全 文 ：之 ,黏土最差 。栽培山兰田间以腐熟猪粪或鹿粪为
底肥对新产生球茎大小有显著影响 ,适宜底肥为猪
粪 。
3.2 　山兰以株距 7 cm,行距 10 cm,以猪粪为底
肥 ,在黑土栽培条件下 , 2年生园鲜品产量为 0.62
kg/1.2m2 ,实栽面积以 440m2计 ,折合亩产(666.7
m2)227 kg。
3.3 　山兰在栽培条件下尚未发现病害 ,如有金龟
子 、毛虫类害虫危害叶片和蒴果 ,用 1 000倍的菊马
乳油或 40%乐果乳化剂 1 500倍液可有效防治。
4 　讨论
4.1 　山兰为阴生草本植物 ,属于地生兰类型 ,在适
宜遮阴条件可进行大田栽培 ,当地农民 、林区职工可
利用林间空地栽培 ,开辟新的增收途径;山兰冬季不
落叶 ,春季开花优雅别致 ,在东北地区园林绿化中可
作为新的花卉品种。
4.2　山兰有有性繁殖和分生繁殖两种方式 ,山兰
一个球茎每年只产生 1 ～ 2个新生球茎 ,繁殖系数
低 ,严重制约山兰扩大生产;山兰一个花序有 20 ～
40朵花 ,每朵花发育成一个蒴果 ,一个蒴果内多达
2 000 ～ 2 500粒种子 ,因此种子繁殖是扩大生产用
苗的有效途径 ,以后应加大这一技术体系的研究 。
4.3　山兰治病的物质基础尚不清楚 ,需要对其化
学成分进行分离和鉴定 ,系统开展药理作用研究 。
参 考 文 献
[ 1] 中国药材公司.中国中药资源志要 [ M] .北京:科学出
版社 , 1994:1553.
[ 2] 周繇 , 朱俊义 ,于俊林 .中国长白山观赏植物彩色图志
[ M] .长春:吉林教育出版社 , 2005:407.
[ 3] 中国科学院北京植物研究所 .中国高等植物图鉴 [ M] .
北京:科学出版社 , 1976:741.
·资源 ·
TaxolContentComparisoninDiferentPartsofTaxusmadia
andTaxuschinensisvar.maireibyHPLC
DONGQuan-feng, LIUJing-jing, YURong-min＊
(ColegeofPharmacy, JinanUniversity, Guangzhou510632, China)
Abstract　Objective:ToanalysisandcomparethetaxolcontentindiferentpartsofTaxusmadiaandTaxuschinensisvar.mairei
byHighPerformanceLiquidChromatography(HPLC).Methods:85% EtOHandCH2Cl2 wereusedfortheextractionoftaxol.By
HPLC, themethodologystudyandtaxolcontentinvestigationwereperformed.Results:Thetaxolwasextractedsuccessfuly.Onesim-
pleandreliablemethodologywasbuiltup.Basingonthese, thetaxolcontentinthesetwoTaxusspp.wereanalysedandcompared, a-
mongofwhichtheleafofTaxuschinensisvar.maireihasthehighesttaxolcontent(5.18×10-5 , w/w).Conclusion:Taxolanditscon-
tentintheoriginalplantscanbesimplyandreliablyextractedandinvestigatedbythesemethods, whichalsocanprovidedthescientific
basisfortherationaldevelopmentofTaxusspp.
