高艷云,張園嬌,張倩,陳建偉*
(1.南京中醫(yī)藥大學(xué) 附屬醫(yī)院,江蘇 南京 210029;2.南京中醫(yī)藥大學(xué) 藥學(xué)院,江蘇 南京 210023)
·基礎(chǔ)研究·
高艷云1,2,張園嬌2,張倩2,陳建偉2*
(1.南京中醫(yī)藥大學(xué) 附屬醫(yī)院,江蘇 南京 210029;2.南京中醫(yī)藥大學(xué) 藥學(xué)院,江蘇 南京 210023)
1.1儀器
Waters2695高效液相色譜儀(Waters2489型PDA檢測(cè)器,Empower工作站);BT1250電子天平(賽多利斯科學(xué)儀器有限公司);KH-500DB型數(shù)控超聲波清洗器(昆山禾創(chuàng)超聲儀器有限公司);SAGA-10TQ易普易達(dá)超純水儀(南京易普易達(dá)科技發(fā)展有限公司)。
1.2試藥
中醫(yī)藥大學(xué)中藥資源學(xué)教研室陳建偉教授鑒定。
對(duì)照品:黃嘌呤(批號(hào):1001279304)、腺苷(批號(hào):101183448)、肌苷(批號(hào):1001271021)、次黃嘌呤(批號(hào):1001392976)、鳥苷(批號(hào):1001662525)、尿苷(批號(hào):1001290893)、胞苷(批號(hào):101069370)購于SIGMA公司。
甲醇(Fisher chemical,批號(hào):155175);磷酸(南京化學(xué)試劑有限公司,批號(hào):13112612030);水為去離子水。
2.1色譜條件
采用WatersXbridgeC18柱(250mm×4.6mm,5μm),以甲醇(A)-0.1%磷酸水溶液(B)為流動(dòng)相,梯度洗脫為0~10min,2%~5%甲醇;10~18min,5%~10%甲醇;18~25min,10%~25%甲醇;25~35min,25%~80%甲醇;流速為0.8mL·min-1,柱溫為30℃,檢測(cè)波長(zhǎng)為260nm,進(jìn)樣量為10μL。
2.2對(duì)照品和供試品溶液的制備
2.2.1混合對(duì)照品溶液制備 精密稱取次黃嘌呤、黃嘌呤、胞苷、腺苷、鳥苷、尿苷、肌苷的對(duì)照品適量,用15%甲醇水溶液配制成質(zhì)量濃度分別為79.84、402.00、120.12、201.80、80.48、80.00、160.00μg·mL-1的混合對(duì)照品溶液。
2.3方法學(xué)考察
2.3.2 線性關(guān)系考察 分別精密吸取混合對(duì)照品溶液0.1、0.5、1.0、1.5、2.0置10mL容量瓶中,加15%甲醇定容至刻度,離心后取10μL注入高效液相色譜儀,按2.1項(xiàng)下色譜條件進(jìn)行分析,以峰面積Y對(duì)進(jìn)樣量X進(jìn)行回歸計(jì)算,回歸方程見表1。
注:1.胞苷;2.次黃嘌呤;3.黃嘌呤;4.尿苷;5.腺苷;6.鳥苷;7.肌苷。圖1 混合對(duì)照品和雞供試品溶液的HPLC圖
對(duì)照品回歸方程線性范圍/μg·mL-1r次黃嘌呤Y=32807X-98354.99~79.840.9999黃嘌呤Y=5166X+5881025.13~402.000.9999胞苷Y=20825X-151137.51~120.120.9997尿苷Y=31414X-2631312.61~201.800.9998腺苷Y=40974X-153965.03~80.480.9998鳥苷Y=28428X-117055.00~80.000.9996肌苷Y=19616X-1586910.00~160.000.9997
2.3.3精密度試驗(yàn) 取2.2.1項(xiàng)下混合對(duì)照品溶液按2.1項(xiàng)下色譜條件分別連續(xù)進(jìn)樣測(cè)定6次,測(cè)定2種嘌呤堿基及5種核苷類化合物的峰面積并計(jì)算其RSD。結(jié)果顯示次黃嘌呤、黃嘌呤、胞苷、尿苷、腺苷、鳥苷、肌苷的RSD分別為0.6%、0.7%、0.5%、0.6%、0.6%、0.6%、0.6%,符合精密度要求。
并計(jì)算RSD。結(jié)果顯示次黃嘌呤、黃嘌呤、胞苷、尿苷、腺苷、鳥苷、肌苷的RSD分別為0.7%、2.1%、1.8%、1.3%、1.4%、2.1%、2.0%。表明供試品溶液在20 h內(nèi)穩(wěn)定性良好。
2.4樣品含量測(cè)定
表2 黃雞中2種嘌呤堿基及5種核苷的加樣回收率試驗(yàn)結(jié)果(n=6)
表3 3種雞中2種嘌呤堿基及5種核苷成分的含量 /mg·g-1
[1] 王鵬飛,何雋,周文,等.蟻巢傘屬真菌研究進(jìn)展[J].微生物通報(bào),2012,39(10):1487-1498.
