Effect of Mesona procumbens Hemsl. extracts on improvement of hyperuricemia and nephropathy

Uric acid is metabolized from purine by the xanthine oxidase (XO) and excreted by urine. High level of serum uric acid leads to hyperuricemia and its complications such as gout, hypertension and chronic renal disease. Hsian-tsao (Mesona) is an annual herbaceous plant belonging to the Labiatae family, which has been reported to possess many beneficial properties, such as, anti-hypertension, hypoglycemic effect, hepatoprotective effect and improvement of diabetic myocardium impairment. However, the inhibitory effects of Hsian-tsao on hyperuricemia and kidney inflammation remain unclear. Therefore, we investigated the potential effect of Hsian-tsao on hyperuricemia and kidney inflammation in vitro and in vivo. We analyzed the active compounds and investigated the effects of Taiwan, Vietnam and China Hsian-tsao extracts after being extracted by water and various concentrations of ethanol (25, 50, 75 and 95%), respectively, on uric acid production and kidney inflammation, the results showed that China Hsian-tsao extracts contained the highest levels of phenolics and flavonoids. The results from HPLC analysis showed that p-hydroxybenzoic acid, caffeic acid and protocatechuic acid (PCA) were detected in Taiwan Hsian-tsao extracts, and the caffeic acid, PCA and hesperidin were detected in Vietnam Hsian-tsao extracts. However, China Hsian-tsao extracts contained caffeic acid only. In addition, both oleanolic acid and ursolic acid, the triterpenoids, presented in Hsian-tsao extracts from Taiwan, Vietnam and China. The in vitro result showed that Taiwan Hsian-tsao extracts exerted the greatest inhibitory effects on uric acid production. We further evaluated the inhibition of Taiwan Hsian-tsao extract on XO activity in THP-1 monocytes. The results showed that Taiwan Hsian-tsao extracts significantly inhibited the XO activity in monosodium urate (MSU)-induced THP-1 monocytes, and the 50% ethanol extracts (EE50) of Taiwan Hsian-tsao exhibited the most potent inhibitory activity. Because chronic kidney inflammation is one of the complications of hyperuricemia, we investigated the alleviative effects of Taiwan Hsian-tsao extracts on kidney inflammation induced by MSU in human embryonic kidney HEK293 cells. The results revealed that Taiwan Hsian-tsao extracts significantly reduced the secretion of pro-inflammatory cytokine interleukin-8. The water extract (WE) of Taiwan Hsian-tsao decreased the superoxide anion generation while EE50 decreased the reactive oxygen species (ROS) generation in MSU-induced HEK 293 cells. Taiwan Hsian-tsao extracts could inhibit uric acid production, reduce XO activity and improve kidney inflammation under MSU condition. These findings indicate that Taiwan Hsian-tsao extracts possess highly potential effects on attenuation of hyperuricemia and the kidney inflammation. We further investigated the effects of Taiwan Hsian-tsao extracts on hyperuricemia and kidney inflammation in vivo by streptozotocin (STZ)-induced SD-rats. The animals