多不飽和脂肪酸除了形成磷脂質構成細胞膜組成分和提供能量來源外，亦是必須脂肪酸的來源。近年來研究顯示，飲食中之脂肪酸攝取過多會增加攝護腺癌、大腸癌與乳癌之危險性。必須脂肪酸中的花生四烯酸在體內是前列腺素（prostaglandin）、前列凝素（thromboxanes）與白三烯素（leukotrienes）的前趨物。 在前列腺中，花生四烯酸可被環氧化酶（cyclooxygenase）代謝成前列腺 素E2（PGE2）刺激攝護腺增生。之前研究中，花生四烯酸被發現可以促進人類攝護腺癌細胞增生，在本研究中，我們亦發現，花生四烯酸在亦可促進人類攝護腺肥大細胞（BPH-1）的增生，且這現象可能與花生四烯酸活化攝護腺肥大細胞內的ERK 與Akt 訊息路徑有關。另外，實驗也發現攝護腺癌負相關的診斷指標GSTP1 蛋白在花生四烯酸長時間處理後表現量會降低。接著我們使用廣存於蔬 果中之類黃酮化合物作篩選，發現其中木犀草素（luteolin）對於BPH-1 細胞之增生具有最強抑制作用，包括抑制因花生四烯酸誘導攝護腺肥大細胞ERK 與Akt的磷酸化，另外花生四烯酸長時間處理BPH-1 細胞造成之GST-P1 蛋白之減少也在經木犀草素處理後恢復。因此，我們認為多元不飽和脂肪酸如花生四烯酸可能對於攝護腺肥大有某種程度之促進作用，而存在蔬果中之天然抗氧化劑如類黃酮 化合物可能對於此種增生作用扮演抑制作用。
並列摘要
PART I The dried fruits of Crataegus pinnatifida, a local soft drink material and medical herb, demonstrated antioxidant effect in our previous study. In the present study, we investigate the anti-inflammatory potential of flavonoid contents from dried fruit of Crataegus pinnatifida (CF-Fs). The preliminary investigation showed that CF-Fs (0.10 - 0.75 mg/ml) decreased the release of PGE2 and nitric oxide as induced by lipopolysaccharide (LPS, an endotoxin) in macrophage RAW 264.7 cells. The in vivo assay showed that pretreatment of rats with CF-Fs (50 mg/kg-200 mg/kg dosed by gavage) for 5 days significantly decreased the serum levels of the hepatic enzyme markers alanine- and aspartate aminotransferase (ALT and AST) induced by the 6-hour treatment with LPS (ip; 5 mg/kg). Histopathological evaluation of the rat livers revealed that CF-Fs reduced the incidence of liver lesions such as neutrophil infiltration and necrosis induced by LPS. Furthermore, we found that pretreatment with CF-Fs decreased the hepatic expression of iNOS and COX-2 induced by LPS in rats. These results demonstrate that CF-Fs present anti-inflammatory potential in vitro and in vivo, and that they may play a role in hepatoprotection. PART II The dried fruits of Crataegus pinnatifida have been used traditionally as oriental medicine and local soft drink material recently. Previously, we demonstrated that C. pinnatifida exhibited anti-oxidation and anti-inflammatory potential. To clarify the active components in anti-transformation and anti-tumor promotion, we collected the polyphenol fraction (CF-TP) of hot-water extracts from dried fruits of C. pinnatifida for the following study. By anchorage-independent transformation assay, CF-TP significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation in JB6 P+ cells. Moreover, we found that CF-TP inhibited the expression of osteopontin (OPN), a transformational marker, and the activation of NF-κB and AP-1 induced by TPA in JB6 P+ cells. In addition, we evaluated the effect of CF-TP on TPA application to ICR mouse skin with measurement of H2O2 production, myeloperoxidase (MPO) activity, edema formation, epidermal thickness and leukocyte infiltration. As a result, CF-TP significantly inhibited the generation of reactive oxygen species (ROS) and the phenomena of inflammation induced by TPA. It also suppressed the expression of COX-2 and iNOS, and the activation of ornithine decarboxylase (ODC). Furthermore, CF-TP inhibited benzo[a]pyrene (B[a]P)/ TPA-induced skin tumor formation and decreased the incidence of tumor. These results indicate that CF-TP possesses potential as a cancer chemopreventive agent against tumor promotion. PART III Dietary fatty acid intake is associated with the risk of development and progression of prostate, colon and breast cancer. Arachidonic acid (AA), an essential polyunsaturated fatty acid, is the major precursor of biological active eicosanoids, which include prostaglandin E2 (PGE2), thromboxanes and leukotrienes. In human prostate, AA was catalyzed to PGE2, the only significant eicosanoid product, by cyclooxygenase (COX) and has demonstrated that PGE2 stimulates prostate cell proliferation and progress to cancer. In previous study, arachidonic acid, an ω-6 fatty acid possess the potential to stimulate the proliferation of human prostate cancer cells. In this study, we demonstrated that arachidonic acid also induced cell proliferation of human benign prostate hyperplasia cells (BPH-1), and this phenomena may involved phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. Moreover the expression of GSTP1, the biomarker in diagnosis of prostate cancer also decreased in arachidonic acid treated BPH-1 cells. The effect of some flavonoids, which are components of fruits, vegetables, and peas, on the cell proliferation of human benign prostate hyperplasia cells (BPH-1) was also investigated in this study. Luteolin inhibited the BPH-1 cell growth and phosphorylation of ERK and Akt. In addition, luteolin induced GST-P1 protein expression. Together, these results suggest that arachidonic acid induced BPH-1 cell proliferation and the ability of anti-proliferation of flavonoids may via inhibited the ERK and Akt signal pathway and Bcl-2 protein expression. The flavonoids could be an improved strategy for prevention and/or treatment of benign prostatic hyperplasia.