全 文 ：№.5　　　　　　　　　　　　　　　 　　　　陕西科技大学学报　　　　　　　　　　　　　　　　　　Oct.2009
Vol.27　　　　　　JOURNAL OF SH AANXI UNIVERSIT Y OF SCIENCE &T ECHNOLOGY　　　　　　·1·
＊ 　文章编号:1000-5811(2009)05-0001-05
HPLC ANALYSIS OF CAMPTOTHECIN AND
10-HYDROXY CAMPTOTHECIN IN CAMPTOTHECA
ACUMINATA DECNE .LEAVES
LI Xiang-li1 , XIAO Kai-jun1 , HU Xian-yun2 , LUO Xin-yi2 , GUO Si-yuan1
(1.Light Industry and Chemical Enginnering Resear ch Institute , South China University of Techno log y ,
Guang zhou　510640 , China;2.The Bio log y Resea rch Institute , Guizhou Academy of Science , Guiyang 　
550009 , China)
Abstract:An HPLC method fo r the simul taneous dete rm inat ion of CPT and HCPT in Camp-
totheca acuminata Decne.leaves w as established.Chromatog raphy w as performed wi th a si-
nochrom ODS column , monitored at 266 nm and the mobile phase w as g radient H 2O/
CH 3OH .The solvent gradient prog ram was as fol low :methanol-water increased linearly
f rom 40% to 50% at the first 6 min and retained for 2 m in w ith 50%, then increased linearly
to 70% in the nex t 32 min.The average recoveries o f CPT in three levels w ere 100.02%,
98.94% and 99.25%, The average recoveries of HCPT w ere 99.13%, 99.66% and
98.97%.Using this assay , CPT and HCPT in leaves of Camptotheca acuminata Decne.in
dif ferent seasons were determined.The resul ts show ed that the accumulation of CPT and
HCPT presented evident relat ivity in the dif ferent grow th stag es.
Key words:HPLC;Camptotheca acuminata Decne.;leaves;camptothecin(CPT);10-hy-
droxy camptothecin(HCPT)
CLC Number:R927.2　　　　Document Code:A
0　Introduction
Camptothecin(CPT)and 10-hydroxy campto thecin(HCPT)are tw o significant secondary metabo-
li tes belonging to terpenoid indole alkaloid produced by the Camp totheca acum inata Decne., a t ree n-
a tive to China
[ 1] .They po ssess st rong anti-cance r bioactivity and the mechanisms as w ell as modes of ac-
tion against cancer is unique which inhibi t topoisomerase I , an enzyme clo sely linked w ith cell divi-
sion[ 2] .Among many CPT and HCPT analogues , i rino tecan and topo tecan have been approved by Uni t-
ed State Food and Drug Administ ration (FDA)fo r application against ref ractory colon-rectum cancer ,
small-cell lung cancer and ovarian cancer[ 3 , 4] .Several othe r analogues are in the process of clinical
test[ 5 , 6] .
In recent y ears , several HPLC me thods have been repo rted to applianaly ze CPT drugs in Camptoth-
eca acum inata Decne.[ 7-11] , but only a few of them dealt wi th the simul taneous o f CPT and HCPT and
analy tical method for the tw o metabo li tes reported only in f ruit and seed of Camp totheca acuminata Dec-
ne.[ 10-12] .
＊收稿日期:2008-08-06
作者简介:李香莉(1980-),女 ,河南省商丘市人 ,在读博士生 ,研究方向:食品化工
基金项目:中法先进研究计划项目(P RA B02-07),广州市科技成果推广项目(2006C13G0011)
陕西科技大学学报 第 27 卷
Fig.1　Structures of Camptothecin(CPT)and 10-hydroxy
camptothecin(HCPT)
In this pape r , we set out to
develop a method for simultaneous
determination of CPT and HCPT in
leaves by HPLC.The method w as
successfully applied to analyze CPT
and HCPT contents in leaves f rom
different seasons in Guiyang city
(China).
1　Experimental
1.1　Reagents and materials
The methanol w as o f HPLC g rade ,water w as ult rapure w ater , all others w ere analy tical g rade.