after induction by STZ were treated by oral gavage 10 mg/kg B.W. allopurinol or various concentrations of Taiwan EE50 (0.25, 0.5 and 1 g/kg) for seven weeks. The results indicated that all dosages of Taiwan EE50 showed no effect on the typical syndrome of type-1 diabetes such as body weight, food intake, water intake, fasting glucose and glucose tolerance in STZ-induced rat. In addition, Taiwan EE50 at 1 g/kg reduced the levels of BUN, kidney pro-inflammatory cytokines IL-1β and TNF-α in STZ-induced rat. In conclusion, Taiwan Hsian-tsao 50% ethanol extracts are enriched in phenolics and triterpenoids compound. Also, EE50 might have the potential effect to inhibit uric acid production, to reduce XO activity and to improve hyperuricemia and kidney inflammation induced by STZ.嘌呤類物質透過黃嘌呤氧化酶 (xanthine oxidase, XO) 代謝為尿酸 (uric acid) 並隨尿液排出體外。當體內尿酸濃度持續過高，會發展為高尿酸血症 (hyperuricemia) 及併發如痛風、高血壓及慢性腎損傷等疾病。仙草 (Mesona) 為唇形科仙草屬一年生之食用草本植物，已被證實富含多種生理活性，如降血壓、降血糖、護肝及改善糖尿病所誘發之心肌損傷，但仙草是否對改善高尿酸血症及減緩腎臟發炎反應之功效尚未釐清。因此，本研究以體外及活體試驗為平台，探討仙草改善高尿酸血症及減緩腎臟發炎反應之潛力。 使用水及不同濃度乙醇 (25、50、75 及 95%) 分別對不同地區仙草 (台灣、越南及大陸) 進行萃取，並分析各萃取物中之活性成分、體外抑制尿酸生成及減緩腎臟發炎反應之功效。活性成分分析結果顯示，大陸仙草萃取物含有最高之總酚及類黃酮含量。經 HPLC 分析結果顯示，台灣仙草萃取物含有 p-hydroxybenzoic acid、caffeic acid 及 protocatechuic acid (PCA)；越南仙草萃取物含有 caffeic acid、 PCA 及 hesperidin；而大陸仙草萃取物僅分析出 caffeic acid。此外，各地區仙草萃取物皆含有三萜類化合物齊墩果酸 (oleanolic acid, OA) 及熊果酸 (ursolic acid, UA)。於抑制尿酸生成試管試驗結果發現，台灣仙草萃取物具有最佳的抑制尿酸生成效果。以細胞模式評估台灣各仙草萃取物抑制 XO 活性能力，結果顯示，台灣仙草各萃取物可顯著抑制尿酸鹽 (monosodium urate, MSU) 誘導 THP-1 胞內 XO 活性上升，其中又以 50% 乙醇萃取物 (50% ethanol extract, EE50) 之抑制效果最佳。 慢性腎臟發炎為高尿酸血症眾多併發症之一，因此以 MSU 誘導 HEK 293 腎胚細胞作為實驗平台，評估台灣仙草各萃取物減緩腎臟發炎反應之功效。實驗結果顯示，台灣仙草各萃取物皆顯著減少促發炎激素介白素-8 (interleukin-8, IL-8) 之分泌量。台灣仙草水萃物 (water extract, WE) 亦可顯著減少 MSU 誘導 HEK 293 腎臟細胞中胞內超氧陰離子含量， 而其 EE50 則可顯著減少胞內 reactive oxygen species (ROS) 之含量。因此，台灣仙草萃取物具有良好抑制尿酸生成、 MSU 誘導之 XO 活性上升與改善腎臟發炎反應，說明台灣仙草萃取物具有改善高尿酸血症及 MSU 誘導的腎臟發炎反應之潛力。 進一步利用動物模式探討台灣 EE50 於活體中改善高尿酸血症及腎臟發炎反應之功效。實驗使用鏈佐黴素 (streptozotocin, STZ) 誘導 Sprague-Dawley 雄性大鼠產生第一型糖尿病、高尿酸血症及腎臟發炎反應，並於誘導一週後分別給予 10 mg/kg B.W. 異嘌呤醇 (allopurinol, AP) 或不同劑量台灣 EE50 (0.25、0.5 及 1 g/kg B.W.)。結果顯示，餵食各劑量台灣 EE50 並無改善 STZ 誘導之大鼠體重降低、攝食飲水量上升、空腹血糖值升高及葡萄糖耐受性下降等糖尿病典型症狀之功效。但餵食中劑量及高劑量台灣 EE50 後，可顯著降低 STZ 誘導之血清尿酸含量及肝臟 XO 活性。此外，餵食高劑量 EE50 亦可顯著降低 STZ 誘導之血中尿素氮、腎臟促發炎激素 IL-1β 及 tumor necrosis factor-alpha (TNF-α) 分泌量上升之效果。 本研究結果證實，台灣仙草 50% 乙醇萃取物富含酚類及三萜類物質，且具有抑制尿酸生成、減緩 XO 活性、改善 STZ 誘導之高尿酸血症及腎臟發炎反應之功效與潛力。目次 全文摘要...............................................i Abstract.............................................iii 目次..................................................vi 圖次..................................................ix 表次..................................................xi 縮寫表................................................xii 前言..................................................1 第一章、文獻整理.......................................3 壹、尿酸 (Uric acid)..................................4 一、尿酸簡介...........................................4 二、尿酸之生合成.......................................4 三、尿酸之排泄及重吸收..................................6 貳、高尿酸血症(Hyperuricemia).........................13 一、高尿酸血症簡介 ....................................13 二、高尿酸血症病因 ...................................13 三、高尿酸血症臨床治療.................................15 四、高尿酸血症併發症...................................16 ?