全 文 ：　　　　 　　天然产物研究与开发　 　　　　　
　　　　　　　　　　 NATURAL PRODUCT RESEARCH AND DEVELOPMENT 2005　Vol.17　No.1
　
　
　
　
　　Received May 27 , 2004;accepted July 16 , 2004
　＊Corresponding author　E-mail:newfourtharmy@163.com
Isolation ,Purification and Identification of Apigenin-
7-O-β-D-glucuronide in Marchantia Convoluta with Silica
Column Chromatography ,RP-HPLC and LC-ESI-MS
ZHU Hua1 ,XIAO Jian-bo2＊ ,ZOU Deng-feng1 ,FU peng1 ,ZHOU Chun-shan
(1.Guangxi Traditional Medical University , Nanning 530001 , China;
2.Department of Chemistry and chemical Engineering , Central South University , Changsha 410083 , China)
Abstract:The methods of silica column chromatography , RP-HPLC , UV , IR and LC-ESI-MS for isolation , purification and iden-
tification apigenin-7-O-β-D-glucuronide in Marchantia convoluta was established in this article.The crude extracts of Marchan-
tia convoluta leaves was obtained after Marchantia convoluta leaves were extracted with 80% ethanol and the extracts were con-
centrated under reduced pressure.The obtained crude extracts dissolved with methanol were subjected to silica gel column and
eluted with the successive eluting solvent mixture of chloroform ,methanol-chloroform1:5 ,methanol-chloroform 2:5 ,methanol-
chloroform 3:5 ,methanol-chloroform 4:5 ,methanol-chloroform 5:5.The eluted fractions were analyzed by RP-HPLC.The fur-
ther purity was analyzed with UV , IR and LC-ESI-MS.The elution chloroform-methanol 5:4 was indicated to consist of only one
kind of constituent by HPLC , whose retention time(tR)was 12.1 min.The HPLC experiment of the co-injections of this fraction
with the standard of apigenin-7-O-β-D-glucuronide resulted in an overlapping and accumulating effect with the similar retention
time.The UV [λmax 336/296(sh)/267 nm] and IR spectra of the test solution were similar to the apigenin-7-O-β-D-glu-
curonide.On the other hand , the LC-ESI-MS experiments indicated that the molecular weight is 446.So the fraction can be i-
dentified as apigenin-7-O-β-D-glucuronide.
Key words:Marchantia convoluta;column chromatography;RP-HPLC;LC-ESI-MS;apigenin-7-O-β-D-glucuronide
硅胶柱色谱/RP-HPLC/LC-ESI-MS分离纯化鉴定拳卷地钱中
芹菜素-7-O-β-D-葡萄糖醛酸甙
朱　华1 ,肖建波2＊ ,邹登峰1 ,傅　鹏1 ,周春山2
(1.广西中医学院 ,南宁 530001;2.中南大学化学化工学院 ,长沙 410083)
摘　要:本文建立了分离纯化鉴定拳卷地钱中芹菜素-7-O-β-D-葡萄糖醛酸甙的硅胶柱色谱/ RP-HPLC/ LC-ESI-MS
方法 。拳卷地钱叶经过 80%乙醇提取后 , 减压蒸馏 , 得粗提物。拳卷地钱叶粗提物用甲醇溶解后上硅胶柱 , 用
不同浓度的甲醇-氯仿溶液洗脱 ,各洗脱液进行 RP-HPLC 分析 , 较纯的洗脱液进行 LC-ESI-MS 分析 , 为制备分离
黄酮类化合物单体提供指导。氯仿与甲醇配比为 5:4的洗脱液经过 RP-HPLC 分析为单一组分 , tR为 12.1 min ,
与对照品芹菜素-7-O-β-D-葡萄糖醛酸甙共注射进行 RP-HPLC实验 ,发现峰高增加 , tR为 12.1 min , UV(λmax为 336/
296(sh)/267 nm)和 IR光谱与芹菜素-7-O-β-D-葡萄糖醛酸甙基本一致。 LC-ESI-MS 测定结果表明 , 与对照品芹菜素
-7-O-β-D-葡萄糖醛酸甙的分子量相同为 446。由此可以鉴定该洗脱组分为芹菜素-7-O-β-D-葡萄糖醛酸甙。
关键词:拳卷地钱;柱层析;RP-HPLC;LC-ESI-MS;芹菜素-7-O-β-D-葡萄糖醛酸甙
中图分类号:R284.1　　　
Marchantia plants(Chinese name Di Qian) is a well- known traditional Chinese medicine and has been exten-
sively used for the treatment of tumefaction of skins[ 1].It
can protect liver and treat hepatitis[ 2].Flavonoids are al-
most universal pigments of plants.They have been de-