[2] 王一心,狄勇,楊桂芝.雞樅菌在大鼠高膽固醇血癥中的抗氧化作用[J].中國(guó)預(yù)防醫(yī)學(xué)雜志,2005,6(1):10-12.
[3] 周繼平,許泓瑜.雞樅菌粉不同組分的體外抗氧化活性研究[J].中國(guó)野生植物資源,2008,27(5):46-49.
[4] 趙云霞,陶眀煊,陸文娟,等.雞樅菌多糖對(duì)小鼠急性酒精性肝損傷的保護(hù)作用[J].食品科學(xué),2014,35(19):260-265.
[5] 邢佳,陶明煊,郭宇星,等.雞樅菌精多糖對(duì)酒精性損傷小鼠腎及免疫器官的抗氧化作用[J].食品科學(xué),2014,35(9):246-249.
[6] 陸奕宇,敖宗華,成成,等.雞樅菌粉提取物鎮(zhèn)痛抗炎作用的研究[J].中成藥,2007,19(12):1742-1745.
[7] 張雪梅,楊豐慶,夏之寧.食品中核苷類成分的藥理作用研究進(jìn)展[J].食品科學(xué),2012,33(9):277-282.
[8] 陳東東,陳亞運(yùn),周萍,等.HPLC同時(shí)測(cè)定延胡索6個(gè)核苷成分含量[J].天然產(chǎn)物研究與開發(fā),2015,27(9):1571-1575.
[9] Ranogajec A,Beluhan S,Smit Z.Analysis of nucleosides and monophosphate nucleotides from mushrooms with reversed-phase HPLC[J].J Sep Sci,2010,33(8):1024-1033.
[10] 夏文娟,曾曉英,袁海龍,等.不同產(chǎn)地冬蟲夏草腺苷含量的測(cè)定[J].中國(guó)中藥雜志,2001,26(8):540-542.
[11] Bekar L,Libionka W,Tian G,et al.Maiken Nedergaard.Adenosine is crucial for deep brain stimulation-mediated attenuation of tremor[J].Nat Med,2008,14(1):75-80.
Determination of Nucleoside and Purine Bases in Three Kinds of Fruiting Body ofTermitomyces
GAO Yanyun1,2,Zhang Yuanjiao2,Zhang Qian2,CHEN Jianwei2*
(1.Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing,210029;2.School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing,210023)
Objective:To establish an HPLC method for the determination of nucleoside components in the fruiting body ofTermitomycesalbuminosus,T.eurrhizusandT.aurantiacus.Methods:The assay was performed on a Waters Xbridge C18(250 mm×4.6 mm,5 μm)column,mobile phase consisted of methanol-0.1% orthophosphoric acid water with gradient elution at the flow rate of 0.8 mL·min-1,column temperature was 30 ℃,and the detection wavelength was set at 260 nm.Results: The three kinds of fruiting body ofTermitomycesall checked out 2 kinds of purine base and 5 kinds of nucleoside,including hypoxanthine,xanthine,cytidine,adenosine,guanosine,uridine,inosine,and they showed good linearity(r≥0.999 6)in the range of 7.51-120.12,4.99-79.84,25.13-402.00,12.61-201.80,5.03-80.48,5.00-80.00,10.00-160.00 μg·mL-1,respectively.The average recoveries(n=6)were within 95.83%-103.01% with RSD≤3.98%.The contents of 2 kinds of purine base and 5 kinds of nucleoside components in the fruiting body ofT.albuminosuswere 0.36,3.13,2.20,3.63,1.11,1.87,1.50 mg·g-1,respectively,they in the fruiting body ofT.eurrhizuswere 0.10,0.78,1.86,3.07,1.48,1.70,0.62 mg·g-1respectively,and they in the fruiting body ofT.aurantiacuswere 0.22,2.53,1.86,2.35,0.78,0.84,0.83 mg·g-1,respectively.Conclusion: This HPLC method was simple,accurate and reducible,and can be used for quality evaluation and quality control ofTermitomyces.The three kinds of fruiting body ofTermitomyceswould have higher study value for medicine,nutrition and health food.
Termitomyces;nucleoside;purine base;HPLC;content determination
江蘇高校優(yōu)勢(shì)學(xué)科建設(shè)工程資助項(xiàng)目(PAPD);我國(guó)水生、耐鹽中藥資源的合理利用研究行業(yè)專項(xiàng)(201407002)
] 陳建偉,教授,研究方向:中藥品質(zhì)評(píng)價(jià)研究;Tel:(025)85811280,E-mail:chenjw695@126.com
10.13313/j.issn.1673-4890.2017.1.014
2016-04-14)
*[