高國(guó)瑞， 潘魯青， 黃 輝， 宋夢(mèng)思(海水養(yǎng)殖教育部重點(diǎn)實(shí)驗(yàn)室(中國(guó)海洋大學(xué))，山東 青島 266003)

黃芪和黃連活性提取物對(duì)凡納濱對(duì)蝦代謝相關(guān)酶的影響?

高國(guó)瑞， 潘魯青??， 黃 輝， 宋夢(mèng)思
(海水養(yǎng)殖教育部重點(diǎn)實(shí)驗(yàn)室(中國(guó)海洋大學(xué))，山東 青島 266003)

本文研究了黃芪和黃連活性提取物在凡納濱對(duì)蝦(Litopenaeusvannamei)體內(nèi)的代謝過(guò)程及損傷效應(yīng)。實(shí)驗(yàn)設(shè)置6個(gè)實(shí)驗(yàn)組：黃芪多糖(黃芪提取物)0.5、1 g/kg，小檗堿(黃連提取物)0.5、1 g/kg，黃芪多糖0.5 g/kg+小檗堿0.5 g/kg，對(duì)照組(投喂配合飼料)。投喂含中草藥的飼料6天后投喂配合飼料10天，檢測(cè)凡納濱對(duì)蝦的代謝酶含量、抗氧化指標(biāo)及組織損傷指標(biāo)。研究表明：黃芪、黃連活性提取物對(duì)凡納濱對(duì)蝦代謝相關(guān)酶影響顯著(P<0.05)。各處理組肝胰腺CYP450含量、GST活性均于第6天時(shí)達(dá)到最大值，停藥3天后仍明顯高于對(duì)照組(P<0.05)；混合處理組CYP450含量、GST活性高于同濃度單獨(dú)處理組。投喂6天內(nèi)，黃芪、黃連活性提取物均顯著提高凡納濱對(duì)蝦T-AOC、SOD活性、GSH含量及GSH/GSSG；混合處理組各指標(biāo)高于同濃度單獨(dú)處理組，各組織抗氧化水平大小為：血淋巴>肝胰腺。黃芪多糖0.5、1 g/kg及黃芪多糖0.5 g/kg+小檗堿0.5 g/kg對(duì)凡納濱對(duì)蝦肝胰腺和鰓脂質(zhì)過(guò)氧化和蛋白質(zhì)羰基化無(wú)顯著影響，而小檗堿0.5、1 g/kg顯著造成凡納濱對(duì)蝦組織損傷；停藥3天后，脂質(zhì)過(guò)氧化程度恢復(fù)至正常水平，而羰基含量保持穩(wěn)定直到實(shí)驗(yàn)結(jié)束。研究結(jié)果表明，各組織DNA損傷與中草藥活性提取物濃度顯著相關(guān)，表現(xiàn)出明顯的時(shí)間劑量效應(yīng)，可作為中草藥活性提取物對(duì)凡納濱對(duì)蝦安全性評(píng)價(jià)的指標(biāo)。

黃芪；黃連；凡納濱對(duì)蝦；代謝酶；抗氧化防御；生物大分子損傷

凡納濱對(duì)蝦(Litopenaeusvannamei)具有較強(qiáng)的適應(yīng)性和較快的生長(zhǎng)速度，是我國(guó)重要的對(duì)蝦養(yǎng)殖種類(lèi)。近年來(lái)，凡納濱對(duì)蝦病害頻繁發(fā)生，嚴(yán)重制約著養(yǎng)殖業(yè)的發(fā)展[1]。防治病害的傳統(tǒng)方法是使用抗生素，但會(huì)導(dǎo)致病菌耐藥性的產(chǎn)生、抗生素殘留等問(wèn)題[2]。中草藥作為綠色天然藥物，具有多功效、不易誘發(fā)病原菌耐藥性等優(yōu)勢(shì)，在水產(chǎn)無(wú)公害養(yǎng)殖病害防治中具有廣闊的應(yīng)用前景[3]。已有研究表明，中草藥能顯著提高水產(chǎn)動(dòng)物免疫力、抗氧化能力及疾病抵抗力等[4-6]。如彭婷等研究發(fā)現(xiàn)飼喂羅非魚(yú)(Oreochromisniloticus)100、200、400 mL/kg黃芪多糖脂質(zhì)體10 d，顯著提高肝臟超氧化物歧化酶(SOD)和谷胱甘肽過(guò)氧化物酶(GSH-Px)活性[7]。Zahran等研究表明，飼喂羅非魚(yú)(Oreochromisniloticus)0.15%黃芪多糖14 d，顯著提高外周血淋巴細(xì)胞吞噬活性、抗菌活性及溶菌酶活性[8]。藥物使用應(yīng)綜合考慮療效、代謝規(guī)律及副作用，然而目前漁用中草藥在應(yīng)用中只注重功效，缺乏在水產(chǎn)動(dòng)物體內(nèi)代謝過(guò)程及副作用的研究。本文研究了黃芪、黃連活性提取物在凡納濱對(duì)蝦體內(nèi)代謝過(guò)程及組織損傷，探討了單一及混合中草藥在凡納濱對(duì)蝦體內(nèi)的代謝規(guī)律及損傷效應(yīng)，為中草藥在水產(chǎn)養(yǎng)殖業(yè)的合理應(yīng)用提供科學(xué)依據(jù)。