、高尿酸血症動物模式.................................23 一、鏈佐黴素 (streptozotocin, STZ) 誘導大鼠第一型糖尿病之機制 ....................................................23 二、鏈佐黴素與高尿酸血症及腎損傷........................24 肆、仙草.............................................25 一、仙草簡介..........................................25 二、中醫食療古代文獻...................................25 三、一般營養成分及機能性成分............................25 四、仙草之特殊生理功效.................................26 伍、研究目的..........................................27 陸、研究架構..........................................28 第二章、仙草萃取物活性成分分析及抑制尿酸生成與發炎反應之功效評估 .....................................................29 摘要.................................................30 Abstract.............................................32 前言.................................................34 材料與方法............................................36 結果.................................................43 一、仙草之萃取........................................43 二、仙草萃取物之活性成分...............................43 三、HPLC 分析台灣、越南及大陸仙草各萃取物中酚酸類化合物含量 ....................................................44 四、HPLC 分析台灣、越南及大陸仙草各萃取物中 OA 及 UA 含量 ....................................................45 五、建立 MSU 誘導 THP-1 細胞 XO 活性實驗模式...........46 六、台灣、越南及大陸仙草萃取物抑制尿酸生成之功效評估......46 七、台灣仙草萃取物抑制 MSU 誘導 THP-1 細胞 XO 活性試驗 ....................................................47 八、台灣仙草各萃取物之細胞毒性及提升 MSU 誘導 HEK 293 細胞存活率下降...............................................47 九、台灣仙草萃取物降低 MSU 誘導 HEK 293 促發炎激素分泌量 ....................................................47 十、台灣 WE 及 EE50 降低 MSU 誘導 HEK 293 氧化壓力.....48 討論.................................................49 第三章、仙草 50 % 乙醇萃取物對以 STZ 誘導大鼠糖尿病及高尿酸血症之改善功效............................................72 摘要.................................................73 Abstract.............................................74 前言.................................................75 材料與方法............................................77 結果.................................................82 一、EE50 對 STZ 誘導 SD 大鼠空腹血糖值、葡萄糖耐受性及胰臟組織病理變化之影響........................................82 二、EE50 對 STZ 誘導 SD 大鼠體重、攝食及飲水量變化之影響 ....................................................83 三、STZ 誘導 SD 大鼠之死亡率...........................83 四、EE50 對 STZ 誘導 SD 大鼠之肝臟及腎臟相對臟器重量之影響 .....................................................84 五、EE50 對 STZ 誘導 SD 大鼠血清尿酸及肝臟XO 活性之影響 .....................................................84 六、EE50 對 STZ 誘導 SD 大鼠血脂生化參數之影響...........85 七、EE50 對 STZ誘導 SD 大鼠腎臟組織病理變化之影響........85 八、EE50 對 STZ 誘導 SD 大鼠血中尿素氮 (blood urea nitrogen, BUN) 及肌酸酐 (creatinine) 生化參數之影響...............85 九、EE50 對 STZ誘導 SD 大鼠腎臟 IL-1β 及 TNF-α 之影響 ......................................................86 討論 .................................................87 總結論 .............................................105 參考文獻..............................................107 附錄..................................................12