Keywords　Taxol;TaxusmadiaCV.Hicksi;Taxuschinensis(Pilger)Rehd.var.mairei(LemeeetLevl.)ChengctL.K.Fu;
HPLC;Contentcomparison
CLCnumber:R284.1　　Documentcode:A　　ArticleID:1001-4454(2010)07-1048-04
HPLC测定比较曼地亚红豆杉和南方红豆杉不同部位的紫杉醇含量
董权锋 ,刘晶晶 ,于荣敏
(暨南大学药学院 ,广东 广州 510632)
　　摘要　目的:分析和比较曼地亚红豆杉和南方红豆杉不同部位的抗癌活性成分紫杉醇的含量。 方法:利用
85%乙醇提取和 CH2Cl2萃取得到相关成分;通过高效液相色谱法进行了方法学研究并对紫杉醇含量进行了测定
和比较。结果:得到了紫杉醇等相关成分 , 建立了一个简单而可靠的方法学研究 , 以此为基础 , 检测分析并比较了
两种植物不同部位中紫杉醇的含量 ,其中以南方红豆杉的树叶中紫杉醇含量最高 , 达到了 5.18 ×10-5(w/w)。结
论:该方法能准确简便地提取红豆杉植物中的紫杉醇并分析其含量 , 可为合理开发红豆杉提供一定的科学依据。
关键词　紫杉醇;曼地亚红豆杉;南方红豆杉;高效液相色谱法;含量比较
收稿日期:2009-08-20＊通讯作者:于荣敏 , Tel:020-85220386, E-mail:tyrm@jnu.edu.cn。
·1048· JournalofChineseMedicinalMaterials　　第 33卷第 7期 2010年 7月
　　Taxolhasbeentheanticancerdrugwithbestac-
tivitiesaroundtheworld.Sofar, themainsourceof
taxolisthenaturalTaxusspp.Forthereasonofthelow
contentoftaxolinthebark, thepathwayofextracting
taxolfromthebarkhascausedacrisisforthenatural
Taxus〔1-3〕.Therefore, basingonthepreviousstudies
anddeterminingthetaxolcontentinotherpartsofTax-
usspp., authorobtainedasubstitutablemethodforex-
tractingtaxol, whichhasthegreatsignificanceforthe
sustainableprotectionofTaxusspp.
　　Taxuschinensisvar.mairei, oneofthevarietiesof
taxuswithfastgrowth, isuniqueinChinaandwidely
spreadinthesouthernandsoutheasternregions〔1, 4〕.
Taxusmadiaisanaturalhybridvarietywiththefemale
parentofTaxuscuspidataandmaleparentofTaxus
bauata.Becauseofthefastgrowth, highutilization,
hugebiomass, strongadaptabilityandstablebiological
characteristics, Taxusmadiahasbeentheprefered
varietyforextractingtaxolapprovedbyFDA.
　　Sofar, thereweremanypapersorreferencesre-
portedtheextractionandcontentdeterminationofTax-
usmadiaorTaxuschinensisvar.maireirespective-
ly〔1-8〕.Inthepreviouspaper〔8〕 , thetaxolcontentin
theleafandbarkofnaturalTaxusmadiaandTaxus
chinensisvar.maireiandtheirtissuecultureswere
triedtobeinvestigated, howeverthetaxolcontentof
naturalTaxuschinensisvar.maireicouldn′tbetested
bytheinvestigatingpathway.
　　Inauthor′sstudy, comparedwiththeprevious
work, authorsuccessfulydeterminedandfurthercom-
paredthetaxolcontentintheleafandbranchofnatural
TaxusmadiaandTaxuschinensisvar.maireiby
HPLC, fromwhichtheresultswilprovidethescientif-
icbasisforindustrialextractionandproductionoftaxol.
1　Materials
1.1　Apparatusandreagents　FA1004 electronic
balance(ShanghaiPrecision＆ ScientificInstrument
Co., Ltd.), DHG-9123Aelectricheatingair-blowing
drier(ShanghaiJinghongLaboratoryInstrumentCo.,
Ltd.), EYELArotaryevaporator(ShanghaiAilangIn-
strumentsCo., Ltd.), Agilent1200 SeriesHPLCIn-
strument, chromatographiccolum:Phenomenex-C18
(250mm×4.6 mm, 5μm)(GuangzhouFLMScientif-
icInstrumentCo., Ltd.), chromatographymethanol,
analysispurityofanhydrousethanol, petroleumether
anddichloromethane.
