全 文 ：HPLC Determination of the Camptothecine in the
Root of Nothapodytes pittosporoides Oliv． Sleum
DENG Shi-ming1，WANG Fa-song1，2* ，ZHOU Da-zhai1，ZHENG Juan2
1． Key Laboratory of the Biological Resource Protection and Utilization of Hubei Province，Enshi，Hubei 445000，China;2． School of Chemistry
and Environmental Engineering，Hubei College for Nationalities，Enshi，Hubei 445000，China
Abstract ［Objective］ The HPLC method for the test of camptothecin in the root of Nothapodytes pittosporoides Oliv sleum was established．
［Method］ In HPLC determination method，the chromatographic column was Diamonsil C 18 (5 μm，250 mm × 4．6 mm) ;the mobile phase consis-
ted of acetonitrile∶water (30∶70) ;the wavelength of UV detector was 254 nm;the flow rate was 1． 0 ml /min and the column temperature was under
the condition of room temperature． ［Result］The calibration curves showed that the instrument was with good precision with its RSD value of 1． 3%;
the testing result had good reproducibility with the RSD value of 0． 87% and the testing processing had good stability with its RSD value of 2． 3%，in-
dicating there relatively was the stability of the camptothecin within 48 hours． The average recovery rate was 99． 2% with the RSD value of 1． 2% in
the sampling and recovering experiment for five repetitions． The content of the camptothecin in the root of the plant from the Enshi of Hubei Province
and the Qianjiang of Chongqing City was 0． 143 6% and 0． 144 8%，respectively． ［Conclusion］ The method was with good isolation efficiency，
strong specificity，rapid analysis，high sensitivity，which could be used as the determination of the camptothecin in the root of Nothapodytes pit-
tosporoides Oliv sleum．
Key words Nothapodytes pittosporoides Oliv sleum，HPLC，Camptothecine，China
Received:December 7，2009 Accepted:January 10，2010
Supported by the Project from the Key Laboratory of the Biological Re-
source Protection and Utilization of Hubei Province(No． 2007023)
and the Project Supporting the Outstanding Young Talent in the Re-
search Plan of Hubei Provincial Department of Education
(Q20082901)．
* Corresponding author． E-mail:zsuwangfasong@yahoo． com． cn
The Nothapodytes pittosporoides (Oliv．)Sleum(N． pit-
tosporoides) ，also known as the Nothapodytes tree，belongs to
Icacinaceae family［1］，and the camptothecin and its methoxy-
camptothecin derivatives are contained in its roots［2］，which
are mainly used for clinical treatment of cancer in digestive sys-
tem［3］． It was reported that the camptothecin in the N． pit-
tosporoides of Olacacea family grown in India was also
found［2］，however，it was not reported that the research on the
determination of the content of camptothecin in it in detail so
far． The determination of the content of camptothecin in the
root of the plant from the Enshi of Hubei Province and the Qian-
jiang of Chongqing City was conducted with HPLC method［4］
and its determination method was studied，which could provid-
ed a theoretical basis of the development of camptothecin from
new plant resource．
1 Material and method
1．1 Material
1． 1． 1 Three samples were collected from the Enshi of Hubei
Province and the Qianjiang of Chongqing City，respectively．
Total of 6 samples were passed through the 60 mesh sieve af-
ter they were naturally dried without direct sunlight and
crushed．
1． 1． 2 The 515 HPLC system(Waters 2487 Dual，U． S． Waters
Corporation) and the chromatography workstation SR 2000
(Shanghai Sanrui Technology Co．，Ltd．)was adopted for the
testing．
1． 1． 3 Main reagent:the reference substance of the Campto-
thecin with the content 98． 1% was from Sigma Corporation;
the acetonitrile(GR)was from Shanghai Chemical Reagent
Company;the water was ultra-pure one and other reagents
were at level of analytical pure．
1． 2 Method
1． 2． 1 The chromatographic condition:the chromatographic
column was Diamonsil C18(5 μm，250 mm × 4． 6mm) ;the
mobile phase was the ration of acetonitrile∶water(30∶70) ;the
detection wavelength was 254 nm; the flow rate was 1． 0
ml /min，the injection volume was 20 μl and the column temper-
ature was normal condition inside room． Under these condi-
