全 文 ：第 31卷第 3期
2011年 6月
林　产　化　学　与　工　业
ChemistryandIndustryofForestProducts
Vol.31 No.3
June2011
OptimizationofUltrasound-assistedExtraction
ofCarnosicAcidfromRosemaryLeaves
　　收稿日期:2010-12-16
　　基金项目:国家林业局 948技术引进项目(2011-4-01);科技部农业科技成果转化资金项目(2008GB24320416)
　　作者简介:李大伟(1985-),男 ,山西怀仁人 ,硕士生 ,从事天然产物化学与利用研究;E-mail:lidawei119@ 126.com
　＊通讯作者:毕良武 ,研究员 ,博士 ,硕士生导师 ,从事天然产物化学与利用研究;E-mail:biliangwu@ 126.com。
LIDa-wei　　
LIDa-wei, BILiang-wu＊ , ZHAOZhen-dong, LIDong-mei, LIUXian-zhang
(InstituteofChemicalIndustryofForestProducts, CAF;NationalEngineeringLab.forBiomass
ChemicalUtilization;KeyandOpenLab.onForestChemicalEngineering, SFA;KeyLab.of
BiomassEnergyandMaterial, JiangsuProvince, Nanjing210042, China)
Abstract:Carnosicacid, thekeybioactivecomponent, wasextractedfromleavesofrosemary(Rosmari-
nusofficinalisL.)usingultrasound-assistedextraction.Thediferentfactorssuchasethanolconcentra-
tion, varietyofacidstabilizer, acidstabilizerdosage, ultrasonicfrequency, ultrasonicpower, ultrasonicextractiontimeandthe
solid-liquidratiowerestudied.Thecontentvariationsofcarnosol, rosmarinicacid, oleanolicacidandursolicacidwerealsoex-
amined.Theexperimentalresultsfor5.0 grosemaryleavesshowedthattheoptimumultrasonicextractionconditionsatroomtem-
peraturewere:ethanolsolutionconcentration75 %, acidstabilizer3 ghydrochloricacid, ultrasonicfrequency28kHz, ultrason-
icpower200W, ultrasonicextractiontime40 min, solid-liquidratio(g∶g)1∶16.Theextractionrateofcarnosicacidreached
18.64mg/gundertheaboveextractionconditions.
Keywords:carnosicacid;rosemary(RosmarinusoficinalisL.);ultrasound-assistedextraction
CLCnumber:TQ351.0　　　　　Documentcode:A　　　　　ArticleID:0253-2417(2011)03-0060-05
迷迭香叶中鼠尾草酸超声波辅助提取的优化
李大伟 , 毕良武 , 赵振东 , 李冬梅 , 刘先章
(中国林业科学研究院林产化学工业研究所;生物质化学利用国家工程实验室;
国家林业局林产化学工程重点开放性实验室;江苏省生物质能源与
材料重点实验室 , 江苏 南京 210042)
摘　要:利用超声波辅助提取法提取迷迭香中的主要生物活性成分鼠尾草酸 ,分别研究了不同条件对鼠尾草酸提取率的
影响。对 5.0g粉碎后的迷迭香叶子进行超声波辅助提取的结果表明 , 室温较适宜的超声波提取条件是:乙醇质量分数
75%、 3g盐酸为酸稳定剂 、超声波频率 28kHz、超声波功率 200W、超声波提取时间 40min、料液比(g∶g)1∶16。在上述
提取条件下 , 鼠尾草酸的得率可达 18.64mg/g。
关键词:鼠尾草酸;迷迭香;超声波辅助提取
Carnosicacidisakindofnaturalantioxidants, whichcanbewidelyusedinmedicine, cosmetics, cream
ofvariousanimalfats, animalfeed, meatandseafood, flour, sauces, seasoning, bakingfood, friedproducts,
naturalpigment, essence, biologicalpesticideandtobacco, etc.[ 1-2] .Especialyitcanbeappliedinthe
treatmentofbloodmalignanttumor, cardiovasculardisease, heartandrespiratorydiseases, inhibitingHIV
infection, inflammation, anti-bacterialtreatment, sorethroat, indigestion, preventingAlzheimer′sdisease,
diabetes, promotingtheformationofnervegrowthfactor, etc.[ 3-4] .Nowadaysmanymethodshavebeenused
inthecarnosicacidextraction, suchasthetraditionalorganicsolventextraction, supercriticalfluidCO2
extractionmethod, homogenateextraction, inorganicalkaliextractionandoverheatwater-jetsubcritical
第 3期 李大伟 ,等:迷迭香叶中鼠尾草酸超声波辅助提取的优化 61　
extraction[ 5-10] .Ultrasound-assistedextractionisoneoftheintensivemethodsforsolventextraction.The
ultrasonicwavegeneratesstrongcavitationandmixingefectwhichcancauseplantcelwalbreakingofand
rapidinfiltrationofsolventtodisolvethetargetantioxidantsquickly, improveextractionyield, shortenextraction
time, decreaseextractiontemperatureandsolventconsumption[ 11-14] .Rosemaryleavescontainseveral
bioactivecompoundsincludingcarnosicacid, carnosol, rosmarinicacid, oleanolicacid, ursolicacid, etc..