The CPT and HCPT standard samples w ere purified by researcher LUO Xin-yi and identif ied by
Ecole N ormale Superieure in Paris.Their puri ty w as bo th 98%.
Four-year old Camptotheca acum inata Decne .in the arbo retum of Guizhou Normal University
(Guizhou , China)was selected for the analy tical sample.The leaf samples to dete rmine the levels o f
CPT and HCPT were col lected in dif ferent seasons.All the samples w ere dried at 60 ℃ till constant ,
crushed and passed through a 60 mesh screen sieve.
1.2　Apparatus and chromatog raphic condit ions
Chromato graphy w ere performed using a P230P HPLC system (Elite , Dalian , China)consisting o f
a P230 gradient pump , a UV230+ UV-VIS detector and a Rheodyne MX7925 injecto r.The analy tical
column used w as sinochrom ODS-BP (250 mm ×4.6 mm I.D., 5.0μm).The separation w as perfo rmed
using a solution of me thanol/distilled w ater mixture wi th a g radient elution (methano l concentration in-
creased linearly f rom 40% to 50%during the first 6 min and stay ed for 2 min w ith 50%, then increased
linearly to 70%in the nex t 32 m in).The flow-rate w as 1 mL/min.The determination w as done at 266
nm and 25 ℃ fo r co lumn.
1.3　Standard solutions
S tandard solutions of CPT and HCPT at 200 and 20 μg/mL were prepared in methanol/N-N- dime-
thylfo rmamide (90∶10 , v/v).Working standard so lutions w ere prepared by diluting these sto ck so lu-
tions wi th the me thanol/N-N-dimethy lformamide mix ture to achieve CPT standard concentrations of 2 ,
20 , 60 , 120 and 200μg/mL and HCPT of 0.1 , 2 , 4 , 6 and 10μg/mL , respectively.A 20μL vo lume o f
each standard solution w as injected in t riplicate onto the HPLC column.The calibration g raphs w ere
const ructed by plo tt ing the peak areas o f CPT and HCPT versus thei r concentrat ions.
1.4　Ex traction and assay procedures
The optimal solvent fo r the simultaneous ex t raction of CPT and HCPT from C.acuminata.D.was
dete rmined.Testing w as done using alcohol , methanol , 70% alcoho l and 70%methanol.
5 g of the leaves pow der w as placed into 50 mL flask , w ith 45 mL of the solvent being tested then
ul trasonically ext racted at 50 ℃for 60 min.Afte r coo ling to room temperature , this solution w as trans-
ferred to a volumetric f lask and 5 mL so lvent w as added to the scale.Finally it w as f ilt rated through 0.
45 μm M illex sy ringe fil ters for HPLC analy sis.
1.5　Recovery
Nine leave samples w ho se content w as known precisely w ere weighed , every set w as 1 g , and divid-
ed into three g roups.The recoveries of CPT and HCPT were calculated by standard addi tion method.
·2·
第 5 期 李香莉等:喜树叶中喜树碱和 10-羟基喜树碱的 HPLC 分析
Fig.2　HPLC chromatogram of CPT and HCPT standards
Fig.3　HPLC chromatogram of a leaf
sample in July
1.6　CPT and HCPT contents in dif ferent seasons
CPT and HCPT contents in Camptotheca acuminata
Decne.leaves f rom dif ferent seasons w ere determined by the
above analyzing method(Fig.2).
2　Results and Discussion
2.1　Chromatog rams
Fig.3 show s representat ive chromatog rams obtained in
our study .The retent ion times o f CPT and HCPT were ap-
proximately 29.47 and 23.59 min , respectively.The repo rts
about simultaneous dete rm inat ion methods o f CPT and
HCPT by HPLC are relatively few in now day s.There are many interfering components in ex t ractive
because of metabolism prosperity of leaves in some month of a y ear.Therefo re , 10-hydroxycamptotheca
can′t be w ell separated wi th neighbor impurity refering to reported condi tions.However , the separating
condi tion w as great ly improved through selecting proper condition of gradient elution.Assay s of Camp-
totheca acuminata Decne.leaf show ed there were no interfering peaks , CPT and HCPT can be separated
Tab.1　Recovery test (n=3)
Sample
Added
/ mg
Ave rage
recovery/ % RSD/ %
CPT
0.5
1.0
1.5
100.02
98.94
99.25
0.60
0.51
0.37
HCPT
0.5
1.0
1.5
99.13
99.66
98.97
0.52
0.47
0.31
sati sfactorily in thirty-five minutes.
