喜树种子中喜树碱和10_羟基喜树碱高效液相色谱法分析_英文_

第25卷第3期2005年9月林　产　化　学　与　工　业Che m istry and I ndustry of Forest Pr oductsVol.25No.3Sep t.2005D ETER M I NAT I O N O F CA M PT O THEC I N AN D102H YDR O X YCA M PT O THEC I N I NCAM PT O TH ECA ACUM I NAT A D.SEE DS B Y HP LC3WANG Y WANG Yang1,Y AN Xiu2feng13,ZHANG Yu2hong2,Y U Tao1,MA M ei2fang1,W E I Huan2yong1(1.College of L ife Sciences,N ortheast Forestry U niversity,Harbin150040,China;2.Key L aboratory of Plant Forestry Ecology,N ortheast Forestry U niversity,Harbin150040,China)Abstract:A si m p le,rap id and sensitive method for the deter m inati on of contents of ca mp t othecin(CPT)and102hydr oxycamp t othecin(HCPT)in Cam ptotheca acum inata Decne.seeds by HP LC was established.The HP LC separati on was perfor med on a KY A H I Q sil C18colu mn(250mm×4.6mm,5μm)at fl ow rate1mL/m in,the s olvent gradient p r ogra m was as f oll ow:acet onitrile2water increased linearly fr om10%t o40% in the first15m in,then decreased linearly t o10%in3m in;at ti m e21m in,st opped the p r ogra m.The alkal oids were detected by a phot odi ode array detect or and quantitatively analyzed at254n m for CPT and266n m f or HCPT.CPT and HCPT were ultras onically extracted by60%alcohol at60℃for50m in.CPT and HCPT contents in different parts of C.acum inata D.seeds were deter m ined by the above analyzing method.The results showed that CPT and HCPT levels were different in the four parts of C.acum inata D.seeds.CPT content was the highest in endos per m,l ower in e mbryonic axis and cotyledon,and the l owest in seed coat,whereas HCPT content was the highest in endos per m,l ower in seed coat,and the l owest in e mbryonic axis and cotyledon.Key words:ca mp t othecin;102hydr oxycamp t othecin;Cam ptotheca acum inata Decne.seeds;ultras onic extracti onCLC nu m ber:T Q283;S792.31 D ocu m en t code:A Arti cle I D:0253-2417(2005)03-0063-04喜树种子中喜树碱和10-羟基喜树碱高效液相色谱法分析王洋1,阎秀峰1,张玉红2,于涛1,马梅芳1,魏焕勇1(1.东北林业大学生命科学学院,黑龙江哈尔滨150040;2.东北林业大学森林植物生态学教育部重点实验室,黑龙江哈尔滨150040)摘　要:建立了一种简便、快速、准确的测定喜树(Cam ptotheca acum inata Decne.)种子中喜树碱和10-羟基喜树碱含量的高效液相色谱分析方法;色谱条件为:采用日本KY A H I Q sil C18柱(250mm×4.6mm,5μm),流速为1mL/m in,梯度洗脱程序为:在前15m in流动相乙腈-水的体积比由10%线性增加至40%,在随后的3m in乙腈-水的体积比线性降至10%并保持恒定3m in,在21m in时停止该程序。

检测器为二极管阵列检测器,喜树碱定量分析波长254n m,10-羟基喜树碱定量分析波长266n m,进样量10μL。

样品制备以60%的乙醇作溶剂,在60℃下超声波提取喜树种子50m in。

利用以上方法分别测定了喜树种子胚乳、胚轴、子叶和种皮中的喜树碱和10-羟基喜树碱含量,喜树碱的含量是胚乳>胚轴,子叶>种皮,10-羟基喜树碱的含量是胚乳>种皮>胚轴>子叶。

关键词:喜树碱;10-羟基喜树碱;喜树种子;超声波提取Cam ptotheca acum inata Decne.(Nyssaceae)is a native tree of South China.Ca mp t othecin(CPT),an3Rece i ved da te:2004-07-08　Founda ti on ite m:国家自然科学基金资助项目(30070086)　B i ography:王洋(1971-),女,黑龙江哈尔滨人,副教授,博士,研究方向为植物次生代谢、植物化学3Correspondence Author:阎秀峰,教授,博士生导师,主要研究领域为植物次生代谢、植物生理生态学。