Theinfluencefactorssuchasethanolconcentration, varietyofacidstabilizer, acidstabilizerdosage, ultrasonic
frequency, ultrasonicpower, ultrasonicextractiontimeandthesolid-liquidratioforextractionofcarnosicacid
togetherwithcarnosol, rosmarinicacid, oleanolicacidandursolicacidfromrosemaryleaveswerestudied.
1　Experimental
1.1　Materialsandmethods
Rosemaryleaveswereobtainedinbulkinadriedform(containingabout10%moisturebymass)froma
randomsampleofthegroundleaves, methanolHPLC-grade;ethanolanalysis-grade;aceticacid, hydrochloric
acid(36%-38%), filterpapers, purewater;KQ-200VDBatthreefrequencycontrolultrasoniccleaner,
KunshanUltrasonicInstrumentsCo., Ltd.;LC-20ATtypeHPLC, SHIMADZU;SHB-Ⅲ Circulatingwater
vacuumpump, HenanTaikangScienceInstrumentFactory;Low-speeddesktopcentrifuge, HunanSaitexiang
InstrumentFactory.
1.2　HPLCanalysis
HPLCanalysiswasperformedonareversedphaseC18HypersilODScolumn(150mm×46mm, 5μm).
TwentyμLsampleswereinjected.Thecolumntemperaturewas40℃.Themobilephasewasprogrammed
withalineargradientfrom90% A(840mLofpurifiedwaterwith8.5mLofaceticacidand150mLof
acetonitrile)and10% B(methanol), to100% Bin30min, remained5min, from100% Bto10%Bin
4min, remained3min, withaflowrateof1.5mL/min.Thesystemwaslefttostabilizefor15minbetween
consecutiveinjections.ThesamplesweredetectedbyPDADat284nm.Duplicateanalyseswereinitialyrun
toestablishreproducibility.Underaboveconditions, theretentiontimeofcarnosicacidwas25.6min, andthe
retentiontimeofrosmarinicacid, ursolicacid, oleanolicacidandcarnosolwere7.5, 29.8, 29.3, 21.9min,
respectively[ 15] .
Carnosicacidstandardsamples(2.6mg)(accordingtotheareanormalization, theaverageproportionof
peakareawas90%)wereaddedinto1.3mLethanol(100%)toconstituteA, 2g/Lofcarnosicacidin
ethanolsolution.2, 3 , 4, 5, 10, 15, 20, 25 , 30 and35μLofmaterAareinjected, respectivelytodetect
theconditionofchromatographicanalysis.Resultsshowedthatthequantityofcarnosicacidat4-70μgshowed
goodlinearrelationship.Thecorelationwas:Y=193 425.9X+120 949.4, R2 =0.999 4.
Withsimilarmethod, thestandardcurvesforcarnosol(6%carnosolinthecarnosicacidstandardsample
ascarnosolstandardsample), rosmarinicacid(accordingtotheareanormalization, theaverageproportionof
peakareawas90%), oleanolicacid(accordingtotheareanormalization, undertheabsorptionwavelengthof
oleanolicacid220nm, theaverageproportionofpeakareawas86%), ursolicacid(accordingtothearea
normalization, undertheabsorptionwavelengthofursolicacid220nm, theaverageproportionofpeakarea
was78%)wereobtained.Thequantityofcarnosolat6-53μgshowedgoodlinearrelationshipandthe
correlationwas:Y=13 946.69X-9 902.924, R2 =0.999.Thequantityofrosmarinicacidat600-3 600μg
showedgoodlinearrelationshipandthecorelationcoeficientwas:Y=29 004.68X+80 497.89, R2 =0.994 7.