The average recove ries of CPT in three levels w ere
100.02%, 98.94% and 99.25% and that o f HCPT w ere
99.13%, 99.66% and 98.97%(Tab.1).Repeatability w as
dete rmined by five-fo ld analy sis of a 4 μg/mL standard solu-
tion and the RSD of CPT and HCPT were 1.76%, 1.55% re-
spectively.
2.2　Lineari ty
The calibrat ion g raphs fo r CPT and HCPT were linear f rom 4 to 200 and from 0.1 to 10μg/mL , re-
spectively.The means of thei r calibration g raphs yielded the fo llowing equations:y =47.61 x+134.77
fo r CPT ,R=0.999 6 , and y =68.552x+32.138 for HCPT , R=0.999 8 , in w hich y is the peak area o f
CPT or HCPT to and x i s the concentration(μg/mL)of CPT o r HCPT .
2.3　Optimal solvent for the sample prepa rat ion
Optimal solvent fo r simultaneous ex t raction of CPT and HCPT from C.acuminata D .leaves w ere
studied.The result show s that ex t racting w i th 60%me thalol solution have a higher ex traction recovery
of CPT and HCPT (Tab.2).
2.4　CPT and HCPT contents in dif ferent seasons
CPT and HCPT contents in dif fe rent seasons w ere analy sized by defined assay and the resul ts w ere
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陕西科技大学学报 第 27 卷
Tab.2　Extraction recovery of CPT and HCPT
with different solvents
Solvent
Ex traction recovery
o f CPT/ ‰
Ex traction recove ry
of HCPT/ ‰
alcoho l 0.708 0.015
methanol 0.729 0.016
70% alcohol 0.903 0.018
70% methanol 0.942 0.020
show n in Fig .4 ～ Fig.7.
C.accum inata D .sprouted new leaves contin-
uously f rom April to November.Liu et al ever re-
po rted that CPT content in leaves descended eleven
percent every month on the everage f rom April to
Octobe r[ 12] .The results o f ZHANG Yu-hong et al
show ed that CPT content exhibited a single apex
curve in young leaves f rom April to November and
i t reached the highest content in July[ 13] ;The results of GUO Yong et al show ed that the content o f
CPT presented tw o peaks curve bo th in tender leaves and older leaves f rom April to N ovember , which
appeared in June and September respectively , it could reach the highest content in June[ 14] .
Fig.4　Variation of CPT contents in 　 　 　　Fig.5　Variation of HCPT contents in
leaves of Camptotheca acuminata 　　　　　　　 leaves of Camptotheca acuminata
in different months　　　　　　　　　　　　　　　　in different months
Fig.6　Variation of CPT and HCPT contents 　Fig.7　Variation of CPT and HCPT contents
in tender leaves of Camptotheca acuminata in　　　in old leaves of Camptotheca acuminata in
　　　　different months　　　　　　　　　　　　　　　　　different months
Howeve r , the results of our test showed that , as can be seen in Fig.4 , in a year , the content o f
CPT presented tw o peaks curve in tender leaves of C.acum inata , which appeared in July and September
respectively and reached the highest point in July;whi le i t declined persistent ly in o lder leaves.
The content of CPT in tender leaves w as obviously highe r than tha t in older leaves af ter June(P<
0.05)which w as in agreement on the w hole wi th the theory tha t the CPT content in tender leaves w as
obviously highe r than that in older leaves as a kind o f chem ical defence material.
As Fig .5 show ed , the content of 10-hydrocampto thecin in tender leaves f luctuated aw fully in dif fer-
ent months , it w as qui te high in April and declined no tably in M ay , then varied between the tw o levels
and assented continuously af ter Octobe r.T he 10-hydro camptothecin content in older leaves increased
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第 5 期 李香莉等:喜树叶中喜树碱和 10-羟基喜树碱的 HPLC 分析
persistent ly except fo r the low est content in July.Bo th in tender leaves and o lder leaves , the content o f
10-hydro campto thecin reached highest point in November and low est in July .