1 材料與方法

1.1 實(shí)驗(yàn)材料

實(shí)驗(yàn)所用凡納濱對(duì)蝦于2014年9月份購(gòu)于青島膠州對(duì)蝦養(yǎng)殖場(chǎng)，體質(zhì)量為(15.2±2.6)g。實(shí)驗(yàn)前暫養(yǎng)含125 L海水的水箱中(72 cm×56 cm×40 cm)一周，暫養(yǎng)條件保持穩(wěn)定：水溫(25±2)℃，鹽度30±1，pH8.2±0.2。暫養(yǎng)期間，連續(xù)充氣，日換水2次，換水量為1/3～1/2，除去殘餌和糞便，投喂配合飼料(粵海飼料集團(tuán)生產(chǎn))，日投喂量為對(duì)蝦體重的5%。

黃芪、黃連均購(gòu)自青島市同仁堂藥店。將中草藥用研磨機(jī)粉碎，取100 g黃芪粉末加入1 L超純水，100℃回流萃取2次，每次2 h，合并上清液，旋轉(zhuǎn)蒸發(fā)濃縮至200 mL，加入800 mL 95%乙醇，靜置24 h，抽濾，沉淀依次用無(wú)水乙醇、丙酮洗滌后在50℃烘箱中烘干。取100 g黃連粉末加入1 L 85%乙醇，超聲震蕩10 min后于80℃回流萃取2 h，重復(fù)萃取2次，合并上清液，并用旋轉(zhuǎn)蒸發(fā)儀濃縮，氮?dú)獯蹈桑占崛∥锓胖迷诿荛]干燥容器中。已有研究表明，黃芪、黃連的主要活性成分分別為：黃芪多糖、小檗堿[8-9]，經(jīng)測(cè)定黃芪提取物中黃芪多糖的含量為28.5%，黃連提取物中小檗堿的含量為40.6%。

1.2 實(shí)驗(yàn)方法

1.2.1 實(shí)驗(yàn)設(shè)置 本研究使用黃芪多糖、小檗堿分別作為黃芪、黃連活性提取物添加量的定量標(biāo)準(zhǔn)，根據(jù)本實(shí)驗(yàn)室之前篩選出的免疫增強(qiáng)劑配方，設(shè)置6個(gè)實(shí)驗(yàn)組，分別為黃芪多糖0.5、1 g/kg，小檗堿0.5、1 g/kg，黃芪多糖0.5 g/kg +小檗堿0.5 g/kg，對(duì)照組(投喂配合飼料)。將各處理組中草藥活性提取物均勻噴灑在配合飼料表面，用魚(yú)油包裹，晾干備用[10]。

實(shí)驗(yàn)前將暫養(yǎng)在自然海水中健康的凡納濱對(duì)蝦隨機(jī)移入各實(shí)驗(yàn)水箱中，每個(gè)梯度均設(shè)3個(gè)平行組，養(yǎng)殖管理與暫養(yǎng)條件相同，實(shí)驗(yàn)期間凡納濱對(duì)蝦無(wú)死亡現(xiàn)象。向?qū)ξr投喂含中草藥飼料6 d后投喂配合飼料10 d，取樣時(shí)間點(diǎn)為0、3、6、7、9、12和16 d。