1.2　Materials　TaxolprovidedbyDr.YANChun-
yan, GuangdongPharmaceuticalUniversity, Taxusma-
diaandTaxuschinensisvar.maireiprovidedbyEngi-
neerQIUChun-ling, ZiboPusonglingMedicinalGar-
den, Shandong, China.
2　Methods
2.1　Testingsolutionpreparation　Certainamount
ofthesamplepowderdriedunder50 ℃ wasprecisely
weighedandextractedby100 mLof85% EtOHfor24
hina250 mLconicalflask.Thesamplesolutionwas
filtrated.Theresiduewasextractedby100mLof85%
EtOHinsuccessionwithultrasonicfor5 min.Thefil-
trateswerecolectedandconcentratedundervacuum
with50℃waterbathtogettheextrac.Afewof40℃
waterwasaddedandsuspendedwiththeextractto50
mL.Theisovolumetricpetroleumetherwasappliedto
extractthesuspendedsolutionfor5 timesandthepe-
troleumetherextractwasremoval.Insuccession, the
suspendedsolutionwasextractedbyisovolumetric
CH2Cl2 for5 times.TheCH2Cl2 extractwascolected
anddriedundervacuumwith50 ℃ waterbath.The
driedextractwasdissolvedwithchromatographyMeOH
to25 mL, andfiltratedby0.45μmfilterpaper.
2.2　Referencesolutionspreparation　8.0 mgof
taxolwaspreciselyweighedanddissolvedina25 mL
volumetricflaskwithchromatographyMeOHtomakea
standardsolutionwithconcentrationof320 μg/mL.
0.5, 1.0, 2.0 , 3.0, 4.0, 6.0, 8.0mLofthestand-
ardsolutionwasrespectivelyaddedintoa25mLvolu-
metricflaskanddilutedwithchromatographyMeOHto
makeaseriesofreferencesolutionswithconcentrations
of6.4, 12.8, 25.6, 38.4 , 51.2, 76.8 and102.4
μg/mL.
2.3　Chromatographicconditions　Chromatograph-
icColum:Phenomenex-C18 (250 mm ×4.6 mm,
5μm), flow rate: 0.8 mL/min, mobile phase:
MeOH-water(gradientelution:0 ～ 10 min, 65%
methanol;13 ～ 25min, 67% methanol;26 ～ 75 min,
100% methanol;76 ～ 80 min, 65% methanol), wave
length:227nm, columntemperature:30℃, injection
volume:20 μL.
2.4　Contentdetermination　Althesampleswere
determinedunderthe“ 2.3” chromatographiccondi-
tions, andthecontentoftaxolwouldbecalculatedby
·1049·JournalofChineseMedicinalMaterials　　第 33卷第 7期 2010年 7月
themethodofexternalstandardcalibrationofpeak
area.
2.5　Methodologicalstudy
2.5.1　Linearrelationship:underthe“2.3” chrom-
atographicconditions, aseriesofreferencesolutions
withdiferentconcentrationsweredeterminedtoobtain
alinearequation.
2.5.2　Precisionstudy:Astandardsamplesolution
withcertainconcentrationwasanalyzedfor5timesun-
derthe“2.3” chromatographicconditions.Therela-
tivestandarddeviation(RSD)ofpeakareaswouldbe
calculatedandanalyzed.
2.5.3　Stabilitystudy:10.0128gofleavesofTaxus
madiawasweighedpreciselyandpreparedforthetes-
tingsolutionundertheabovepreparationmethods.The
testingsolutionwasanalyzedfor5 timesunderthea-
bovechromatographicconditionswithintervalof2 h.
RSDofpeakareaswouldbecalculatedandanalyzed.
2.5.4　Repeatabilitystudy:5 portionsofleavesof
Taxusmadiawereweighedpreciselyandpreparedfor
thetestingsolutionsunderthe“2.1” preparationmeth-
ods.Thetestingsolutionswereanalyzedunderthea-
bovechromatographicconditions.RSDofpeakareas
wouldbecalculatedandanalyzed.
