Toxicolgical Studies of Grass Carp Bile Juicews(II)

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Project title

Code/計畫編號

DOH89-TD-1084

Translated Name/計畫中文名

草魚膽汁之毒性學研究(II)

Project Coordinator/計畫主持人

Deng-Fwu Hwang

Funding Organization/主管機關

Environmental Protection Administration

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=592161

Year

2000

Start date/計畫起

01-07-1999

Expected Completion/計畫迄

01-06-2000

Bugetid/研究經費

840千元

ResearchField/研究領域

生物科學
化學

為知草魚膽汁中之膽酸、膽酸鹽和膽醇硫酸鹽之組成，乃首先從草魚膽汁中純化出5α-cyprinol sulfate，其過程為將草魚膽汁藉PHP-LH-20和silica gel兩種管柱層析純化後，取其酒精溶出物，經減壓濃縮除去溶劑後，以氰甲烷做結晶，將此結晶物經紅外光譜儀 (FT-IR)、氫原子 (1H) 及碳-13 (13C) 核磁共振光譜儀 (NMR) 和質譜儀 (MS) 之化學分析，結果發現5α-cyprinol sulfate之分子式為C27H48SO8，分子量532，其在草魚膽汁係以5α-cyprinol sulfate存在。同時研發高效能液相層析法 (HPLC) 分析膽酸和膽鹽之方法，發現以ODS逆向層析管，溶煤系統0.3 % ammonium carbonate/ acetonitrile (73: 27, v/v) 10min→(68: 32) 10min→(50: 50) 10min，即可將14 種膽酸、膽酸鹽和膽醇硫酸鹽分離。其中deoxycholic acid 和glycodeocholic acid 會在18分鐘時同時溶出，但另以溶出條件buffer 0.3 % ammonium carbonate/ acetonitrile (73: 27, v/v) 即可區分此兩種膽酸成分。經採樣14種魚類膽汁，經以HPLC方法分析膽酸、膽酸鹽和膽醇硫酸鹽，得知草魚和鯉魚膽汁中之5α-cyprinol sulfate之含量為膽酸、膽酸鹽和膽醇硫酸鹽全量之94 %以上，而鱒魚、鰻魚和鯖魚等12種魚類膽汁中膽酸主要成分為taurocholic acid和taurochenodeoxycholic acid，而香魚膽汁中膽酸主要成分為cholic acid和taurocholic acid。除草魚和鯉魚膽外，其它魚類膽汁中都未檢出5α-cyprinol sulfate，在陸地脊椎動物方面雞膽主要成份為glycolithocholic acid和taurocholic acid，而鴨膽主要膽汁成份為taurochenodeoxycholic acid、cholic acid和ursodeoxycholic acid。為知5α-cyprinol sulfate 之毒性，乃以口胃管餵食老鼠生理食鹽水 (0.6ml)、草魚膽汁 (100 mg/ 0.6ml) 、 5α-cyprinol sulfate (100 mg/ 0.6ml)、butanol extract (100 mg/ 0.6ml) 和5α-cyprinol (100 mg/ 0.6ml)，6小時後，發現草魚膽汁、butanol extract、5α-cyprinol和5α-cyprinol sulfate組之老鼠，其血漿中BUN 和creatinine顯著上升 (P<0.05)，顯示除對照組之外，其他各組老鼠腎功能都受損。但5α-cyprinol組之老鼠，氨基酸轉氨酵素AST和ALT未見變化，顯示5α-cyprinol之毒作用器官為腎臟，而草魚膽汁和5α-cyprinol sulfate之毒作用器官為肝臟和腎臟。 To elucidate the responsible toxic components in the bile juice of grass carp, the bile alcohol salt from grass carp bile juice was purified and identified. By IR、1H-NMR, 13C-NMR and MS spectrometry analyses, a compound with molecular formula of C27H48SO8 and molecular weight of 532 was obtained from grass carp bile juice and identified as 5α-cyprinol sulfate. In the meantime, the determination method for bile acids in fish bile juices was developed. It was found that high performance liquid chromatography (HPLC), with a reversed ODS column and a buffer system of 0.3 % ammonium carbonate/ acetonitrite (73: 27, v/v) 10min→(68: 32) 10min→(50: 50) 10min, clearly separated 14 bile acids. Among them, deoxycholic acid and glycodeoxycholic acid appeared at the same time. These two components were further separated by using other buffer 0.3 % ammonium carbonate/ acetonitrite (73: 27, v/v). Based on the analyses of above HPLC method, 14 samples of bile juices obtained from 14 fish species were determined. It was found that the major component of bile juices was 5α-cyprinol sulfate for grass carp (Ctenopharyngodon idellus) and common carp (Cyprinus carpio). The bile juices of rainbow trout (Bodianus oxycephalus), rock trout (Salmo salar), japanese eel (Anguilla japonica), spanish mackerel (Aixos maru), right eyed flounder (Pleuronectes cornutus), rudder fish (Girella punctata), black scraper (Thamnaconus modestus), tongue sole (Paraplagusia bilineata), japanese jack (Trachurus japonicus) and mackerel (Scomberomorus commerson) contained mainly taurocholic acid and taurochenodeoxycholic acid, but that of ayu (Plecoglossus altirelis) fish contained mainly cholic acid and taurocholic acid. Chicken (Pectoralis major) bile juice was mainly composed of glycolithocholic acid and taurocholic acid, but duck (Anas platyhynchos var. domestica) bile juice was mainly inclusive of taurochenodeoxycholic acid, cholic acid and ursodeoxycholic acid. To compare the toxicity of 5α-cyprinol sulfate and grass carp bile juice, the rats were separately administered with saline (0.6 ml), grass carp bile juice (100 mg/ 0.6ml), 5α-cyprinol sulfate (100 mg/ 0.6 ml), butanol extract (100 mg/ 0.6ml) and 5α-cyprinol (100 mg/ 0.6ml). After 6 hr, the plasma of rats were examined. The value of BUN and creatinine in the plasma of rats treated with grass carp bile juice, 5α-cyprinol sulfate, butanol extract and 5α-cyprinol were significantly higher than that of saline group. The function of liver was also affected by raw bile juice and 5α-cyprinol sulfate, but not by 5α-cyprinol.

Keyword(s)

草魚
鯉魚
膽汁
膽酸
膽醇鹽
Grass carp
Common carp
Bile juice
Bile acids
Bile alcohol

DSpace CRIS

Toxicolgical Studies of Grass Carp Bile Juicews(II)

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