　林　产　化　学　与　工　业第25卷64indole alkal oid,was is olated fr o m the woody parts by W all,et al in1966[1].Because of its high anti2tu mor activity in the Leuka e m ia1210mouse leuke m ia assay,CPT attracted i m mediate attenti on as a potential che motherapeutic agent against cancer[2].The devel opment of CPT as an anti2tu mor drug was deterred due t o a variety of unaccep table side effects on hu man[3]until CPT′s unique mechanis m of acti on as an inhibit or of t opois omerase I was discovered by L iu,et al.in1985[4].102Hydr oxyca mp t othecin(HCPT),a natural derivative of CPT in C.acum ina ta D.,has been sho wn efficacy against lung,breast and uterine cervical cancers[5]thr ough the sa me mechanis m of inhibiti on of t opois omerase I.Among many CPT and HCPT anal ogues,which have been investigated over more than30years,irinotecan[6]and t opotecan[7]have been app r oved by United State Food and D rug Ad m inistrati on(F DA)f or app licati on against col on2rectum cancer and ovarian cancer.The deter m inati on methods of CPT[8-10]and HCPT[11]by HP LC have been reported separately,but the si m ultaneous deter m inati on of CPT and HCPT hasn′t been discussed s o far,and the op ti m al conditi ons f or the si m ultaneous extracti on of CPT and HCPT in C.acum ina ta D.t o p repare analytic sa mp le has not been referred.W hen ultras ound passes thr ough a p lant cell at sufficiently high intensities,cavities are f or med because of the alternating p ressure changes in the cell.These cavities gr ow in size and then collap se vi olently.U ltras onic extracti on is a kind of methods that can achieve analytical sa mp les in one2ti m e2extracti on thr ough keep ing p lant materials in extractant.It is a rap id,si m p le and accurate method t o p repare analytical sa mp les.I n this paper, the op ti m al extracti on f or the p reparati on of analysis sa mp les by HP LC was syste matically discussed.1　Materials and methods1.1　Pl an t ma ter i a ls and equ i p m en ts1.1.1　Plant materials　C.acum ina ta D.seeds were bought fr om J intang,Sichuan Pr ovince in January 2004.Seven2day seedlings were ger m inated fr om seeds t o study the p reparati on of the analytical sa mp le.To deter m ine the levels of CPT and HCPT in seeds,seeds were separated int o f our different parts,i.e. endos per m,e mbryonic axis,cotyledon,and seed coat.A ll sa mp les were dried at80℃for24h,and gr ound t o60mesh.1.1.2　Equi pments and reagents　W aters HP LC syste m with1525binary HP LC pu mp,2996phot odi ode array detect or and717p lus aut osa mp ler;B i ofuge22R high s peed centrifugal machine;B104electr onic balance;B2321ultras onic oscillat or;Multite mpⅢcycle water2bath.Acet onitrile is chr omat ographically pure; W ater is ultrapure water;others are all analytically pure.The CPT(purity99%)and HCPT(purity96%) standard sa mp les were kind gifts fr om The Stehlin Foundati on f or Cancer Research(Houst on,TX,US A).1.2　St andard soluti on and st andard curveD iss olved6mg CPT and6mg HCPT by acet onitrile in a100mL2measuring flask as mother s oluti on. Shifted1,2,3and4mL of mother s oluti on int o4different5mL2measuring flasks,res pectively,then added acet onitrile t o the scale and shook up,res pectively.Under the selective HP LC conditi ons,standard sa mp les were analyzed in tri p licate.The standard curves were perf or med bet w een concentrati on(mg/mL)of CPT/X1 HCPT/X2and peak area/Y.Y1=54715X1-16186,R1=0.9991Y2=37622X2-8895.8,R2=0.99901.3　Prepara ti on of ana lyti ca l s am plesT o study the op ti m al conditi ons f or the si m ultaneous extracti on of CPT and HCPT fr om C.acum inata D. seeds,experi m ents were p ref or med by3fact ors and11levels of unif or m design(Table1).The3fact ors were第3期王洋,等:喜树种子中喜树碱和10-羟基喜树碱高效液相色谱法分析65　concentrati on of alcohol,extracti on te mperature and extracti on ti m e .Accurately measured 1g seedling powder int o 25mL measuring flask,according t o the designed experi 2mental conditi ons,added 20mL of different concentrati on of aq .alcohol and ultras onically extracted at the corres ponding te mperature f or definite ti m e .Then cooled t o r oom te mperature,added aq .alcohol t o the scale,and shook up.One mL of supernatant was centrifuged at 12500g f or 10m in,and deter m ined by HP LC .2　Results and disscussi on2.1　HP LC cond iti on sColu mn:KY A H I Q sil C18colu mn (4.6mm ×250mm ,5μm;Japan );fl ow rate:1mL /m in;sa mp le injecti on:10μL;the s olvent gradient p r ogra m was as f oll ow:acet onitrile 2water of 10%increased linearly t o 40%in the first 15m in;then decreased