2.5.5　Samplerecoverystudy:5portionsofleavesof
Taxusmadiawereweighedprecisely.1mLofstandard
samplesolutionwithconcentrationof76.8μg/mLwas
respectivelyaddedintoeverysample.Then, theywere
preparedforthetestingsolutionsunderthe“ 2.1 ”
preparationmethods.Thetestingsolutionswereana-
lyzedunderthe“ 2.3 ” chromatographicconditions.
RSDofpeakareasforthemwouldbecalculatedand
analyzed.
2.6　Contentdetermination　10.0088 gofbran-
chesofTaxusmadia, 10.0165 gofleavesofTaxus
chinensisvar.maireiand10.0042 gofbranchesof
Taxuschinensisvar.maireiwerepreciselyweighedand
preparedforthetestingsolutionsundertheaboveprep-
arationmethods.Thetestingsolutionswereanalyzed3
timesrespectivelyundertheabovechromatographic
conditions, andgottheaveragevalues.
3　ResultsandDiscussion
3.1　Linearrelationship　Thelinearequation, Are-
a=35.228Amt+6.589(r=0.99993)(Amt:μg/
mL), wouldbegotenbytheanalysisofreferenceso-
lutionsunderthe“ 2.3” chromatographicconditions.
Theequationindicatedthatthelinearrelationshipbe-
tweenconcentrationoftaxolanditspeakareawasgood
intherangeof6.4 ～ 102.4μg/mL.
3.2　Precisionstudy　TheRSDof5 peakareaswas
0.23%, whichindicatedthegoodprecisionofdeter-
minedmethods.
3.3　Stabilitystudy　TheRSDof5 peakareaswas
0.65%, whichindicatedthatthedeterminationprocess
wasstablefor8h.
3.4　Repeatabilitystudy　TheRSDof5 peakare-
as, corespondingtothesamplesandcontent, which
indicatedthegoodprecisionofthemethodology, RSD
=0.61%.
3.5　Samplerecoverystudy　TheRSDofpeakare-
asforthemwasshowninTable1, whichindicatedthe
goodreliabilityofthemethodology.Triplicateexperi-
mentshavebeendone.
3.6　Contentdetermination　Forthetaxolcontent
ofeverysample, thedeterminedresultswereshownin
Table2.Theanalyzedresultsindicatedthatthecon-
tentoftaxolintheleafwashigherthanthatinthe
branch, andthehighestcontentoftaxolwasintheleaf
ofTaxuschinensisvar.mairei, whichwas5.18×10-5
(w/w).
　Table1　 Resultsofsamplerecoverystudy
Samples/g ContentofTaxol/mg AddedTaxol/μg
Theoreticalconc.ofTaxol/(μg/mL)
Practicalconc.ofTaxol/(μg/mL)
Recovery/%
Averagerecovery/% RSD/%
10.0026 0.3831 76.8 92.124 93.283 101.26
10.0019 0.3831 76.8 92.124 94.102 102.15
10.0025 0.3831 76.8 92.124 93.102 101.11
10.0040 0.3832 76.8 92.128 91.224 99.02
10.0103 0.3834 76.8 92.136 92.536 100.43
100.79 1.16
·1050· JournalofChineseMedicinalMaterials　　第 33卷第 7期 2010年 7月
　Table2　　Resultsofcontentdetermination(n=3)
VarietiesofTaxus Contentoftaxol(w/w)×10
-5
Leaf Branch
Taxuschinensisvar.mairei 5.18 1.37
Taxusmadia 3.85 3.42
4　Conclusion
　　ThecrudeextractfromTaxusspp.usualycon-
tainssomesubstanceswithlowpolarity, suchaschlo-
rophyl, sterol, resin, andsoon, whichcanbere-
movedbyorganicreagentswithlowpolarity〔9〕 .For
thiscase, authorusedpetroleumethertoremovethem.
