全 文 ：　　　　 　　天然产物研究与开发　 　　　　　
　　　　　　　　　　 NATURAL PRODUCT RESEARCH AND DEVELOPMENT 2005　Vol.17　No.2
　
　
　
　
　　Received May 11 , 2004;Accepted May 31 , 2004
　＊Corresponding author　E-mail:newfourtharmy@163.com
Determination of the Content of Flavonoids in
Marchantia convolute L.
XIAO Jian-bo ,CAO Hui ,XIANG Hai-yan ,ZHOU Chun-shan＊
(College of Chemistry and Chemical Engineering , Central South University , Changsha 410083 China)
Abstract:Methods for determining flavonoids from Marchantia convolute were established in this article.The contents of total
flavones in Marchantia convolute were determined through developed visible spectrophotometry.The reversed phase high perfor-
mance liquid chromatography was set up to determine the contents of flavonoids in Marchantia convolute.In visible spectropho-
tometry Beer s law was obeyed in range of 1.6 ～ 16 μg/ mL and the recoveries were in the range of 97.61～ 101.59%.In
HPLC apigenin , luteolin and quercetin were quantitated by calibration curve method at detection wavelength of 350 nm.The re-
sult shows that the average recovery is 96.79 ～ 101.13%, RSD is 0.66 ～ 1.52%.These methods were used to determine
flavonoids in Marchantia convolute with satisfactory results.
Key words:Marchantia convolute;flavonoids;determination
拳卷地钱中黄酮类化合物的含量测定
肖建波 ,曹　慧 ,向海艳 ,周春山＊
(中南大学化学化工学院 ,长沙 410083)
摘　要:本文建立了拳卷地钱中黄酮类化合物的含量测定方法。采用改进的铝盐显色分光光度法测定拳卷地
钱总黄酮含量;采用反相高效液相色谱法测定拳卷地钱黄酮类化合物中芹菜素 、槲皮素和木犀草素的含量。 分
光光度法表明:总黄酮含量在 1.6 ～ 16μg/mL范围内 ,线性关系良好 ,加标回收率在 97.61 ～ 101.59%, 相对标准
偏差 0.16%;反相高效液相色谱法表明平均加标回收率 96.79～ 101.13%,相对标准偏差 0.66～ 1.52%。本方法
用于拳卷地钱中黄酮类化合物含量的测定准确可靠 , 结果令人满意。
关键词:拳卷地钱;黄酮类化合物;测定
中图分类号:R282.710.3　
Introduction
Marchantia convoluta L.(Chinese name Quan Juan Di-
Qian)belongs to family Liverworts and genus Marchantia
L.[ 1] .In China , there are a large number of broyophytes
plants , especially in Guangxi Province and Guizhou
Province.It s about 3000 kinds of broyophtes paints that
have been reported.But less than 1.8% of them were
used as herbal drugs.Compendium of Materia Medica
recorded Marchantia L.could be used as medicine.But
there are few reports about their chemical constituents and
determination of the medical bioactive ingredients of
Marchantia.L[ 2-8] .According to the resources investiga-
tion and literature systematization , Marchantia convoluta
is a main specie of Marshantia plant in Guangxi.In this
paper abundant flavonoids were found in Marchantia con-
voluta by qualitative analysis , colorimetry and HPLC.
These methods were used to determine flavonoids in
Marchantia convoluta L.with satisfactory result.
Materials ,Reagents and Apparatus
Experimental materials
Marchantia convoluta collected in Shangling City of
Guangxi Province was identified by Zhou Zi-jing , a botany
professor of Biology department of Guangxi Chinese Medi-
cal University.The whole plant , after washing with water
and drying in the shade for several days ,was powered.
Principal reagents
Rutin , apigenin , luteolin and quercetin were provided from
Chinese Medicine Checking Institute;Other reagents are
A.R.
Principal apparatus
756MC UV-Vis photometer (Shanghai);BT8100 liquid
chromatograph with BT8200 UV detector and C-R6A data
186
DOI :10.16333/j.1001-6880.2005.02.018
processing computer(Biotronic).
Experimental
Extraction of total flavones
After carry out an orthogonal test to study the affectors of
extraction rate , the best extraction condition for total
flavones in Marchantia convoluta was discovered as fol-
lowing:temperature , 70 ℃;time , 30 min;times , 3;sol-
vent ,80% alcohol;material and ratio of solvent , 1:15.
The whole plant (63.2144 g)was extracted with 80% al-
cohol(1000 mL)for 30 min at 70 ℃.The solvent was dis-
tilled off under reduced pressure.The residue was constant
volume to 250 mL by methanol.Accurately measuring 2.5
mL above solution to 100 mL measuring flask was constant
volume to 100 mL by methanol.Then the test solution used
for quantitative analysis was prepared.
Preparation of standard sample
Accurately weighting certain quantity of apigenin , luteolin
and quercetin were dissolved in methanol and constant
volume to 100 mL by methanol to prepare standard solu-
tion.In this fixed solution the concentration of apigenin ,
luteolin and meletin were 25 μg/mL , 30 μg/mL and 26
μg/mL.Accurately weighting rutin that had been constant
weight in vacuum condition at -5 ℃ 40 mg was dis-
solved in alcohol and constant volume to 100 mL by alco-
hol to prepare standard solution of rutin.
