逆沒食子酸對於體內和體外N-乙醯轉移酵素活性影響的研究; In vivo and in vitro effects of ellagic acidon N-acetyltransferase activity

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Title:	逆沒食子酸對於體內和體外N-乙醯轉移酵素活性影響的研究;In vivo and in vitro effects of ellagic acidon N-acetyltransferase activity
Authors:	何蓁蓁;Ho, Chin-Chin
Contributors:	中國醫藥學院中國藥學研究所
Keywords:	逆沒食子酸;N-乙醯轉移酵素;ellagic acid;N-acetyltransferase
Date:	1999
Issue Date:	2009-11-26 11:59:58 (UTC+8)
Abstract:	本研究係藉acetyl-CoA recycling mixture assay及高效液層析儀(high performance liquid chromatography HPLC)以檢測不同劑量之並沒食子酸-ellagic acid (EA)對於正常Sprague Dawley (SD)大鼠之血液、肝臟、膀胱、大腸及白血球(淋巴球,單核球)等組織，及人類大腸癌細胞株(human colon tumor cell line colo 205)對於芳香胺類(arylamine) N-乙醯轉移酵素 (N-acetyltransferase NAT)活性及其動能常數(kinetic constant)改變，和2-AF及其代謝物之影響。結果顯示EA對於大鼠之血液、肝臟、膀胱、大腸之組織胞質液 (cytosol)及對於單核白血球、人類大腸癌細胞株之完整細胞(intact cells)，均是降低其NAT活性及降低動能常數之Km和V max值之作用，對NAT酵素而言，EA是uncompetitive inhibitor (非競爭性抑制劑)。藉由流式細胞儀(flow cytometry analysis)檢測不同濃度的EA在不同培養時程的細胞週期各期DNA含量之細胞總數，以探討EA對人類大腸癌細胞之細胞週期的影響。結果顯示EA 5 µM、50µM、100 µM、250 µM 及500 µM培養12、18、24、48、72小時對人類大腸癌細胞之細胞週期之合成期 (S phase)有統計意義之抑制 (p<0.05)， 500 µM 培養12小時，100 µM、250 µM 及500 µM 24小時，很多細胞停留於G0/G1期(G0/G1 arresting phase p<0.05)，因而進入S phase會減少。檢測EA對於SD大鼠灌食2-aminofluorene (2-AF)之乙醯化及代謝的影響部分，如果EA給藥24小時後再灌2-AF，再經24小時後，則膀胱組織之2-AAF及其代謝產物顯示有意義減少(p<0.05)；若EA與2-AF同時灌食24小時後，其小便及大便的代謝物亦均有意義減少(p<0.05)。而NAT活性受抑制之效果依序為同時灌食2-AF與EA>灌食EA經24小時後再灌食2-AF>控制組(只灌食2-AF)。綜合以上的結果，顯示並沒食子酸(EA)具有減低N-乙醯轉移酵素的活性；並對大腸癌細胞之細胞週期之複製期(S phase)有抑制作用。; The determinations of N-acetyltransferase (NAT) activities in cytosol and intact cells were analyzed by using acetyl-CoA recycling mixture assay and high performance liquid chromatography (HPLC). The effects of ellagic acid (EA) on arylamine N-acetyltransferase (NAT) activities and kinetic constant changes of NAT enzyme from blood, liver, bladder, colon, urine, feces, and leukocytes (i.e. lymphocytes and monocytes) of Sprague-Dawley (SD) rats and also in human colon tumor cell line (colo 205) were determined. The results indicated that there were a decreasing of NAT activities and kinetic constant changes of NAT associated with increased level of EA in the cytosol or intact cells of examined tissues or cells. EA was an uncompetitive inhibitor for examined NAT enzyme. The cell cycle of human colon tumor cell (colo 205) treated with various concentrations of EA for different time was determined. The cell cycle analysis was based on the cell numbers of DNA content by using flow cytometry analysis. The results also showed that the treatment of 5,50,100,250 and 500 µM EA for 12,24,48 and 72 hours in human colon tumor cells resulted in the inhibition of S phase which may be via the arresting cell cycle at G0/G1 (p<0.05). The effects of EA orally on 2-aminofluorene (2-AF) N-acetylation and metabolism in SD rats were examined. It was indicated that 24 hours after EA treatment then oral treatment of 2-AF for 24 hours, The 2-AAF and 2-AF metabolites from bladder tissues showed a significant difference between control and experimental groups. But the treatment of EA and 2-AF at the same time to the rats, the decrease in 2-AF metabolites from urine and feces showed a significant difference between control and experimental groups. The inhibitory level of NAT by EA from experimental groups was treatment of EA and 2-AF treatment at the same time > 24 hours after EA treatment then treated with 2-AF > 2-AF treatment alone (control). In conclusion, these results indicated that EA could decrease NAT activity and inhibit S phase of cell cycle of human colon tumor cells.
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