tions，the retention time of camptothecin was 12．85 minutes．
1． 2． 2 The standard curve:the standard solution with the con-
centration of 0． 046 mg/ml was made with 2． 3 mg reference
substance of the Camptothecin being dissolved in the methanol
with final constant volume of 50 ml． 1． 0，2． 0，3． 0，4． 0 and
5．0 ml standard solution were added in the methanol in 10 ml
flask with the constant volume of 10 ml，which were shaken to
even，and then，the solutions of 20 μl were injected and meas-
ured，respectively．
1． 2． 3 The test of precision:the RSD value of camptothecin
was calculated after 5 consecutive times test from same solu-
tion of the reference substance under the chromatographic
condition．
1． 2． 4 The test of reproducibility:the content of each compo-
nent in 5 repetitions of same sample，which were precisely bal-
anced at the level of 0． 000 1 mg，was measured according to
Medicinal Plant /药用植物研究 2010，1(2):37 －39 Responsible editor:Xia Jing Responsible translator:Huang Zhongxiang
the determination method of sample．
1． 2． 5 The test of stability:the sample solutions precisely
taken were measured for five consecutive tests at 2，4，8，24
and 48 under the chromatographic condition，respectively，and
the content in the sample was calculated．
1． 2． 6 The test of recovery rate:1 ml testing solution of five
samples，which were precisely taken，was added into 1ml
standard solution of 0． 044 mg/ml and measured under the
chromatographic condition;then，the average recovery rate of
them was calculated．
1． 3 Test of sample 1． 0 g dried powder of N pittosporoides
root precisely balanced and 25 ml methanol were put into 50 ml
conical flask，which was coldly soaked for 12 hours，then ex-
tracted by means of the technique of ultrasonic treatment for 30
minutes，and finally，the extractive solution of the sample was
made after the sample treated by ultrasonic method was taken
out，filtered，and finally its constant volume was 50 ml with
methanol． 20 μl standard solution and sample solution，which
were precisely drawn respectively，were injected into the liquid
chromatography for continuous three times，then，the data of
the peak area for each times test was recorded，and finally，
the content of camptothecin in each sample was calculated
through the application of the linear regression equation．
2 Results and analysis
2．1 The verification of the determination method of the
content of camptothecin in N． pittosporoides root with
HPLC
2． 1． 1 The standard curve:the chromatograms of reference
sample and testing sample were as follows (Fig． 1 －2)． It can
be seen from the Fig． 1 and Fig． 2，there was a clear peak of
camptothecin absorption at 12 － 15 minutes． The peak area
(Y)being taken as the ordinate and the concentration of camp-
tothecin(X) (mg/ml)being taken as the abscissa，a linear re-
gression equation:Y = 289．08X + 0．379 3，with the correla-
tion coefficient R2 value of 0． 999 5，was formed． The linear
range of camptothecin was 0． 046 －0．23 mg/ml．
Fig． 1 HPLC Chromatogram of the Reference Sample of
Camptothecine
2． 1． 2 The testing results of the precision，reproducibility，sta-
bility and recovery rate． The good precision of the instrument，
the good reproducibility and the good stability of the determina-
tion appeared based on the method from 1． 2． 2 －1． 2． 5 men-
tioned above，with the RSD value of 1． 3%，0．87% and 2．3%，
Fig． 2 HPLC Chromatogram of the Testing Sample of
Camptothecine
respectively，which indicated that the camptothecin was in the
relatively stable state within 48 hours． The experiment in the re-
covery rate indicated the average recovery rate was 99．2%
with the RSD value of 1． 2% based on five repetitions，showing
a good recovery rate． Therefore，the method was with good
isolation efficiency，strong specificity，rapid analysis，high sen-
sitivity，which could be used as the determination of the camp-
tothecin in the root of N． pittosporoides．
2． 2 The testing results of the sample The average con-
tent of camptothecin in the root of N． pittosporoides from the
Enshi of Hubei Province and the Qianjiang of Chongqing City
was 0． 143 6% and 0．144 8%，respectively，(Seen in the Ta-