Thequantityofoleanolicacidat1-7μgshowedgoodlinearrelationshipandthecorelationcoeficientwas:
Y=1 027 184.38X+204 604.5, R2 =0.996 5.Thequantityofursolicacidat2-28μgshowedgoodlinear
relationshipandthecorelationcoeficientwas:Y=2 529 707.50X-5 482 132, R2 =0.999 1.
62　 林　产　化　学　与　工　业 第 31卷
1.3　Theassaysfortheextractioncontent
Theextractionsolutionwasfilteredby3 piecesoffilterpaper.Thevolumeoffiltratewasmeasured.Then
thefiltratewascentrifugatedatroomtemperatureand4 500r/minfor6min.Takesupernatantofeachsample
byHPLCforthreeparaleltests.Theaveragedpeakareawassubstitutedintotheregressionequationtocalcu-
latethecontentofcarnosicacidandothercomponents.
Theformulasforcalculatingthecomponentsofcarnosicacidamount(M1), carnosolamount(M2), rosma-
rinicacidamount(M3), ursolicacidamount(M4)andoleanolicacidamount(M5)areasfolows.
M1 =(Y-120 949.4)÷193 425.9 ÷20×0.9×V
M2 =(Y+9 902.924)÷13 946.69 ÷20×0.06×V
M3 =(Y-80 497.89)÷29 004.68 ÷20×0.9×V÷1 000
M4 =(Y+5 482 132)÷2 529 707.5 ÷20×0.78×V
M5 =(Y-204 604.5)÷1 027 184.38 ÷1.2×0.86×V
Where:Y—peakarea;V—extractionsolutionvolume.
1.4　Theselectionofultrasonicextractionconditions
Thegroundrosemaryleaves(5.0g)wereaddedintoaqueousethanolsolution.Therosemaryleaveswere
extractedbyethanolunderultrasonicconditions.Afterfiltrationandcentrifugation, supernatantofeachsample
wastakentodoHPLCforthreeparaleltests.Theaveragedpeakareawassubstitutedintotheregresionequa-
tiontocalculatethecontentofcarnosicacidandothercomponents[ 8] .
Thesingle-factorextractionexperimentwasdone, aswel.Thediferentfactorssuchasethanolconcen-
tration(40%, 50%, 60%, 70%, 80%), varietyofacidstabilizer(aceticacidandhydrochloricacid),
acidstabilizerdosage(0.1, 0.5, 1, 2, 3, 4, 6g), ultrasonicfrequency(28, 45, 100kHz), ultrasonicpow-
er(120, 140, 160, 180 , 200W), ultrasonicextractiontime(20, 30, 40, 50 , 60min)andthesolid-liquid
ratio(g∶g)(1∶6, 1∶8, 1∶10 , 1∶12, 1∶14, 1∶16 , 1∶18, 1∶20)werestudied.
Fortheorthogonaltestofextraction, theL18(36)orthogonaldesignandtest(ethanolconcentration, acid
stabilizerdosage, ultrasonicfrequency, ultrasonicpower, ultrasonicextractiontime, thesolid-liquidratio)
weregiven.5.0 gofrosemarysamplewasusedineachtest.
2　ResultsandDiscussions
2.1　Optimizationofultrasound-assistedextraction
Ultrasonicextractionofcarnosicacidfromrosemaryleaveshastheadvantagesofrelativelyhigherextrac-
tionrate, shorterextractiontime, lowertemperature.Theextractionprocessissimpleandadaptable[ 16-17] .