Ontheotherhand, sincethecrudeextractalsocon-
tainssomethingwithlargepolarity, suchastannin, a-
minoacid, salt, andsoon, authoruseddichlorometh-
anetoobtaintaxol.Inordertoavoidemulsification
duringtheextractionofcrudeextract, authorchosethe
leavesofTaxusmadiaandthesamplewasextractedby
85% EtOHconsultingwiththerelatedreference〔5〕.
Meanwhile, authorprocessedthesamplebyultrasonic
extractionbecausethemethodcanreducetheextracted
period, improvetheeficiencyandavoidthetransform
oftaxolintoothersubstances〔9〕.
　　Fortheinfluencescausedbymobilephase, col-
umntemperatureanddetectionwavelength, basingon
thereferences〔2, 4, 5〕 andourpractices, authormade
surethatthewavelengthwas227 nm, thecolumntem-
peraturewas30 ℃, theflowratewas0.8 min/mL,
andthemobilephaseshouldbeMeOH-waterwithgra-
dientelution(0 ～ 10 min, 65% methanol;13 ～ 25
min, 67% methanol;26 ～ 75 min, 100% methanol;
76 ～ 80min, 65%methanol).Inthisway, authorcan
reducetheanalysisperiodandobtainthegoodsepara-
tionoftaxol, whichwouldbeconvenientfortheexperi-
men.
　　Foraword, authorstudywilprovidesomescien-
tifictheoriesonthemedicinalvalueresearchofTaxus
madiaandTaxuschinensisvar.maireiandindustrial
productionoftaxol.
Acknowledgments:ThankQIUChun-lingandDr.YANChun-yan
somuchforfriendlyprovidingTaxusmadia, Taxuschinensisvar.marrei
andtaxolstandardsample.
References
[ 1] WangCW, TongC, LiWJ, etal.Efectsofshadingon
Taxuschinensisvar.maireigrowthanditstaxolcontent
[ J] .ChineseJournalofEcology, 2008, 27(8):1269-
1273.
[ 2] WangZG, ZhouCS, YanJL, etal.Determinationof
paclitaxelcontentinTaxusmadiabyHPLC[ J] .Applied
ChemicalIndustry, 2007, 36(1):84-86.
[ 3] ZhangDY.Studyonthetaxolcontentindifferentpartsof
TaxusgrowinginChina[ J] .JournalofFujianCollegeof
Forestry, 2003, 23(2):160-163.
[ 4] ZhouXL, PengQC.DeterminationoftaxolinTaxusby
HPLC[ J] .LishizhenMedicineandMateriaMedicaRe-
search, 2008, 19(6):1434-1436.
[ 5] LiuXQ, ZhangXD, ZhuYL, etal.Structureidentifica-
tionofbiflavonesanddeterminationoftaxolfromTaxus
madia[ J] .JournalofChineseMedicinalMaterials, 2008,
31(10):1498-1501.
[ 6] ZengZP, WangSQ.HPLCdeterminationofpaclitaxelin
Taxuschinensisvar.Maireiwithdiferentgrowthyears
[ J] .CentralSouthPharmacy, 2008, 6(1):56-57.
[ 7] LiXL, NanYL, ZhangYQ, etal.Determinationoftax-
olinCanadianyewbyHPLC[ J] .ChineseJournalofEx-
perimentalTraditionalMedicalFormulae, 2006, 12(3):
9-10.
[ 8] DuYT, XuJY, BuXY, etal.Thedeterminationoftax-
olcontentinthenaturalmaterialsandthetissueculturesof
TaxusbyHPLC[ J] .JournalofChemicalIndustryofFor-
estProducts, 2005, 39(1):17-20.
[ 9] ZhaoGH, ZhangPZ.Studyonextractionandpurification
technologyoftaxol[ J] .FoodandFermentationIndustries,
2008, 34(5):138-142.
《中药材 》杂志为国内外权威的中药科技学术期刊 ,欢迎投稿 !
·1051·JournalofChineseMedicinalMaterials　　第 33卷第 7期 2010年 7月