郭宗甫臺灣大學：獸醫學研究所莊雅玲Chuang, Ya-LingYa-LingChuang2007-11-282018-07-092007-11-282018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/59974血中尿酸值會藉由尿酸的過度製造或排泄過低而增高，並造成高尿酸血症（hyperuricemia）。高尿酸血症的發生，可能引起尿酸鹽沈積於關節而引發疼痛性炎症反應而造成痛風，且已有許多報告指出其和動脈硬化、高血壓、心血管疾病、糖尿病及腎臟疾病相關。黃嘌呤酶會將次黃嘌呤及黃嘌呤代謝成尿酸，故其為引發高尿酸血症之關鍵酵素。活性氧屬除已知會造成脂質過氧化而導致食物變性外，也有許多文獻指出其與慢性心衰竭、老化及呼吸性窘迫症候群有關。而由於藥品使用的副作用及保健食品的盛行，使得自天然物中找尋無副作用或副作用較低的有效成分之研究受到重視，其中又以黃嘌呤酶抑制劑及抗氧化劑等為目前研究重心。而雖然大部分的天然物是安全的，但長期性且大量的服用是否會影響肝腎功能，或懷孕動物高劑量的服用是否會影響胚胎發育或導致胎兒畸形，仍須實驗進行探究。因此本實驗選用傳統中醫藥用於降低血中尿酸值之植物白花水竹草，進行其萃取物之生物活性試驗及毒性試驗。 新鮮白花水竹草降低血中尿酸值試驗是使用尿酸酶抑制劑oxonate，以腹腔注射方式製成高尿酸血症模式大鼠，在分別口服白花水竹草之葉粗萃液、莖粗萃液或磷酸緩衝液後，每隔90分鐘採取血液進行血液學及血液生化學檢測，並同時監測血壓變化。萃取物降低血中尿酸值試驗是使用高尿酸血症模式大鼠，分別口服由白花水竹草乾燥葉片以正己烷、乙醯乙脂、丁醇及蒸餾水所萃取出之HE、EAE、BE及WE產物，並每隔30分鐘採血進行尿酸值檢測。促尿酸排泄試驗以高尿酸血症模式大鼠，分別口服磷酸緩衝液或WE產物，並於口服後之30、90、150及210分鐘採取尿液，進行尿酸值檢測。DPPH自由基清除效力及抗氧化活性試驗是以不同濃度的四種分劃層萃取物與試劑反應後，分別於517 nm及500 nm進行吸光值檢測。另外分別於懷孕第1~4天或8~11天口服50倍降低血中尿酸值之有效劑量的白花水竹草乾葉（625 mg/kg），以瞭解其是否影響胎兒著床及致畸形。亞急性毒性試驗則連續28天使大鼠口服10倍降低血中尿酸值之有效劑量的白花水竹草乾葉（125 mg/kg），並以血液學、血液生化學及組織病理學評估毒性。 研究結果顯示新鮮白花水竹草葉片可降低血中尿酸值，其四種分劃層萃取產物也具同樣效果，且最後一個評估時間點以WE之降低血中尿酸值能力最佳。四種分劃層萃取產物也可抑制黃嘌呤酶，且同樣以WE之抑制力最強。促尿酸排泄試驗結果顯示WE無促進尿酸排泄效力。DPPH自由基清除試驗及抗氧化活性試驗，則以BW之抗氧化力最強。毒性試驗方面，結果顯示懷孕期間口服50倍降低血中尿酸值之白花水竹草乾葉不會造成懷孕母鼠之胚胎著床、致畸形性及耗損等影響；亞急性毒性試驗之血液學檢測結果顯示Hb、HCT及RBC值顯著上升（p＜0.05），血液生化學檢測結果顯示ALKP及BUN值顯著下降（p＜0.05）。組織病理學檢查結果顯示肝細胞腫脹及輕微膽管增生，其餘肺臟、脾臟及腎臟則無顯著變化。 綜合本研究之實驗結果，可以得到以下結論：白花水竹草可藉由抑制黃嘌呤酶而降低血中尿酸值，且可清除自由基及預防脂質過氧化而達抗氧化之功能。雖懷孕期間口服五十倍降低血中尿酸值劑量之白花水竹草並不會影響懷孕母鼠之著床，也不會造成胎仔出現畸形或耗損之情形，但28天且高量的口服白花水竹草會造成肝細胞腫脹及輕微膽管增生情形，表示白花水竹草也許不適合長期且大量的服用。Serum uric acid was be elevated because of urate overproduction and/or underexcretion, and it will cause hyperuricemia. Hyperuricemia may cause the deposition of uric acid in joints and lead to painful inflammation. There were also many researches indicated that hyperuricemia was related with atherosclerosis, hypertension, cardiovascular diseases, diabetes mellitus and renal disease. Xanthine oxidase (XO) will catalyze the hypoxanthine and xanthine to uric acid, which is the main enzyme in hyperuricemia. The current agent used to treat the hyperuricemia is XO inhibitor, but the allopurinol used in clinical will cause side effect and prompt the development of XO inhibitor. Reactive oxygen species (ROS) had been known to cause lipid peroxidation which induce nutritional degradation of food products. ROS were also related to chronic cardiac failure, ageing, acute respiratory distress syndrome. Because of the side effect of the medicine and the fashion of healthy food, many scholars start to pay attention to the research of non side effect or low side effect compound from traditional medicine. Among these researches, most scholars are focusing on XO inhibitor agents and antioxidant. Although the natural antioxidant is safe in general, daily usage may cause potential complication, for example it may affect the function of liver and kidney or it may also impact on fertilized egg implantation and fetal organogenesis when the rats were oral administration with high dosage during the period of pregnancy. Therefore this study was investigated to the bioactivity and toxicity of extractive from the leaves of Tradescantia albiflora Kunth, which is a Chinese medicine plant to reduce serum uric acid level on rat. The experiment was designed to use oxonate to be injected intraperitonelly to induce rat in hyperuricemic model, then these rats were oral administration crude extractive from the leaves, stem of T. albiflora or PBS respectively. Blood samples were collected to process hematology and biochemistry study every 90 mins, and monitored the change of blood pressure in the same time. We have used hexane, ethyl acetate, butanol and distilled water to extract the dry leaves of T. albiflora then got hexane extractive (HE), ethyl acetate extractive (EAE), butanol extractive (BE) and water extractive (WE) respectively. The experiment of decrease the plasma uric acid (PUA) level with extractives used by rat in hyperuricemic model to oral administration with HE, EAE, BE and WE extractive respectively, and bloods samples were collected to process PUA test every 30 mins. The experiments of eliminated uric acid were utilized by rat in hyperuricemic model to oral administration with the PBS or WE respectively, and urine samples were collected to process urine uric acid (UUA) test after 30、90、150 and 210 mins. These tests of the efficiency for scavenge of DPPH and