From Fig.6 and Fig.7 we can seen that the accumulation o f campto thecin and 10-hydrocamptothecin
presented evident relativi ty in the different g row th stages.The campto thecin and 10-hydrocamptothecin
contents in tende r leaves w ere bo th high in early stages.After June , the changes of the two alkaloids ex-
hibi ted good mutual effects.In a year , the campto thecin accumulation decreased persistent ly in older
leaves , while the 10-hydrocampto thecin content in olde r leaves increased continuously w hich w as oppo-
site to campto thecin except for the low est content in July w hen the campto thecin acumuni tion in tender
leaves reached the highest contents.
3　Conclusions
In this study , we established an HPLC method fo r the simultaneous determination of CPT and
HCPT in Camptotheca acuminata Decne.leaves.The method w as succe ssfully applied to analy ze CPT
and HCPT contents in leaves in dif ferent seasons.Our resul ts show that the accumulation of CPT and
HCPT pre sented evident rela tivity in the dif fe rent grow th stages.T he contents of CPT and HCPT in
tender leaves w ere bo th high in early stages.Afte r June , the changes of the tw o alkaloids exhibited good
mutual ef fect.The campto thecin accumulation decreased pe rsistently in older leaves fo r all the g row th
period , while the 10-hydrocamptothecin content in olde r leaves w as increasing continuously w hich w as
opposi te to the decreasing of camptothecin accumula tion.This results suggest tha t the change fashion
between them was probably no t only f rom campto thecin to 10-hydro campto thecin but also f rom 10-
hydro campto thecin to camptothecin.
References
[ 1] Wall M.E., Wani M.C., C ook C.E., et al.Plant anti tumor agents.The isolat ion and s tru cture of camp tothecin , a n ovel alkaloid-
al leukemia and humo r inhibi ti or f rom Camp totheca acuminata[ J] .Am Chem Soc , 1966 , 88(16):3 888.
[ 2] Sabry M.Attia , Abdulaziz M.Aleisa , Saleh A.Ba , et al.M olecular cy togenetic evalu at ion of the mechanism of micronuclei for-
mation indu ced by camptoth ecin , topotecan , and i rinotecan[ J] .Envi ronm ental and Molecular Mutagenesis , 2009 , 50:145-151.
[ 3] A rm st rong DK , Sp rigg s D , Levin J , et al.H em atologic safety and tolerabi lit y of topotecan in recurrent ovarian cancer and sm al l cell
lu ng can cer:An integ rated analy sis[ J] .Oncologis t , 2005 , 10:686-694.
[ 4] Punt CJ , Koopm an M.Capeci tabine an d i rinotecan as f irs t-line t reatment of advan ced colorectal cancer[ J] .J Clin Oncol , 2008 , 26:
1 907- 1 908.
[ 5] Milan P., H erb ert P..Camptoth ecins:New Anticancer Agents[ M] .Boca Raton:CRS Pres s , 2000:67.
[ 6] Keh rer , D.F.S., Soepenb erg , O., Loos , W.J., et al.M odulation of cam ptoth ecin analog s in th e t reatment of cancer:a review
[ J] .Ant i-C ancer Dru gs , 2001 , 12(2):89-105.
[ 7] ZHANG Yan-hong , LIU Zhan-mei , SH EN H ui-fang , eta l.Determination of camp tothecin in f ruit s of camptotheca acuminata dec-
ne by reversed-ph ase high performance liquid chromatog raph[ J] .C hinese Journal of Ch rom atography , 2003 , 21(2):162-164.
[ 8] MA Mei-f ang , YU T ao , DAI Shao-ju n , et al.Determination of con tents of 10-hydrocycam ptothecin in Cam ptotheca acuminata by
high-performan ce liquid ch rom atogram[ J] .Journal of Fores t ry Research , 2002 , 13(2):144-146.
[ 9] YAN Xiu-f eng , WANG Yang , YU Tao , et al.Determination of camptoth ecin in leaves of camp totheca acuminata decne.by HPLC
[ J] .J ou rnal of Ins t rumen tal Analysis , 2002 , 21(2):15-17.