38
scribed and occur mainly in plants as O-, C-glyco-
sides[ 3] .They have been found to be an important part of
the human diet and are considered as active principles of
many medical plants.Apigenin-7-O-β-D-glucuronide
(Fig.1)has many bioactivity such as antioxidants , anti-
inflammatory and anticarcinogen.No reports about con-
stituents of Marchantia convolute of China , to our knowl-
edge ,have been performed.In this article apigenin-7-O-β-
D-glucuronide has been isolated , purified and identified
with silica column chromatography ,RP-HPLC , IR/UV and
LC-ESI-MS.It afforded evidences for using Marchantia
convoluta which is abundant in Guangxi Province.
Fig.1　Formula of apigenin-7-O-β-D-glucuronide
Experimental
Materials , reagents and apparatus
LC-ESI-MS spectra were run on a Finnigan instru-
ments.IR spectra were carried out on a Bruker Vector22
spectrophotometer in KBr pellets.UV spectra were record-
ed on a Shimadzu UV-2501PC spectrophotometer.HPLC
spectra were obtained from BT8100 liquid chromatograph
with BT8200UV detector and C-R6A data processing com-
puter(Biotronic).Silica gel(100 ～ 200 mesh), Ocean
Chemical Factry of Qingdao.All reagents were of analyti-
cal pure grade.Marchantia convoluta collected in
Shangling City of Guangxi Provincewas identified by Zhou
Zi-jing , a botany professor of Biology department of
Guangxi Chinese Medical University.The whole plant , af-
ter washing with water and drying in the shade for several
days ,was powered.The standard sample of apigenin-7-O-
β-D-glucuronide was provided from Sigma.
Condition of chromatography , infrared spectra , ultra-
violet spectra and mass spectra
Column chromatography
Open-column used grass column , 68cm ×25mm.Solid
phase is silica gel after pre-treatment.The following elu-
tion order was observed , to obtain 50 mL solution in each
case , chloroform ,methanol-chloroform 1:5 ,methanol-chlo-
roform 2:5 ,methanol-chloroform 3:5 ,methanol-chloroform
4:5 ,methanol-chloroform 5:5.
RP-HPLC
The reversed phase high performance liquid chromatogra-
phy was set up to determine the content of flavonoids in
Marchantia convolute.Column , Kromasil RP-C18(250×
4.6 mm i.d , 5 μm , Hanbon Science &Technology Co.,
Ltd).Mobile phase , V(methanol):V(acetonitrile):V
(acetic acid):V(phosphoric acid):V(H2O)= 200:
100:10:10:200.Detecting wavelength 350 nm.Flow rate ,
0.60 mL/min.Column temperature , room temperature.
The quantity of injecting sample ,6.0μL.
Infrared spectrap
IR spectra were carried out on a Bruker Vector22 spec-
trophotometer.A blankKBr pellet was made first.Then the
test solution was dropped to the pellet.At last the pellet
was dried under IR lamp before determining.
Ultraviolet spectra
The quartz sample cells were employed.The test solution
was scanned in spectrophotometer using corresponding
reagents as blank solution.If the absorbency is too high ,
the solvent need to be diluted.Scanning wavelength is
from 190 nm to 400 nm.The rate of scanning is 200 nm/
min.Absorbency is in range of 0 ～ 3.000.