Fig.1　Curve of linear range
Fig.2　Stability curve of coloration system
Results and Discussion
Determining the content of total flavones by colorime-
try
Determining method
By studying the factors that affect the determination , the
optimun condition for this experiment were found:Adopt-
ing NaNO2-AlCl3-NaOH coloration system determined the
content of total flavones with rutin as standard sample.
Test solution accurately quantified to 25 mL measuring
flask was added 0.5mol/L NaNO2 1.0mL , shook up , laid
5 min , then added 0.3 mol/L AlCl3 1.0 mL , shook up ,
added 1.0 mol/L NaOH 5.00 mL , finally make the con-
tent volume with 60% alcohol ,determined the absorbency
at 525 nm with corresponding reagents as blank solution.
Standard cure
Accurately measuring standard solution of rutin 2.0 ,3.0 ,
4.0 ,5.0 ,6.0 , 7.0 mL detected absorbency according to
the method in Determining method , the regression equa-
tion of concentration X(μg/mL)against absorbency Y was
Y=-0.0153+0.03003X , r=0.9996 , RSD=0.16%.
Linear range
Accurately measuring standard solution of rutin 0.25 ,
0.50 , 1.00 , 2.00 , 3.00 , 5.00.7.00 , 9.00 , 10.00 ,
11.00 , 12.00 ,13.00 mL detected absorbency according to
the method in Determining method.The figure of concen-
tration against absorbency was Fig.1.From Fig.1 we
found the linear range was 1.6 ～ 16 μg/mL in 25 mL
measuring flask.In this range Beer s law is obeyed.
Stability of coloration system
Accurately measuring standard solution of rutin 4.50 mL
detected absorbency for every 5 min according to the
method in Determining method.The figure of time against
absorbency was Fig.2.
We found the coloration solution is stable within 15 min ,
but it will decrease after 20 min.
Experiment of standard addtion recovery
The results of adding standard solution of rutin to test so-
lution of known concentration were listed in Table 1 , the
recovery was in range of 97.61 ～ 101.59%, the average
recovery was 99.28%.
Table 1　The recovery of standard addition obtained by col-
orimetry
Sample
quantity(μg)
Rutin
added(μg)
Determining
(μg) Recovery
Average
recovery
120.1075 40 159.1465 97.61%
120.1075 80 199.8005 99.62% 99.28%
120.1075 120 242.0125 101.59%
120.1075 160 278.3285 98.89%
1872005　Vol.17　No.2 肖建波等:拳卷地钱中黄酮类化合物的含量测定 　
Results of total flavones in Marchantia convoluta
The determination results of total flavones in Marchantia
convoluta L.were listed in Table 2 , the average content of
the test solution is 4.8043μg/mL ,RSD=1.1%.
Table 2　Results of determination
NO.号 Determining (μg) Average(μg) RSD
1 4.7452
2 4.8772
3 4.7891 4.8043 1.1%
4 4.7768
5 4.7334
Determining the content of flavonoids by HPLC
Chromatographic conditions
The reversed phase high performance liquid chromatogra-
phy was set up to determine the content of flavonoids in
Marchantia convoluta.Column ,Kromasil RP-C18;Flowing
phase ,V(methanol):V(acetonitrile):V(acetic acid):V
(phosphoric acid):V(H2O)=200:100:10:10:200;De-
tecting wavelength 350 nm;Flow rate ,0.60 mL/min;Col-
umn temperature , room temperature;The quantity of in-
jecting sample ,6.0μL.
Effect of concentration of methanol in flowing phase to de-
termination
Accurately measuring different volume of methanol fixed
with 10 mL acetic acid , 100 mL acetonitrile ,10 mL phos-
phoric acid and 200 mL H2O as flowing phase , then the
fixed standard solution was sucked off 6.0 μL to carry out
HPLC analysis in terms of the condition in Chro-
matographic conditions.The relationship of retention time
to concentration of methanol in flowing phase was Fig.3.
We found the concentration of methanol has an important
effect on the retention time.With increasing the concen-
tration of methanol , the retention time decreased.
Effect of concentration of acetic acid in flowing phase to
determination
Accurately measuring different volume of acetic acid fixed
with 200 mL methanol ,100 mL acetonitrile ,10 mL phos-
phoric acid and 200 mL H2O as flowing phase , then the
Fig.3　The relationship of retention time to concentration of
methanol in flowing phase
(1.apigenin 2.luteolin 3.quercetin)
fixed standard solution was sucked off 6.0μL to carry out
HPLC analysis in terms of the condition in Chro-
matographic conditions.The relationship of retention time
to concentration of acetic acid in flowing phase was Fig.
4.We found little quantity of acetic acid can change the
retention time of these three compounds largely.