ble 2)and there was not difference in the content of camptothe-
cin in the root of N． pittosporoides between two places，based
on the results of 6 samples-testing and according to the method
of 1． 3 mentioned-above．
Table 1 The Testing Result of the Content of Camptothecine in
N． pittosporoides Root from Two Places
No． Origin Content∥% Meant∥%
1 Enshi* 0． 143 2
2 Enshi 0． 147 8 0． 143 6
3 Enshi 0． 139 8
4 Qianjiang＊＊ 0． 144 2
5 Qianjiang 0． 145 2 0． 144 8
6 Qianjiang 0． 145 0
Note:n =3． * the Enshi of Hubei Province and＊＊ the Qianjiang of
Chongqing City
3 Discussions
It was reported by Zhang Yuhong et al that the content of
the camptothecin in common camptotheca fruit was generally in
the range of 0． 10% －0．13%［5］ and by Huang Xiaowu et al that
the content of camptothecin in common camptotheca fruit was
0． 135%［4］;while the content of the camptothecin in the N． pit-
tosporoides root from the Enshi of Hubei Province and the
Qianjiang of Chongqing City was higher than these in common
camptotheca fruit，with average content of 0． 143 6% and
0．144 8%，respectively，and it was not clear what reason re-
sulted in the difference and the differences maybe result from
the measurement methods． There was richening resource of
the plant in the Wuling Mountain area with relatively concentrat-
ed distribution，which could be used for the study and utilization
in-depth of new plant resource of camptothecin-containing．
83 Medicinal Plant 2010
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檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪
44．
HPLC法测定马比木根中喜树碱的含量
邓仕明1，王发松1，2* ，周大寨1，郑 涓2 (1．生物资源保护与利用湖北省重点实验室，湖北恩施 445000;2．湖北民族学院化学与环境工
程学院，湖北恩施 445000)
摘要 ［目的］建立 HPLC测定马比木根中喜树碱含量的方法。［方法］HPLC法测定，色谱柱为 Diamonsil C 18 柱 (5 μm，250 mm ×4． 6 mm) ，流
动相为乙腈∶水(30∶ 70) ，检测波长 254 nm，流速 1． 0 ml /min，柱温为室温。［结果］喜树碱含量在 0． 046 ～0． 23 mg /ml 范围内线性关系良好;仪器
精密度良好，其 RSD值为 1． 3%;试验的重现性良好，其 RSD值为 0． 87%;试验的稳定性良好，其 RSD值为 2． 3%，说明喜树碱在 48 h内比较稳
定。加样回收率试验，重复 5次，计算得到平均回收率为 99． 2%，RSD值为 1． 2%。湖北恩施和重庆黔江产马比木根中的喜树碱含量分别为
0． 143 6%和 0． 144 8%。［结论］该方法分离效果好、专属性强、分析速度快、灵敏度高，可作为马比木根中喜树碱含量测定的方法。
关键词 马比木;HPLC;
欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁欁
喜树碱
(From page 36)
the side effect and drug tolerance［5］． However，the apoptosis
mechanism of medicine inducement is not clear and needs a
further research．
References
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ticancer drugs［J］． Biochem Pharmacol，1990． 40:2353
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I as an intracellular target of the anticancer drug camptothecin［J］．
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in cancer and cancer therapy［J］． Cancer，1997，73:2013．
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sis research［J］． Cancer，2000(19):1173 －1176． (in Chinese)．
［6］LIU CJ，SHI HM，ZHANG ZZ，et al． Study on optimization of extrac-
tion technology of tanshinone IIA from Salvia miltiorrhiza Buhge with
ultrasonic extraction method based on HPLC［J］． Journal of Anhui
Agricultural Sciences，2010，38(9):204 －205． (in Chinese)．
［7］CAO R，ZHANG CH，WANG Z，et al． RP －HPLC determination of
content of aristolochic add in different processing with manchurian
dutchmanspipe stem［J］． XIANDAI NONGYE KEJI，2009(10):14 －
15． (in Chinese)．
［8］LI H，ZHANG J，ZHANG SG，et al． Determination of chlorogenic
acid in compound Shilintong capsule by HPLC［J］． Journal of Anhui
Agricultural Sciences，2010，38(9):177 －178，180． (in Chinese)．
［9］WANG GF，XIONG Y，LIU WZ． Comparison of HPLC and FPIA
method in determination of carbamazepine in human serum［J］．
Journal of Tropical Medicine，2006，6(2):185 －187． (in Chinese)
檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪
．
羟基喜树碱对人白血病 K562 细胞增殖和凋亡的影响
邵淑丽，吴 敏，武广慧 (齐齐哈尔大学生命科学与工程学院，黑龙江齐齐哈尔 161006)
摘要 ［目的］研究羟基喜树碱对人白血病 K562细胞增殖与凋亡的影响。［方法］通过光学显微镜、荧光显微镜、电子显微镜、琼脂糖凝胶电泳、
流式细胞术等方法，探讨不同浓度羟基喜树碱作用不同时间后，K562细胞凋亡的情况。［结果］羟基喜树碱能够诱导 K562 细胞凋亡，作用 48 h
的最佳浓度为 8． 0 μ g /ml。羟基喜树碱可影响 K562细胞增殖周期中的 S期，即 DNA合成期，细胞在此期停滞，诱导其发生凋亡。［结论］为将
羟基喜树碱应用于临床治疗白血病提供前期试验依据。
关键词 白血病;K562细胞;羟基喜树碱;细胞增殖;细胞凋亡
93DENG Shi-ming et al． HPLC Determination of the Camptothecine in the Root of Nothapodytes pittosporoides Oliv． Sleum