2.1.1　Thesingle-factorextractionexperiment　Itwasshownthatwithethanolconcentrationincreasing, the
extractionamountofcarnosicacidreachedthemaximumatethanolconcentrationof60%, whiletheamountof
ursolicacidwastheleast.Whenadding1gofhydrochloricacid, theextractionamountofcarnosicacid
reachedthemaximum(whileotherfourcomponentswerenotinhigherlevels).Theamountofcarnosicacidat
28kHzwaslarger, whiletheamountsofothercomponentswerelower.Withultrasonicextractiontimeincreas-
ing, theamountofcarnosicacidfirstincreasedandthendecreased.Whentheultrasonicextractiontime
reached50minoreven60min, theultrasonicbathtemperatureroseto50 even60℃.Whentheextraction
timewas40min, theultrasonictemperaturewasabout40℃ whichproducedthehighestamountofextraction.
Theamountofcarnosicacidreachedthebiggestwhentheratio(g∶g)reached1∶18.Theamountofcarnosic
acidatultrasonicpower120Wwasrelativelyhigherwhiletheamountofoleaonlicacidwaslower.Thesingle-
factorextractionexperimentalresultsshowedthattheoptimumultrasonicextractionconditionsatroomtempera-
turewere:ethanolsolutionconcentration60%, acidstabilizer1ghydrochloricacid, ultrasonicfrequency
第 3期 李大伟 ,等:迷迭香叶中鼠尾草酸超声波辅助提取的优化 63　
28kHz, ultrasonicpower120W, ultrasonicextractiontime40min, solid-liquidratio(g∶g)1∶18.
2.1.2　Theorthogonaltestofextraction　TheL18 (36)orthogonaldesignandtestresultsweregivenin
Table1.Eachtestused5.0gofrosemarysample.ItwasshownthattheimpactfactorsonextractionwereA>
E>B>F>C>D.Ethanolconcentrationwasthefirstfactorinfluencingtheextractionofcarnosicacid.The
optimumcombinationwasA3B3C3D1E3F2 , i.e.ethanolconcentration75 %, hydrochloricacid3g, solid-
liquidratio(g∶g)1∶16, ultrasonicfrequency28kHz, ultrasonicpower200W, ultrasonicextractiontime
40min.Throughrepeatedexperimentsfor3timesundertheoptimumconditions, theaverageextractionrateof
carnosicacidwas18.64mg/g.
Table1　Resultsoforthogonaltest
No.
A
ethanolmass
fraction/%
B
hydrochloric
aciddosage/g
C
solid-liquid
ratio(g∶g)
D
ultrasonic
frequency/kHz
E
ultrasonic
power/W
F
ultrasonic
time/min
extraction
rateof
CA/(mg·g-1)
1 45 1 1∶8 28 120 20 9.620
2 45 2 1∶12 45 160 40 11.282
3 45 3 1∶16 100 200 60 13.936
4 60 1 1∶8 45 160 60 15.714
5 60 2 1∶12 100 200 20 16.664
6 60 3 1∶16 28 120 40 18.892
7 75 1 1∶12 28 200 40 18.612
8 75 2 1:16 45 120 60 14.210
9 75 3 1∶8 100 160 20 17.856
10 45 1 1∶16 100 160 40 14.760
11 45 2 1∶8 28 200 60 10.628
12 45 3 1∶12 45 120 20 7.612
13 60 1 1∶12 100 120 60 11.572
14 60 2 1∶16 28 160 20 13.494
15 60 3 1∶8 45 200 40 15.746
16 75 1 1∶16 45 200 20 15.912
17 75 2 1∶8 100 120 40 12.752
18 75 3 1∶12 28 160 60 16.316
k1 11.306 14.365 13.719 14.594 12.443 13.526
k2 15.347 13.172 13.676 13.413 14.904 15.341
k3 15.943 15.060 15.201 14.590 15.250 13.729
R 4.637 1.888 1.524 1.181 2.807 1.814
3　Conclusions
Ultrasonicextractionwasusedtoincreasetheextractioneficiencyofcarnosicacidfromrosemaryleaves.
Thefactorsafectingtheamountofcarnosicacidwerestudied.Theoptimumconditionsfor5.0grosemary
leavesatroomtemperaturewere:ethanolsolutionconcentration75%, hydrochloricacid3g, solid-liquidratio
(g∶g)1∶16, ultrasonicfrequency28kHz, ultrasonicpower200W, ultrasonicextractiontime40min.The
extractionrateofcarnosicacidreached18.64mg/g.Theresearchcouldguidethefuturepilotexperimentand
providethebasisforthesubsequentpurificationofcarnosicacidproduct.
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