1.2.2 樣品制備 各處理組取8尾蝦，用預(yù)先吸入0.3 mL預(yù)冷抗凝劑(0.01 mol/L HEPES、0.01 mol/L氯化鉀、0.45 mol/L氯化鈉、0.01 mol/L EDTA二鈉，滲透壓為750 mOsm/kg，pH=7.00)的1 mL無(wú)菌注射器，按照1∶1的比例在腹血竇處采血。于4℃下800g離心10 min，取上清液-80℃保存?zhèn)溆?。于冰上解剖分別取肝胰腺和鰓，液氮研磨，分裝于離心管中(每管裝80～100 mg)，-80℃保存?zhèn)溆谩?/p>

1.2.3 測(cè)定方法 細(xì)胞色素P450(CYP450)含量的測(cè)定參照Omura和Sato的方法[11]。

谷胱甘肽硫轉(zhuǎn)移酶(GST)活性的測(cè)定參照Habig的方法[12]。GST活性以2,4-二硝基苯谷胱甘肽的增速表示，酶活力單位為nmol/mg蛋白。

總抗氧化活力(T-AOC)使用試劑盒(南京建成生物科技有限公司)進(jìn)行測(cè)定。

超氧化物歧化酶(SOD)的測(cè)定參照Marklund和Marklund的方法[13]。SOD活力單位定義為：25℃下每分鐘抑制鄰苯三酚自氧化速率到50%時(shí)所需要的酶量。

還原性谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)含量的測(cè)定參照Anderson的方法[14]。

DNA損傷的測(cè)定采用堿解旋的方法[15]。提取各實(shí)驗(yàn)組肝胰腺和鰓DNA并各分為3份，分別測(cè)定雙鏈、單鏈及堿解旋的熒光值。DNA損傷程度用F值表示，F(xiàn)值越小，表明DNA損傷程度越大，其計(jì)算公式為：

蛋白質(zhì)羰基含量的測(cè)定參照Lund等的方法[16]。

脂質(zhì)過(guò)氧化(LPO)程度采用測(cè)定丙二醛(MDA)含量的方法[17]。

蛋白含量的測(cè)定采用考馬斯亮藍(lán)法[18]，并以牛血清蛋白溶液為標(biāo)準(zhǔn)溶液作標(biāo)準(zhǔn)曲線。

1.3 數(shù)據(jù)處理與分析

所有實(shí)驗(yàn)數(shù)據(jù)采用SPSS17.0進(jìn)行分析。實(shí)驗(yàn)結(jié)果以3個(gè)平行組數(shù)據(jù)的平均值±標(biāo)準(zhǔn)差(Means±SD)表示，并應(yīng)用單因素方差分析(One-Way ANOVA)和Duncan檢驗(yàn)法進(jìn)行統(tǒng)計(jì)分析。P<0.05代表差異顯著，P<0.01代表差異極其顯著。

2 實(shí)驗(yàn)結(jié)果

2.1 中草藥活性提取物對(duì)凡納濱對(duì)蝦肝胰腺代謝活性的影響 由圖1可知，黃芪、黃連活性提取物對(duì)凡納濱對(duì)蝦肝胰腺CYP450含量及GST活性影響顯著(P<0.05)，而對(duì)照組無(wú)明顯變化。各處理組肝胰腺中CYP450含量及GST活性在12d內(nèi)呈明顯的峰值變化，于6 d時(shí)達(dá)到最高值，停藥3 d后仍顯著高于對(duì)照組水平(P<0.05)。提取物混合處理組肝胰腺中CYP450含量及GST活性高于同濃度單獨(dú)處理組。停止投藥后，各處理組肝胰腺中CYP450含量及GST活性逐漸下降，于停藥6 d后恢復(fù)正常水平。