[ 10] WANG Yang , YAN Xiu-f eng , ZHANG Yu-hong , et a l.Determination of camptothecin and 10- hydrocycamptothecin in Camop-
totheca acumin ta D.seeds by H PLC[ J] .Chemist ry and In dust ry of Forest Produ cts , 2005 , 25(3):64-66.
[ 11] LIU Wen-zhe , QIN H ai-yan , SUO Zhi-rong.HPLC analysis of cam ptoth ecin and 10- hydroxycamp tothecin in f ruit of Camp totheca
acuminata decne[ J] .C hin J Pharm Anal , 2005 , 25(2):168-170.
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第 5 期 黄建蓉等:响应曲面法优化超声提取柚皮中总黄酮工艺
响应曲面法优化超声提取
柚皮中总黄酮工艺
黄建蓉1 , 2 , 李　琳1 , 张　霞1 , 李　冰1
(1.华南理工大学轻工与食品学院 , 广东 广州　510640;2.广东药学院食品科学学院 , 广东 中山　528458)
摘　要:在单因素试验的基础上 ,利用响应曲面法对超声辅助提取柚皮中总黄酮的工艺参数进
行了优化.以乙醇浓度 、超声时间 、提取温度为自变量 ,总黄酮提取率为响应值 ,采用 Box-Be-
hnken设计试验方案 ,利用 SAS 软件对实验数据进行回归分析 ,得到黄酮提取率的二次多项
式预测模型.在固液比为 1∶30的条件下 ,优化的工艺参数为:乙醇浓度 67.0%,超声时间
28.0 min ,提取温度 50.0 ℃.在此工艺条件下柚皮总黄酮提取率为 5.301%.
关键词:柚皮;黄酮;超声辅助提取;响应曲面法
中图分类号:R284.2;TQ028.8　　　　文献标识码:A
(上接第 5页)
[ 12] Liu Z., Carpenter S.B., Bourgeois W.J., et al.Variations in the secondary metaboli te camptothecin in relat ion to ti ssue age and
season in Cam ptotheca acuminata[ J] .Tree Physiology , 1998 , 18:265-270.
[ 13] ZhANG Yu-hong , Z U Yuan-gang.Study on camptothecin content in leaves of Camp totheca acuminata f rom dif feren t region s and
seasons[ J] .Chinese Bul let in of Botany , 2003 , 20(5):572-575.
[ 14] GUO Yong , LIN Gui-yun , SUN Yan-xia , et a l.Studies on the seasonal ch anges of camptothecin con tent in th e leaves of Cam pto-
theca acumina ta[ J] .S outhw es t China Journal of Agricu ltu ral S cien ces , 2006 , 119(16):143-145.
喜树叶中喜树碱和 10-羟基
喜树碱的 HPLC分析
李香莉1 , 肖凯军1 , 胡先运2 , 罗心毅2 , 郭祀远1
(1.华南理工大学轻化工研究所 , 广东 广州　510640;2.贵州科学院生物研究所 , 贵州 贵阳　550009)
摘　要:建立了同时测定喜树叶片中喜树碱含量的高效液相色谱分析方法.采用 sinochrom
ODS C18分析柱(250 mm×4.6 mm BP , 5μm),以甲醇/水为流动相 ,检测波长为 266 nm ,梯度
洗脱程序为:在前 6 min流动相甲醇-水的体积比由40%线性增加至 50%, 并保持恒定 2 min ,
在随后的 32 min甲醇-水的体积比线性增至 70%,在 40 min时停止该程序;喜树碱在低 、中 、
高 3个量的平均加样回收率分别为 99.66%, 100.3%,99.97%;10-羟基喜树碱在低 、中 、高 3
个量的平均加样回收率分别为 99.87%,99.98%,100.4%.对不同季节的喜树嫩叶和成熟叶
进行了喜树碱与 10-羟基喜树碱含量的分析 ,结果表明喜树碱和 10-羟基喜树碱的含量变化呈
现明显的相关性.
关键词:高效液相色谱法;喜树;叶片;喜树碱;10-羟基喜树碱
中图分类号:R927.2　　　　文献标识码:A
·11·