Fig.4　The relationship of retention time to concentration of
acetic acid in flowing phase
(1.apigenin 2.luteolin 3.quercetin)
Separation of flavonoids in HPLC
The HPLC figure of test solution of Marchantia convoluta
L.was Fig.5(A).The HPLC figure of fixed standard sam-
ple was Fig.5(B).We found almost all constituent of
flavonoids in Marchantia convoluta L.were separated from
each other in terms of the condition in Chromatographic
conditions.Rs>1.5.
Fig.5　HPLC chromatogram of the extractive from Marchantia convoluta(A)and standard samples(B)
(1.quercetin 2.luteolin 3.apigenin)
188 天然产物研究与开发　　　　　　　　　　　　　 　　　2005　Vol.17　No.2
Standard curve
Accurately measuring fixed standard solution 1.0 , 2.0 ,
3.0 ,4.0 ,5.0 mL to five 10mL measuring flasks was con-
stant volume by methanol , each was sucked off 6.0 μL to
carry out HPLC analysis in terms of the condition in
Chromatographic conditions , by regression analysis of
each peak area X against concentration Y(μg/mL), the
regression equation obtained was listed in Table 3.
Table 3　Standard curve of HPLC
Sample
Linear range
(μg/mL)
Regression
equation
Correlation
coefficient
Detection
limit(μg)
apigenin 0.4-30.0 Y=-1127+7484X 0.9997 0.2
quercetin 1.0-20.4 Y=-1204+4212.5X 0.9992 0.4
luteolin 0.6-26.8 Y=-2986+13792X 0.9995 0.2
Precision of method
Test solution was sucked off 6.0 μL to carry out HPLC
analysis in terms of the condition in Chromatographic
conditions for 5 times.RSD of apigenin , luteolin and
meletin were 2.49%,1.21% and 0.33%.
Recovery experiment
Accurately measuring certain volume of test solution of
known content to three 25 mL measuring flask was added
different volume of fixed standard solution , then we pre-
pared them to be the analyzing sample for HPLC , finally
we detected the recovery rate.The results were showed in
table 4.The results indicated that the average recovery of
apigenin , luteolin and quercetin were 101.13%, 97.52%
and 96.79%,RSD were 1.52%,0.66%and 0.89% re-
spectively.This method can be used for exact analysis of
the extraction of Marchantia convolute.
Table 4　The recovery of standard addition obtained by HPLC(n=9)
Sample Quantity(μg) Added(μg) Determined(μg) Recovery (%) Average (%) RSD
apigenin 4.91 1.04 2.08 4.16 5.92 7.21 9.21 98.72 103.19 101.48 　　101.13 1.52%
luteolin 3.58 1.20 2.40 4.80 4.66 5.85 8.09 97.53 98.46 96.57 97.52 0.66%
quercetin 8.57 1.00 2.00 4.00 9.18 10.37 12.11 95.92 98.10 96.34 96.79 0.89%
Results of HPLC analysis
Test solution was sucking off 6.0 μL to carry out HPLC
analysis in terms of the condition in Chromatographic
conditions.The sample was determined three times.The
results were showed in Table 5.The average content of
apigenin , luteolin and quercetin were 0.0047%,
0.0035% and 0.0081%,RSD were 2.83%, 2.80% and
3.62% respectively.
Table 5　Results of HPLC analysis
Sample Determined Percentage(%) Average
found(%) RSD(%, n=3)
apigenin 0.0045 0.0048 0.0049 0.0047 2.83
luteolin 0.0034 0.0036 0.0036 0.0035 2.80
meletin 0.0077 0.0085 0.0082 0.0081 3.62
Conclusion
Two methods determining the flavonoids were established
in this article.(1)Assaying of total flavones.The contents
of total flavones in Marchantia convolute were determined
through developer visible spetrophotometry.By studying
the factors that affected the determination , the optimum
conditions for this experiment were found:NaNO2-ALCl3-
NaOH , color-developing agent;15 minutes , color time;525
nm ,wavelength.The data of the content and absorbance
formed a standard curve , namely Y =- 0.0153 +
0.03003X;the recovery of the samples was between
97.61% and 101.59%;the average recovery was
99.28%, and the precision was high(RSD=1.13%,N
=5).The operation was easy , fast and reliable , and it can
be used to determine the assaying of total flavones.(2)
Analysis of flavonoids by HPLC.The reversed phase high
performance liquid chromatography was set up to deter-
mine the content of flavone compound in Marchantia con-
volute.The material steps were as follows:taking acetic
acid-methanol-acetonitrile-phosphoric-acid as the multi-
component mobile phase and eluting comparably , then the
flavone extract was separated and determined directly with
30 minutes.The detection wavelength was 350 nm , the
flow velocity was 0.60mL/min , and the quantitative anal-
ysis was conducted with calibration curve method.The re-
sult indicated that the average recovery of apigenin , lute-
olin and quercetin were between 96.79%and 101.13%,
the relative standard deviation was between 0.66% and
1.52%.This method can be used for exact analyses of the
extraction of Marchantia convolute.
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1892005　Vol.17　No.2 肖建波等:拳卷地钱中黄酮类化合物的含量测定 　
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194 天然产物研究与开发　　　　　　　　　　　　　 　　　2005　Vol.17　No.2