|Abstract: ||龍鬚菜(Gracilaria spp.)在日本一般做為生魚片配菜的食用藻類，但與龍鬚菜有關的中毒案例在日本已發生四件，共有8位患者，其中3人死亡，患者將生鮮龍鬚菜與生魚片一起食用，而產生中毒的症狀，並且發現女性的症狀較男性嚴重，但因中毒案例不多，因此只能初步推測此類中毒對女性傷害較大，一般在將食物吃進體內30~60分鐘之後就有症狀產生，一般的症狀為噁心、嘔吐及腹瀉等，嚴重的情況下會導致血壓過低因而休克、死亡。經研究發現造成中毒的龍鬚菜樣本含有高量的前列腺素E2 (prostaglandin E2 , PGE2)，此外，龍鬚菜及患者體內的環氧合酶(cyclooxygenase, COX)會與生魚肉中的花生四烯酸(arachidonic acid, AA)作用生成PGE2，造成短時間在患者體內累積高量的PGE2，並且在藥理學上PGE2所具有的副作用與中毒症狀相似，因此推測此類中毒可能與PGE2有關 (Sajiki and Kakimi, 1998；Noguchi et al., 1994)。為了解台灣本地龍鬚菜藻類之食用安全性，本研究首先發展出一套HPLC分析方法來測定龍鬚菜藻類中前列腺素的含量，所檢測的前列腺素包括前列腺素A2 (prostaglandin A2 , PGA2)、PGE2及前列腺素F2α (prostaglandin F2α , PGF2α)，移動相為乙腈(acetonitrile)與0.017 M的磷酸，分析條件為0至30分鐘，乙腈以線性梯度由35%提高至60%，流速為1 mL/min，偵測波長為196 nm。依此條件所製作出前列腺素(PGA2、PGE2及PGF2α)的檢量線R2值皆大於0.999，顯示有良好的線性關係，並且其變異數相關係數低(coefficient of variation, CV%小於4.7)，表示儀器及標準品的穩定度高，PGA2及PGE2檢測極限為0.5 μg/mL，PGF2α的檢測極限為2.5 μg/mL，PGA2、PGE2及PGF2α的回收率分別為82.2±4.7%、91.3±9.2%及76.5±5.2%。接著探討反應條件對龍鬚菜中PGE2生成之影響及不同季節與不同品種龍鬚菜PGE2含量及生成量之差異，實驗結果顯示，水溶性花生四烯酸鈉鹽的添加量為龍鬚菜藻體重量(wet wt)之0.02% (w/w)時PGE2的生成量可達最大值，氧氣也會影響PGE2的生成，在反應中攪拌增加溶氧量，PGE2的生成量會比在厭氧(充氮氣密封)的條件下高23%。溫度的影響方面，龍鬚菜萃取液加入花生四烯酸鈉鹽在25oC反應其PGE2之生成量最高。pH的影響方面，在pH 8.5反應其PGE2之生成量最高，此外，龍鬚菜中PGE2的含量及生成量會因不同季節及不同品種而有差異，並且也發現龍鬚菜萃取液在4oC下放置6小時後，PGE2的生成量為控制組(25oC)的23%，由於PGE2的生成是花生四烯酸經COX及PGE2 synthase的作用而產生，因此推測龍鬚菜中與PGE2生成有關之酵素(COX及PGE2 synthase)相當的不安定。環境因子(包括鹽度、溫度、光度、離水、金屬離子)對龍鬚菜中PGE2生成之影響方面，實驗結果發現，改變龍鬚菜的生長環境會造成龍鬚菜中PGE2生成量的改變，其中低溫、鹽度與光度的改變、離水及鈣離子皆會促進PGE2的生成，但aspirin (非專一性COX抑制劑)及nimesulide (專一性COX-2抑制劑)都能顯著的抑制龍鬚菜中PGE2的生成，顯示當生長環境改變，龍鬚菜受到緊迫後會誘發產生COX-2使PGE2的生成量增加。綜合結果顯示，龍鬚菜生成PGE2之產量受種屬、季節、環境因子和反應條件所影響。|
Gracilaria is a kind of edible red algae (Japanese name, “ogonori). People are accustomed to take raw fish with Gracilaria spp. in Japan. However, some poisoning cases occurred due to ingesting seaweed Gracilaria spp. in Japan. Those victims ate raw fish and raw seaweed. The common symptoms in the patients are nausea, vomiting and diarrhea appearing 30-60 min after ingestion. In extreme cases, they developed to very low blood pressure, followed by shock and death. In addition, the symptoms in female were more serious than those in male. The possible reason of seaweed poison cases was supposed that raw seaweed contained plenty of prostaglandin E2 (PGE2) and cyclooxygenase (COX). The enzyme (COX) in the seaweed and/or the body tissues of the victim may be acting on the arachidonic acid (precursor of PGE2) in the raw fish or in the seaweed producing PGE2. Therefore great amount of PGE2 was accumulated at short time in the victim’s body (Sajiki and Kakimi, 1998；Noguchi et al., 1994). In this study, a HPLC method was developed for quantifying prostaglandins in the seaweed. Prostaglandins included prostaglandin A2 (PGA2), PGE2 and prostaglandin F2α (PGF2α) were detected in this study. The mobile phase was gradient acetonitrile (35%~60%) and 0.017 M phosphoric acid at flow rate of 1 mL/min and 196 nm within 30 min. The standard curves of prostaglandins (PGA2, PGE2 and PGF2α) were extremely linear (R2 > 0.999) with low correlation coefficients (less than 4.7) in the range of 5~50 μg/mL. Those data implicate the standard solutions and HPLC assay system was all quite stable. The detection limit of PGA2 and PGE2 was 0.5 μg/mL and that of PGF2α was 2.5 μg/mL. The average recoveries of PGA2, PGE2 and PGF2α were 82.2±4.7%, 91.3±9.2% and 76.5±5.2%, respectively. The next section of this study is to investigate the effects of reaction conditions on PGE2 production in the seaweed. Maximum amount of PGE2 was producted as the ratio of arachidonic acid sodium salt and seaweed (wet wt) was 0.02% (w/w). Due to oxygen affecting PGE2 production, more amount of PGE2 was produced in aerobic condition than in anaerobic condition. The optimal temperature of PGE2 production was 25oC and the optimal pH was 8.5. There were different PGE2 level in different Gracilaria species and the season variation of PGE2 level was also found. The PGE2 level was 23% comparing to control when the extract solution of the seaweed was placed in 4oC after 6 hr. Arachidonic acid was metabolized to PGE2 by COX and PGE2 synthase. Therefore this result indicated that the enzymes of related PGE2 production in the extract solution were not stable. Effects of environmental factors on PGE2 production in the seaweed were also investigated. The effects of environmental conditions caused to change the PGE2 production in the seaweed. Some special environment factors including low temperature, salinity, irradiance and Ca2+, promoted PGE2 production in the seaweed, but aspirin and nimesulide inhibited. Hence, PGE2 production in the seaweed was affected by species, season, environmental factors and reaction conditions.