2.2 中草藥活性提取物對(duì)凡納濱對(duì)蝦抗氧化防御指標(biāo)的影響 由圖2、3所知，黃芪、黃連活性提取物對(duì)凡納濱對(duì)蝦血淋巴和肝胰腺中抗氧化指標(biāo)影響顯著(P<0.05)，而對(duì)照組無(wú)明顯變化。各處理組血淋巴和肝胰腺中T-AOC、SOD活力、GSH含量及GSH/GSSG在12 d內(nèi)呈明顯的峰值變化，于6 d時(shí)達(dá)到最高值，停藥3 d后仍顯著高于對(duì)照組水平(P<0.05)。投喂活性提取物6 d內(nèi)，黃連提取物處理組中各抗氧化指標(biāo)高于同濃度黃芪提取物處理組；混合處理組血淋巴和肝胰腺中各抗氧化指標(biāo)高于同濃度單獨(dú)處理組。停止投藥后，各處理組血淋巴和肝胰腺中各抗氧化指標(biāo)逐漸下降，于停藥6 d后恢復(fù)正常水平。

(“*”代表與對(duì)照組差異顯著(P<0.05)，“**”代表與對(duì)照組差異極其顯著(P<0.01)。Significant differences from control in the sametime of sampling are indicated with an asterisk atP<0.05,and with two asterisks atP<0.01)

圖1 中草藥活性提取物對(duì)凡納濱對(duì)蝦肝胰腺CYP450含量(A)及GST活性(B)的影響
Fig.1 Effects of herb extracts on CYP450 content(A)and GST activity(B)in hepatopancreas ofL.vannamei

(“*”代表與對(duì)照組差異顯著(P<0.05)，“**”代表與對(duì)照組差異極其顯著(P<0.01)。Significant differences from control in the same time of sampling are indicated with an asterisk atP<0.05,and with two asterisks atP<0.01.)

圖2 中草藥活性提取物對(duì)凡納濱對(duì)蝦血淋巴T-AOC(A)，SOD(B)，GSH(C)及GSH/GSSG(D)的影響
Fig.2 Effects of herb extracts on T-AOC(A),SOD activity (B),GSH content(C)and the ratio of GSH/GSSG(D)in hemolymph ofL.vannamei

2.3 中草藥活性提取物對(duì)凡納濱對(duì)蝦組織損傷效應(yīng)的影響 由圖4、5可知，黃芪、黃連活性提取物對(duì)凡納濱對(duì)蝦鰓和肝胰腺DNA損傷影響顯著(P<0.05)，而對(duì)照組無(wú)明顯差異。各處理組F值在12 d內(nèi)呈明顯的峰值變化，均于6 d達(dá)到最低值，且與中草藥活性提取物濃度呈明顯的負(fù)相關(guān)(P<0.05)，停藥3 d后仍顯著低于對(duì)照組水平。黃連活性提取物單獨(dú)作用對(duì)凡納濱對(duì)蝦鰓和肝胰腺脂質(zhì)過(guò)氧化及蛋白質(zhì)羰基化影響顯著(P<0.05)，而黃芪活性提取物處理組、混合處理組與對(duì)照組相比無(wú)明顯變化。各處理組丙二醛及羰基含量分別在9、16 d內(nèi)呈峰值變化，均于6 d達(dá)到最高值，停藥3 d后，各處理組丙二醛含量恢復(fù)正常值，而羰基含量保持穩(wěn)定，直到實(shí)驗(yàn)結(jié)束。

(“*”代表與對(duì)照組差異顯著(P<0.05)，“**”代表與對(duì)照組差異極其顯著(P<0.01)。Significant differences from control in the same time of sampling are indicated with an asterisk atP<0.05,and with two asterisks atP<0.01.)

圖3 中草藥活性提取物對(duì)凡納濱對(duì)蝦肝胰腺T-AOC(A)，SOD活性(B)，GSH含量(C)及GSH/GSSG(D)的影響
Fig.3 Effects of herb extracts on T-AOC(A),SOD activity (B),GSH content(C)and the ratio of GSH/GSSG(D)in hepatopancreas ofL.vannamei

(“*”代表與對(duì)照組差異顯著(P<0.05)，“**”代表與對(duì)照組差異極其顯著(P<0.01)。Significant differences from control in the same time of sampling are indicated with an asterisk atP<0.05,and with two asterisks atP<0.01.)

圖4 中草藥活性提取物對(duì)凡納濱對(duì)蝦鰓DNA損傷(A)、脂質(zhì)過(guò)氧化(B)、蛋白質(zhì)羰基化(C)的影響
Fig.4 Effects of herb extracts on DNA damage(A),lipid peroxidation(B)and protein carbonylation(C)in gill ofL.vannamei

(“*”代表與對(duì)照組差異顯著(P<0.05)，“**”代表與對(duì)照組差異極其顯著(P<0.01)。Significant differences from control in the same time of sampling are indicated with an asterisk atP<0.05,and with two asterisks atP<0.01.)