LC-ESI-MS
Mobile phase of LC-ESI-MS is similar to RP-HPLC.LCQ
Electrospray mass spectrometry was used.The ESI source
potentials were:Capillary , 3KV;lens , 0.5KV;extractor ,
4v;the cone volatage(CV)was changed during analyses
from 30 to 65 V as describled below-higher CV values al-
lowed increased CID of protonated or deprotonated
molecules.Mass range of scanning:150 ～ 180 m/z.
Skimmer:-40 volt.Cut Off:50 m/z.Differentiate rate is
13000 m·z-1/ sce.Nitrogen was used as the nebulishing
and desolvation gas at flow rates of 100 and 60 L/h re-
spectively.
Extraction and separation
Extraction and separation
The whole plant collected in Guangxi province after wash-
ing with H2O and drying in the shade for several days
were extracted with 80%ethanol for 1 hour at 70 ℃.The
solvent was distilled off under reduced pressure to afford a
dark green oil.The extract was dissolved in methanol and
mixed with about five times its weight of silica gel(100 ～
200 mesh).The mixture is evaporated in rotary evaporator
at 30 ～ 40 ℃.The resulting powder is distributed on the
top of the open column , in which some pre-treatment silica
gel were added.Before eluting a shallower of glass beads
covered the top of the open-column.The following elution
order was observed , to obtain 50 mL solution in each
case , chloroform , methanol- chloroform 1:5 , methanol-
chloroform 2:5 , methanol-chloroform 3:5 ,methanol-chlo-
roform 4:5 ,methanol-chloroform 5:5.The eluted fraction
was analyzed by RP-HPLC.The further degree of purity
was analyzed with Infrared Spectra ,Ultraviolet Spectra and
392005　Vol.17　No.1　　朱　华等:硅胶柱色谱/RP-HPLC/ LC-ESI-MS分离纯化鉴定拳卷地钱中芹菜素-7-O-β-D-葡萄糖醛酸甙 　
Mass Spectra.
Preparation of the solution for determining
The standard sample dissolved with methanol.The solvent
of elution was distilled off under reduced pressure , then
dissolved with methanol.The solution was separated with
centrifugal machine.The superstratum solution was used
as the test solution.
Identification of apigenin-7-O-β-D-glucuronide
The test solution was determined by RP-HPLC.The fur-
ther purity was analyzed with UV , IR and LC-ESI-MS.
Results and Discussion
Methods for introducing the sample into the chro-
matographic column
In this article a solid introduction was carried out.The
packing material is rejected after the separation and impu-
rities left on the stationary phase are also disposed of.Dry-
chromatography is rapid and very little solvent is re-
quired[ 4].
Analyzing the test solution by HPLC
The chromatograms of the extract and the elution were
Fig.1 and Fig.2 respectively.The elution of methanol-
chloroform 4:5 gave only one chromatogram peak.Its re-
tention time is 12.1 min compare to the chromatogram
peak No.1 of the crude extract.The HPLC experiment of
the co-injections of this fraction with the standard of api-
genin-7-O-β-D-glucuronide resulted in an overlapping and
accumulating effect with the similar retention time.From
Fig.2 we found this elution has one constituent that can
be eluted by silica gel column chromatography by one
step.
t/min
Fig.2　HPLC of crude extracts(A)and the test solution(B)
Ultraviolet spectroscopy of the test solution
The ultraviolet spectrum of the test solution is Fig.3.
Fig.3-1 was scanned using methanol as solvent , but
Fig.3-2 was scanned using methanol-NaOH as solvent.
Fig.3-1 showed two intense bands at 267 nm and 336
nm.In Fig.3-2 these peaks changed very much because
the base had an important effect on the UV spectrum of
flavonoids.The ultraviolet spectrum of the test solution is
similar to the standard sample.