the ferric thiocyanate (FTC) have utilized different concentration of extractives belonging to 4 layers react with agents, and tested the absorption with 517 and 500 nm respectively. Crude extractives were divided into three groups. One was the control group (oral phosphate buffer). Second group was tested for oral consumption of 50 times the effective dosage of reducing plasma uric acid level (625 mg/kg) for 1~4 days during pregnancy. Third group was tested during 8~11 days of pregnancy. Subacute toxicities were tested the consumption of the 10 times effective dosage of reducing plasma uric acid level (125 mg/kg) for 28 days and blood samples were collected to be conducted hematology, biochemistry and histology study. The results of these studies indicated that T. albiflora can decrease the level of blood uric acid, these 4 different types of extractive had the same ability, but the WE had the best affect in last estimative point (240 min). These 4 different types of extractive can also inhibit the XO, and WE had the best efficacy. The result of eliminated uric acid test revealed that WE hadn’t affect to eliminate uric acid. The BE had better efficacy in the scavenge of DPPH testing and the FTC testing. The toxicological result showed that fertilized egg implantation rate, fetal organogenesis and resorption rate were not be impacted on the femate rat after oral administrateion with 50 times the effective dosage extractives of reducing plasma uric acid level (625 mg/kg) from the leaves of T. albiflora extractive during the pregnant period.The test of subacute toxicity revealed the Hb, HCT and RBC had significant increase (p＜0.05); the ALKP and BUN had significant decrease (p＜0.05) compared with control group. This study has shown that T. albiflora can decrease the blood uric acid by inhibiting XO, and it also can eliminate free radical by preventing fatty oxidantion to achieve antioxidant. Female rat oral administrated high dosage during the period of pregnancy may not impact fertilized egg implantation, fetal organogenesis and resorption, but oral administrated T. albiflora for long and high dosage may cause hepatic cell swelling and slightly hyperplasia of bile duct, it could suggest that T. albiflora is not suitable to medicate in long and high dosage.英文縮寫對照表---------------------------------------------------------------------------------I 目錄----------------------------------------------------------------------------------------------III 圖次---------------------------------------------------------------------------------------------VII 表次--------------------------------------------------------------------------------------------XIII 中文摘要--------------------------------------------------------------------------------------XIX 英文摘要--------------------------------------------------------------------------------------XXI 第一章 文獻回顧------------------------------------------------------------------------------1 第一節 白花水竹草介紹---------------------------------------------------------------1 1.1 學名------------------------------------------------------------------------------1 1.2 原產地及用途-------------------------------------------------------------------2 第二節 尿酸與高尿酸血症------------------------------------------------------------2 2.1 尿酸生合成---------------------------------------------------------------------2 2.2 高尿酸血症原因---------------------------------------------------------------4 2.3 高尿酸血症與疾病------------------------------------------------------------5 2.3.1 高尿酸血症與痛風----------------------------------------------------5 2.3.2 高尿酸血症與心血管疾病-------------------------------------------6 2.3.3 高尿酸血症與腎臟傷害----------------------------------------------6 2.3.4 高尿酸血症與其他疾病----------------------------------------------7 2.4 高尿酸血症及痛風流行病學調查------------------------------------------7 2.4.1 台灣人高尿酸血症及痛風流行病學調查-------------------------7 2.4.2 其他地區高尿酸血症及痛風流行病學調查----------------------9 2.5 高尿酸血症治療---------------------------------------------------------------9 2.5.1 尿酸生成抑制劑-------------------------------------------------------9 2.5.2 尿酸排泄促進劑-----------------------------------------------------10 2.6 黃嘌呤酶抑制劑之天然物-------------------------------------------------11 2.7 高尿酸血症動物模式-------------------------------------------------------12 2.8 黃嘌呤酶之特性-------------------------------------------------------------13 2.9 黃嘌呤酶缺血再灌流傷害之關係----------------------------------------13 第三節 