圖5 中草藥活性提取物對(duì)凡納濱對(duì)蝦肝胰腺DNA損傷(A)、脂質(zhì)過(guò)氧化(B)、蛋白質(zhì)羰基化(C)的影響
Fig.5 Effects of herb extracts on DNA damage (A),lipid peroxidation(B)and protein carbonylation(C)in hepatopancreas ofL.vannamei

3 討論

3.1 中草藥活性提取物在凡納濱對(duì)蝦體內(nèi)代謝過(guò)程的影響 中草藥活性提取物經(jīng)口服后，從對(duì)蝦胃中輸送到肝胰腺，通過(guò)解毒代謝酶系統(tǒng)進(jìn)行代謝和消除[19]。CYP450在I相代謝中催化外源性化合物進(jìn)行單加氧反應(yīng)[20]。GST是II相代謝中重要的代謝酶，可催化谷胱甘肽(GSH)與外源物質(zhì)及活性代謝產(chǎn)物結(jié)合，形成水溶性較強(qiáng)的代謝物排出體外[21-22]。李小彥等研究發(fā)現(xiàn)投喂100 mg/kg黃芩苷7 d，顯著提高中國(guó)對(duì)蝦(Fenneropenaeuschinensis)肝胰腺中CYP1A、CYP2及GST活性[23]。本研究表明投藥6 d，各處理組肝胰腺CYP450含量及GST活性顯著升高，混合處理組中CYP450含量及GST活性高于單獨(dú)處理組；停藥后各處理組肝胰腺CYP450含量及GST活性顯著減少，混合處理組中CYP450含量及GST活性高于單獨(dú)處理組，各指標(biāo)均于停藥第6天恢復(fù)正常水平，這與上述研究結(jié)果基本類(lèi)似。由此可見(jiàn)，與單獨(dú)作用相比，黃芪、黃連活性提取物混合作用誘導(dǎo)了CYP450含量及GST活性的升高；凡納濱對(duì)蝦能提高機(jī)體CYP450含量增強(qiáng)轉(zhuǎn)化化合物的功能，并提高GST活性以強(qiáng)化中草藥活性提取物與GSH的結(jié)合能力，最終促進(jìn)中草藥活性提取物的代謝。作者認(rèn)為肝胰腺中CYP450含量及GST活性能綜合反映中草藥活性提取物在對(duì)蝦體內(nèi)的代謝規(guī)律，可為中草藥活性提取物療程的應(yīng)用提供科學(xué)依據(jù)。

3.2 中草藥活性提取物對(duì)凡納濱對(duì)蝦抗氧化防御指標(biāo)的影響 中草藥活性提取物在代謝過(guò)程中產(chǎn)生活性氧(ROS)，對(duì)蝦可通過(guò)調(diào)節(jié)機(jī)體的抗氧化防御系統(tǒng)如抗氧化酶活力等來(lái)適應(yīng)增長(zhǎng)的活性氧[24]，但活性氧的含量超過(guò)機(jī)體自身消除能力就會(huì)造成DNA損傷、脂質(zhì)過(guò)氧化、蛋白質(zhì)羰基化等氧化損傷[25]。T-AOC代表機(jī)體整體抗氧化水平。SOD是對(duì)蝦抗氧化防御系統(tǒng)重要的酶，可以通過(guò)還原氧化金屬離子活性位點(diǎn)來(lái)催化有毒的超氧陰離子快速反應(yīng)生成分子氧和過(guò)氧化氫[26]。GSH具有與體內(nèi)自由基結(jié)合的能力，轉(zhuǎn)化為易代謝酸物質(zhì)，加速自由基的清除的作用，其含量的增加可反映機(jī)體抗氧化能力的提高[27]。在氧化應(yīng)激反應(yīng)下，機(jī)體內(nèi)谷胱甘肽還原型逐漸減少，GSSG含量逐漸上升[28]。已有研究表明，黃芪活性提取物能顯著提高水產(chǎn)動(dòng)物抗氧化能力。如樊英等研究發(fā)現(xiàn)向刺參(Stichopusjaponicus)體腔中注射0.6 mg/頭的無(wú)菌黃芪多糖溶液3 d，顯著提高體腔液中SOD活力[29]。白東清等研究發(fā)現(xiàn)飼喂黃顙魚(yú)(Pelteobagrusfulvidraco)600～1200 mg/kg黃芪多糖56 d，顯著提高各組織SOD和過(guò)氧化氫酶(CAT)活力[30]。凡納濱對(duì)蝦血淋巴各指標(biāo)代表機(jī)體整體水平，而肝胰腺是主要的代謝器官。本研究表明，黃芪、黃連活性提取物單獨(dú)及混合作用均顯著提高凡納濱對(duì)蝦T-AOC，SOD活性，GSH含量及GSH/GSSG，各組織抗氧化能力大小為：血淋巴>肝胰腺，與上述研究結(jié)果基本類(lèi)似。黃連提取物處理組中各抗氧化指標(biāo)高于同濃度黃芪處理組，表明黃連活性提取物具有較高的抗氧化增強(qiáng)作用。