Fig.3　UV Spectra of test solution
Infrared spectroscopy of the test solution
Infrared spectroscopy of the test solution is Fig.4(KBr
pellet).3421 cm-1 a broad band means O-H stretch-
ing.The value of the O-H stretching frequency has been
used as a test for measure the strength of hydrogen
bonds.2958 cm-1 ,2925 cm-1 and 2855 cm-1 showed the C-
H bands stretching of saturated hydrocarbons.But there
are no saturated hydrocarbons in apigenin.So this showed
the saturated hydrocarbons in glucuronide.The presence of
a carbonylgroup is immediately apparent in the IR spec-
trum with its very strong band at 1736 cm
-1
and 1625
cm
-1.1736 cm-1 is a strong band showing the C =O
stretching of -COOH in glucuronide.But 1625 cm-1 means
the C =O stretching of apigenin.The carbonyl group in
apigenin is at lower frequency than the carbonyl group in
glucuronide.1167 cm-1 and 1122 cm-1 mean the presence
of -O-.
Fig.4　IR Spectra of the test solution
LC-ESI-MS of the test solution
The on-line detection of flavonoid glycosides by mass
spectrometry is now supplanting current spectrometry de-
tection and the use of tandem mass spectrometry allows us
to observe the corresponding aglycone as a fragment.The
spectra produced in Fig.5-A is positive ion electrospray
ionization tandem mass spectrometry , obtained at low elec-
tron beam energies so that secondary f ragmentations are
minimized and multi-step fragmentations avoided.In the
40 天然产物研究与开发　　　　　　　　　　　　　 　　　2005　Vol.17　No.1
LCQ-ESI-MS the molecular ion is fairly abundant and rel-
atively few fragment ions are observed.The secondary frag-
mentation of m/z 270 in Fig.5-A involved losing the glu-
curonide.In positive ion electrospray ionization tandem
mass spectrometry the peak of [M+Na] + and [M+K] +
were often produced.The ion weight of [ M+K] + , sub-
tract by the ion weight of [M+Na] + , is 16[ 5].According
this theory ,we can find the molecular ion peak , then can
conclude the molecular weight.From Fig.5-A , we found
the most abundant ion(base peak)in the positive mass
spectra is 446.9 m/z and the exist of [ M+Na] + and
[M+K] +.So we concluded the molecular weight of the
main constituent in test solution was 446.The molecule
weight of apigenin-7-O-β-D-glucuronide is 446 exactly.
Fig.5　LC-ESI-MS diagrams of the test solution
A.positive ion　B.negative ion
469-446.9=22.1(Na+);
485-446.9=38.1(K+);
485-469=16(MNa+-MK+)。
The negative ion electrospray ionization tandem mass
spectrometry(Fig.5-B) can only afford molecular ion
peaks.So it is complement to the positive mass spec-
tra.From Fig.5-B we found the molecular ion peak of the
main constituent in test solution was 445[ M-H]-.So we
can conclude the molecular weight of the test solution was
446.
Conclusion
The crude extracts was obtained after leaves were extract-
ed with 80% ethanol , then the obtained crude extracts
dissolved with methanol were subjected to silica gel col-
umn and eluted with the successive eluting solvent mixture
of methanol-chloroform.The elution chloroform- methanol
5:4 was indicated to consist of only one kind of con-
stituent by HPLC , whose retention time(tR)was 12.1
min.The HPLC experiment of the co-injections of this
fraction with the standard of apigenin-7-O-β-D-glu-
curonide resulted in an overlapping and accumulating ef-
fect with the similar retention time.The UV and IR spec-
tra of the test solution were similar to the standard sam-
ple.The LC-ESI-MS experiments indicated that the molec-
ularweight is 446.So the fraction can be identified as api-
genin-7-O-β-D-glucuronide.Apigenin-7-O-β-D-glu-
curonide can be obtained from the Marchantia convolute
by one step.
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412005　Vol.17　No.1　　朱　华等:硅胶柱色谱/RP-HPLC/ LC-ESI-MS分离纯化鉴定拳卷地钱中芹菜素-7-O-β-D-葡萄糖醛酸甙