抗發炎中藥材之研究 安石榴對誘導型一氧化氮合成? 抑制之成份研究

Anti-inflammation Activity of Chinese Medicines Inducible Nitric Oxide Synthase Inhibition of Punica granatum Linne

李佳蓉(Chia-Jung Lee)

指導教授 ： 王靜瓊

臺北醫學大學/藥學院/生藥學研究所/碩士(2005年)

摘要

本研究利用革蘭氏陰性菌細胞壁的脂多糖(Lipopolysaccharide , LPS)誘導老鼠巨噬細胞RAW 264.7釋放一氧化氮(NO)，並以Griess reaction 分析方法來偵測其含量變化。經由篩選26種70% 丙酮萃取之中藥材，結果顯示：以蛇床子、花椒、安石榴皮、萊菔子、敗醬等具有明顯抑制NO產生之作用，IC50 分別為13.57、75.37、78.12、95.38及99.56μg/ml。進而檢測其化學組成，顯示安石榴皮富含多酚類，並利用SNP(Sodium Nitroprusside)作為NO供應者，檢測樣品是否具有直接清除NO之作用。結果發現這五種中藥皆無法直接清除NO。進而以Carrageenan誘導小鼠足掌發炎之動物實驗，檢測安石榴萃取物之抗發炎作用，結果顯示：餵服100 mg/kg之實驗組，於誘導後第2、4、5、6小時皆為有統計意義上的抑制足腫發炎反應。因此利用NO抑制的活性追蹤法，分離安石榴之抗發炎活性成分。經由Diaion HP-20、TSK HW-40 (C)、ODS等不同分離管柱，純化得到4個主要抗發炎成分活性成分，分別為Punicalagin、 Punicalin、Strictinin A、 Granatin B。再以體內、外抗發炎試驗評估其效用，顯示： Punicalagin、 Punicalin、Strictinin A、Granatin B皆會抑制NO產生，其IC50 分別為69.8、78.6、63.1以及33.6 μM。此外，也發現這4種主要的抗發炎活性成分具有抑制 iNOS的表現，但對iNOS活性並無明顯影響。其中以Granatin B 效果最好，且於Carrageenan誘導的小鼠足掌發炎反應，劑量為10 mg/kg時亦可有統計意義上的抑制。進一步分析其血清，此成分也能降低血清中之PGE2產生。因此推測，安石榴的主要抗發炎活性成分可能是Granatin B，此結果可做為未來品質管制安石榴皮及開發抗發炎藥之參考。 目前紅石榴常被當作水果食用，我們利用HPLC指紋圖譜及抗發炎抗氧化活性分析，結果顯示藥用安石榴與食用紅石榴具有不同的指紋圖譜與活性，因此鑑定其與藥用的安石榴為不同品系的石榴。

關鍵字

安石榴 ； 抗發炎

並列摘要

In this study, 26 kinds of 70% acetone extract of traditional medicines were screened Nitric oxide (NO) production which induced by LPS-stimulated RAW 264.7 macrophage cell line and quantitatively detected the amount of NO production by Griss reaction. Results illustrated that Punnica granatum, Raphni semen, Patrinia villosa, Fructus Cnidii and Zanthoxylum simulans showed more potential than the other herbs. And IC50 of each extracts were 78.12、95.38、99.56、13.57、75.37 μg/ml, respectively. Furthermore, we analyzed the chemical components of these herb extracts and found that Punnica granatum was rich in polyphenol. We used Sodium Nitroprusside (SNP) as a NO donor to detect the direct NO-clearance ability and we discovered that these 5 herb extracts had no NO-clearance function. Punnica granatum is a traditional Chinese herb and widely used in clearing heat and eliminating parasites. Therefore, the anti-inflammatory effect of the Punnica granatum extract was evaluated by Carrageenan – induced paw edema in mice. The results showed the Punnica granatum extract could significantly decreased the paw edema after treatment at 100 mg/kg for 3 hr. Furthermore, we used column chromatography combined with the decreased NO bioassay-guided fractionation. The 20% and 40% MeOH fractions eluted by Diaion HP-20 gel had stronger potency in inhibition NO production than the other fractions. Finally, four polyphenols including punicalagin, punicalin, strictinin A, and granatin B were isolated from the 20% and 40% MeOH eluted fractions. Moreover, the NO inhibition IC50 of each compounds were 69.8、78.6、63.1 and 33.6 μM . The NO inhibitory effect of the granatin B was stronger than the others. These four compounds both had iNOS inhibition ability. Pomegranates have several specises. “Red” pomegranate is wildly used as a jucie fruit. However, the constituents of “red” pomegranate are different from Punnica granatum by HPLC. Then Punnica granatum had the different active with the red pomegranate. Moreover, the anti-inflammation effects of Punnica granatum were better than red pomegranate. In summary, two pomegranates of the same name were different specises.

並列關鍵字

Punnica granatum ； Anti-inflammation

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