3.3 中草藥活性提取物對(duì)凡納濱對(duì)蝦組織損傷效應(yīng)的影響 DNA損傷是作評(píng)估基因毒性的生物標(biāo)志物之一[31]，已有研究表明，農(nóng)藥、污染物、環(huán)境突變均可引起機(jī)體DNA損傷[32-34]。如養(yǎng)殖水域pH由7.3突變至5.6和9.3均造成凡納濱對(duì)蝦血細(xì)胞DNA損傷[35]。Chang等研究發(fā)現(xiàn)暴露于4.25 mol/L氯化鎘環(huán)境中，凡納濱對(duì)蝦血細(xì)胞DNA損傷顯著，暴露6 h后，DNA損傷逐漸恢復(fù)[36]。LPO是評(píng)價(jià)氧化損傷的重要指標(biāo)。已有研究表明，飼喂羅非魚(yú)(Oreochromisniloticus)0.15%黃芪多糖14 d，血漿中脂質(zhì)過(guò)氧化無(wú)明顯變化[8]。蛋白質(zhì)羰基化被廣泛用來(lái)評(píng)價(jià)氧化應(yīng)激和損傷效應(yīng)[37-38]。已有研究表明，暴露于1 μg/L溴氰菊酯4 d，顯著提高斑節(jié)對(duì)蝦(Penaeusmonodon)蛋白質(zhì)羰基化含量[39]。Parvez和Raisuddin研究發(fā)現(xiàn)農(nóng)藥脅迫顯著增加鯉魚(yú)(Channapunctata)各組織中蛋白質(zhì)羰基化含量[40]。本研究表明黃芪活性提取物處理組及混合處理組中脂質(zhì)過(guò)氧化及蛋白質(zhì)羰基化無(wú)顯著變化；黃連活性提取物處理組中DNA損傷、脂質(zhì)過(guò)氧化及蛋白質(zhì)羰基化均于6 d達(dá)到最大值，且與中草藥活性提取物濃度呈明顯的正相關(guān)(P<0.05)，各組織損傷程度大小為：肝胰腺>鰓；停藥3 d后，各處理組脂質(zhì)過(guò)氧化恢復(fù)正常值，而羰基含量保持穩(wěn)定，直到實(shí)驗(yàn)結(jié)束，這與上述結(jié)果基本類(lèi)似。黃連活性提取物(小檗堿0.5、1 g/kg)處理組對(duì)凡納濱對(duì)蝦組織損傷影響顯著，說(shuō)明黃連提取物在代謝過(guò)程中產(chǎn)生的活性氧含量超出凡納濱對(duì)蝦自身的抗氧化防御能力，因此造成氧化損傷。此外，黃芪、黃連活性提取物混合作用對(duì)凡納濱對(duì)蝦各組織損傷無(wú)顯著影響，說(shuō)明黃芪活性提取物可降低黃連活性提取物對(duì)凡納濱對(duì)蝦的組織損傷。各組織DNA損傷與中草藥活性提取物濃度顯著相關(guān)，表現(xiàn)出明顯的時(shí)間劑量效應(yīng)，可作為中草藥活性提取物對(duì)凡納濱對(duì)蝦安全性評(píng)價(jià)的指標(biāo)。