linearly t o 10%in 3m in;at ti m e 21m in,st opped the p r ogra m (ready f or next injecti on ).Qualitative analysis:CPT and HCPT were detected by 2996phot odi ode array detect or at retenti on ti m e 18.8m in and 15.5m in,res pectively,and the peak purity was compared with the standard sa mp le thr ough the UV s pectru m.Quantitative analysis:wavelength 254nm f or CPT,266n m f or HCPT .2.2　O pti m a l cond iti on s for the s am ple prepara ti onTable 1　Exper i m en t cond iti on s and results of un i for m desi gnNo .extracti onte mp./℃X 1extracti onti m e /m inX 2alcohol concn ./%X 3yield 1)/(mg ・g -1)(dry weight )CPT Y 1HCPTY 211045600.9460.12021520300.3420.0413*******.4600.0744*******.4830.0455********.3620.03463530200.2630.028********.3950.0458455100.1090.01495015700.6490.0891*******.0750.013116035500.7420.095　1)Y 1=-0.4079021-0.0103298X 1+0.0157990X 2+0.0277084X 3+0.0003949X 21-0.0002646X 1X 2-0.0001646X 1X 3-0.0000835X 22-0.0001677X 23R =0.9909;Y 2=-0.0398635-0.0019719X 1+0.0029073X 2+0.0026236X 3+0.0000585X 21-0.0000619X 1X 2+0.0000037X 22-0.0000105X 2X 3-0.0000184X 23R =0.9998Table 2　O pti m a l exper i m en t a l cond iti on s of a lka lo i dssa mp les te mp./℃ti m e /m in alcohol concn ./%CPT 551065.4HCPT 605055.6op ti m al conditi on605060Experi m entresultsofunif or mdesign were dis p layed in Table 1.U sing extracti on te mperature (X 1),extracti on ti m e (X 2),and alcohol concentrati on (volu me rati o,sa me as in the f oll owing )(X 3)as independent variables,CPT (Y 1),and HCPT (Y 2)as dependent variables,the results were analyzed by stepwiseregressi onanalysis methodusing unifor m design s oft w are .Based on the regressi on equati ons,the op ti m al conditi ons f or the extracti on of CPT and HCPT were calculated res pectively within the scopes of selected ex peri m ental conditi ons (Table 2).Comp rehensively analyzed the condi 2ti ons,the op ti m al conditi ons for the si m ultaneous extracti on of CPT and HCPT were t o extract C.acum inatamaterials by 60%alcohol at 60℃for50m in undersupers onicextracti onmethod .Among the three affectingfact ors,the most significant fact or wasalcohol concentrati on,then extracti on te mperature,and the least was the extracti on ti m e .2.3　Prec isi on exper i m en tI njected a CPT and HCPT standard sa mp le for 5ti m es,res pectively,relative standard deviati on (RS D )of CPT was 3.32%,and that of HCPT was 4.04%.66　林　产　化　学　与　工　业第25卷2.4　Coeff i c i en t of recoveryMeasured 0.5g ×6sa mp le powder int o 25mL 2measuring flasks,res pectively,then 0.5mg of CPT standard sa mp le and 0.1mg of HCPT standard sa mp le were added t o each flask .Extracted and deter m ined according t o the above extracti on and analyzing conditi ons .The average recovery and RS D of CPT and HCPT were 95.23%,3.78%;and 93.56%,5.10%,res pectively .2.5　CPT and HCPT con ten ts i n d i fferen t parts of C.acum ina ta D.seedsTable 3　CPT and HCPT con ten ts i n d i fferen t parts ofC.acum ina taD.seedsmg/gparts CPT content (dry weight )HCPT content (dry weight )endos per m 2.5550.082e mbryonic axis 0.7410.021cotyledon 0.7450.010seed coat0.3600.048CPT and HCPT contents in different parts of C.acum inata D.seeds were deter m ined by the above analyzing method .Theresultsshowed that CPT and HCPT levels were different in the f our parts of C.acum ina ta D.seeds .CPT content wasthehighestinendos per m ,l ower in e mbryonic axis and cotyledon,and the l owest in seed coat,whereas HCPT content was the highest in endos per m ,l ower in seed coat,and the l owest in e mbryonic axis and cotyledon (Table 3).3　Conclusi on3.1　A rap id and si m p le HP LC analyzing method for the deter m inati on of contents of CPT and HCPT in C.acum inata D.seeds was established .3.2　U sing unif or m experi m ental design,the si m ultaneous extracti on of CPT and HCPT was studied,and the op ti m al conditi ons were t o extract C.acum inata D.materials by 60%alcohol (volume rati o )at 60℃f or 50m in under supers onic extracti on .3.3　CPT and HCPT levels were different in the f our parts of C.acum ina ta D.seeds .CPT content was the highest in endos per m ,l o wer in embryonic axis and cotyledon,and the l owest in seed coat,whereas HCPT content was the highest in endos per m ,l ower in seed coat,and the l owest in e mbryonic axis and cotyledon .References:[1]WALL M E,WAN IM C,COOK C E,et al .Plant antitumor agents .Ⅰ.The is olati on and structure of camp t othecin,a novel alkal oidalleukem ia and tumor inhibit or fr om Cam ptotheca 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