活性氧-------------------------------------------------------------------------14 3.1 活性氧的定義----------------------------------------------------------------14 3.2 活性氧種類-------------------------------------------------------------------15 3.3 活性氧來源-------------------------------------------------------------------18 3.4 體內抗氧化機制-------------------------------------------------------------19 3.5 抗氧化活性測定-------------------------------------------------------------20 3.6 應用於抗氧化之天然物----------------------------------------------------21 第四節 毒性評估----------------------------------------------------------------------21 4.1 毒性試驗項目----------------------------------------------------------------21 4.2 臨床毒性試驗時機----------------------------------------------------------24 第二章 緒言----------------------------------------------------------------------------------26 第三章 材料與方法-------------------------------------------------------------------------28 第一節 白花水竹草樣本製備-------------------------------------------------------28 1.1 白花水竹草粗萃液之製備-------------------------------------------------28 1.2 白花水竹草成分之製備----------------------------------------------------28 第二節 高尿酸血症動物模式建立-------------------------------------------------29 2.1 實驗動物----------------------------------------------------------------------29 2.2 頸動脈埋管操作-------------------------------------------------------------29 2.3 實驗設計----------------------------------------------------------------------30 2.4 高尿酸血症模式大鼠之操作----------------------------------------------30 2.5 血漿採集方法----------------------------------------------------------------31 2.6 資料分析與統計-------------------------------------------------------------31 第三節 高尿酸血症模式大鼠研究白花水竹草降低血中尿酸值效力試驗-31 3.1 新鮮植物效力試驗----------------------------------------------------------31 3.2 成分效力試驗----------------------------------------------------------------32 3.3 抽離成分促進尿酸排泄試驗----------------------------------------------33 3.4 資料分析與統計-------------------------------------------------------------33 第四節 生物活性篩選---------------------------------------------------------------33 4.1 黃嘌呤酶抑制試驗----------------------------------------------------------33 4.2 自由基2,2-Diphenyl-1-picrylhydrazyl（DPPH）清除能力----------34 4.3 抗氧化性測定----------------------------------------------------------------35 第五節 高劑量白花水竹草對大鼠毒理學之探討-------------------------------35 5.1 白花水竹草對懷孕大鼠受精卵著床之影響----------------------------35 5.2 白花水竹草對懷孕大鼠致畸形性試驗----------------------------------36 5.3　亞急性毒性試驗-------------------------------------------------------------36 5.4 組織病理學檢查-------------------------------------------------------------37 5.5 資料分析與統計-------------------------------------------------------------37 第四章 結果----------------------------------------------------------------------------------38 第一節　白花水竹草樣本製備------------------------------------------------------38 1.1　白花水竹草乾濕比----------------------------------------------------------38 1.2 白花水竹草各層萃取成分比例-------------------------------------------38 第二節 高尿酸血症動物模式建立------------------------------------------------38 2.1 高尿酸血症模式大鼠的建立----------------------------------------------38 2.2 新鮮白花水竹草降低血中尿酸值效力結果----------------------------39 2.3 新鮮白花水竹草對高尿酸血症模式大鼠各組血液學檢測結果----39 2.4 新鮮白花水竹草對高尿酸血症模式大鼠各組血液化學檢測結果-41 2.5 新鮮白花水竹草對高尿酸血症模式大鼠各組血壓檢測結果-------42 2.6 萃取成分降低血中尿酸值效力結果-------------------------------------43 2.7 抽離成分促尿酸排泄作用-------------------------------------------------44 第三節 生物活性篩選結果---------------------------------------------------------44 3.1 黃嘌呤酶抑制試驗----------------------------------------------------------44 3.2 自由基2,2-Diphenyl-1-picrylhydrazyl（DPPH）清除能力----------45 3.3 抗氧化性測定----------------------------------------------------------------45 第四節 高劑量白花水竹草對懷孕大鼠毒理學之探討------------------------46 4.1 白花水竹草對懷孕大鼠受精卵排卵及著床之影響------------------46 4.2 白花水竹草對懷孕大鼠致畸形性之影響------------------------------46 4.3 亞急性毒性試驗------------------------------------------------------------46 4.3.1 組織病理學檢查------------------------------------------------------47 第五章 討論----------------------------------------------------------------------------------48 第六章 參考文獻----------------------------------------------------------------------------52 附錄----------------------------------------------------------------------------------------------642148287 bytesapplication/pdfen-US高尿酸血症黃嘌呤酶抑制劑白花水竹草抗氧化劑hyperuricemiaxanthine oxidase inhibitorTradescantia albiflora Kunthantioxidant[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59974/1/ntu-94-R92629018-1.pdf