4 結(jié)語(yǔ)

黃芪、黃連活性提取物單獨(dú)及混合作用均可顯著提高凡納濱對(duì)蝦肝胰腺CYP450含量及GST活性，血淋巴和肝胰腺中T-AOC、SOD活性、GSH含量及GSH/GSSG；混合處理組各抗氧化指標(biāo)高于同濃度單獨(dú)處理組，各組織抗氧化水平大小為：血淋巴>肝胰腺；黃芪多糖0.5、1 g/kg及黃芪多糖0.5 g/kg+小檗堿0.5 g/kg對(duì)凡納濱對(duì)蝦各組織脂質(zhì)過(guò)氧化和蛋白質(zhì)羰基化無(wú)顯著影響，而小檗堿0.5、1 g/kg顯著造成凡納濱對(duì)蝦各組織損傷。各組織DNA損傷可作為中草藥活性提取物對(duì)凡納濱對(duì)蝦安全性評(píng)價(jià)的指標(biāo)。

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責(zé)任編輯 朱寶象

Effects ofAstragalusradixandCoptischinensisExtracts on Metabolic Enzymes Activities of White Shrimp (Litopenaeusvannamei)

GAO Guo-Rui, PAN Lu-Qing, HUANG Hui, SONG Meng-Si
(The Key Laboratory of Mariculture (Ocean University of China), Ministry of Education, Qingdao 266003, China)

The effects ofAstragalusradixandCoptischinensisextracts on metabolic enzymes, antioxidant defense and biomolecule damage of shrimp (Litopenaeusvannamei) were investigated. Six diets containingA.radixextract (Astragaluspolysaccharide) (0.5, 1 g/kg),C.chinensisextract (berberine) (0.5, 1 g/kg), the combination ofAstragaluspolysaccharide (0.5 g/kg) and berberine (0.5g/kg), and a control were used. Shrimps were fed with the extract containing diets for 6 days followed by a period feeding the common diet for 10 days. Results showed thatA.radixandC.chinensisextracts had significant effects on metabolic enzymes, antioxidant parameters and biomolecule damage ofL.vannamei(P<0.05). CYP450 content and GST activity in hepatopancreas of the experimental groups increased in 12 days, and then recovered to those of the control in 6 days of feeding the common diet. CYP450 content and GST activity in composite extracts group were higher than those in single extract groups.A.radixandC.chinensisextracts significantly improved the T-AOC, SOD activity, GSH content and the ratio of GSH to GSSG in hemolymph and hepatopancreas of shrimpL.vannameiin 6 days of feeding. These parameters could provide the scientific basis for the application of herb extracts in aquaculture. F values in single extract groups showed peak changes in 12 days, reached the minimum on day 6 and recovered to that of control after 6 days of feeding the common diet. No significant DNA damage was observed in composite extracts group. The MDA and PC contents in hepatopancreas and gill ofC.chinensisextract groups reached the maximum on day 6, and MDA content recovered to that of the control after 3 days of feeding the common diet. There was no significant MDA content and PC content inA.radixextract groups and composite extracts group. Our findings indicated that DNA damage in hepatopancreas and gill was significantly dose and time dependent, and could be used as a safety evaluation index of herbal extracts.

Astragalusradix;Coptischinensis;Litopenaeusvannamei; metabolic enzyme; antioxidant defense; biomolecule damage

海洋公益性行業(yè)科研專(zhuān)項(xiàng)經(jīng)費(fèi)項(xiàng)目(201305005)資助 Supported by the National Marine Public Industry Research Project(201305005)

2016-04-19；

2017-01-11

高國(guó)瑞(1988-)，男，博士生。E-mail:gaoguoruily@163.com

?? 通訊作者：E-mail：panlq@ouc.edu.cn

S96

A

1672-5174(2017)06-061-08

10.16441/j.cnki.hdxb.20160135

高國(guó)瑞，潘魯青，黃輝，等. 黃芪和黃連活性提取物對(duì)凡納濱對(duì)蝦代謝相關(guān)酶的影響研究[J]. 中國(guó)海洋大學(xué)學(xué)報(bào)(自然科學(xué)版), 2017, 47(6): 61-68.

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