Euphorbia helioscopia L is widespread in China and has a large number of flavonoids. Quercetin glycosides, having useful biological activities, are abundant in this plant, and no validated analytical method has so far been developed for their determination. We, therefore, standardized a reversedphase highperformance liquid chromatography (RPHPLC) assay for quercetin detection. For this, the plant was locally procured and identification was confirmed based on its morphohistological characteristics. Ethyl acetate extracts of leaves, stems, and roots were analyzed by RPHPLC using Agilent 1120 HPLC TCC18 column (250 9 4.6 mm; 5 lm) with UVdetector system. The mobile phase of methanol0.2% phosphoric acid (65:35) solution was used with the flow rate of 1.0 ml min1 at 30C, and the detection was performed at 360 nm wavelength. Our data show that the linear range of quercetin was 0.025–0.150 mg.ml1 (r = 0.9995; n = 6) with the recovery rate of 97.50–103.30% (average 100.40%; RSD = 2.28%). The target component was baseline separated during only the period of 9 min. The repeatability of RPHPLC analysis was demonstrated with an RSD of 1.77% (n = 6), and the highest quercetin content (average 1.42 mg g1dryweight) was present in leaves. It was, therefore, concluded that RPHPLC is a simple, rapid, accurate, and sensitive method for the detection of quercetin from Euphorbia helioscopia L.
Trang 1O R I G I N A L P A P E R
Quantitative Analysis of Quercetin in Euphorbia helioscopia L
by RP-HPLC
Hai Peng Liu•Xiao Feng Shi •You Cheng Zhang•
Zhong Xin Li• Lin Zhang•Zhe Yuan Wang
Published online: 17 February 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Euphorbia helioscopia L is widespread in
China and has a large number of flavonoids Quercetin
glycosides, having useful biological activities, are
abun-dant in this plant, and no validated analytical method has so
far been developed for their determination We, therefore,
standardized a reversed-phase high-performance liquid
chromatography (RP-HPLC) assay for quercetin detection
For this, the plant was locally procured and identification
was confirmed based on its morpho-histological
charac-teristics Ethyl acetate extracts of leaves, stems, and roots
were analyzed by RP-HPLC using Agilent 1120 HPLC
TC-C18 column (250 9 4.6 mm; 5 lm) with UV-detector
system The mobile phase of methanol-0.2% phosphoric
acid (65:35) solution was used with the flow rate of
1.0 ml min-1at 30°C, and the detection was performed at
360 nm wavelength Our data show that the linear range of
quercetin was 0.025–0.150 mg.ml-1 (r = 0.9995; n = 6)
with the recovery rate of 97.50–103.30% (average
100.40%; RSD = 2.28%) The target component was
baseline separated during only the period of 9 min
The repeatability of RP-HPLC analysis was demonstrated
with an RSD of 1.77% (n = 6), and the highest quercetin
content (average 1.42 mg g-1dry-weight) was present in
leaves It was, therefore, concluded that RP-HPLC is a
simple, rapid, accurate, and sensitive method for the
detection of quercetin from Euphorbia helioscopia L
Keywords Chinese herb Euphorbia helioscopia L Quercetin RP-HPLC
Introduction Euphorbia helioscopia L (Family Euphorbiaceae) is widely distributed in most parts of China [1] It has been tradi-tionally used for the prevention, improvement, or cure of various diseases such as liver cancer, esophagus cancer, nasopharyngeal carcinoma, bronchitis, acute glomerulone-phritis, and epidemic parotitis in China for centuries [2 7] Phytochemical studies reveal that Euphorbia helioscopia L contains a variety of secondary metabolites Terpenoids [8 15], flavonoids [16, 17], tannins [18,19], steroids and lipids [9] were isolated from this plant during the twentieth century Some of the compounds were found to have tumor [20–22], bacteriostatic [23], allergic and anti-asthmatic [24], insecticidal [25], and inhibitory effect on mushroom tyrosinase in vitro [26] The recent studies have shown that terpenoids and flavonoids are the main biologi-cally active substances The flavonoids isolated from Euphorbia helioscopia L mainly include quercetin [17], kaempferol, and flavonoid glycosides such as quercetin-3-D-galactosidase [17], quercetin 3-b-glucoside, quercetin 3-b-galactosidase, quercetin 3-b-galactosidase-2-gallic acid [27], and quercetin-5, 3-2-D-galactosidase [16]
Quercetin glycosides appear to predominate in Euphorbia helioscopia L Quercetin is known to have a variety of biological activities and may play an important role in the medicinal effects of Euphorbia helioscopia L To our knowledge, no validated analytical method has been so far reported for determining quercetin contents in Euphorbia helioscopia L Therefore, in this study, we developed a rapid, simple and selective reversed-phase high-performance
H P Liu Y C Zhang (&) Z X Li L Zhang Z Y Wang
Department of General Surgery, Lanzhou University Second
Hospital, 82 Cuiyingmen, Chengguan District,
Lanzhou 730030, Gansu, China
e-mail: zhangychmd@yahoo.com.cn
X F Shi
Institute of Materia Medica, Gansu Academy of Medical
Sciences, Lanzhou 730050, Gansu, China
DOI 10.1007/s12013-011-9161-0
Trang 2liquid chromatography (RP-HPLC) method for the detection
of quercetin contents in Euphorbia helioscopia L
Materials and Methods
Samples
The plant of Euphorbia helioscopia L was procured from
Dingxi, Gansu Province in China during the months of
June, July and August, 2008 The plant identification was
authenticated based on its specific morphological and
his-tological characteristics by the School of Life Sciences,
Lanzhou University The plant was air-dried immediately
after collection, and some of them were first separated into
roots, stems and leaves and then preserved The samples
were stored in bags and kept in a dry and cool place for
later use
Instrumentation
Analytical balance (AE260, Mettle Company,
Switzer-land), Rotary Evaporators (BU¨ CHI AG, Switzerland), and
Agilent 1120 High-Performance Liquid Chromatography
system (Agilent Technologies, Santa Clara, CA, USA)
were used in this study
Chemicals and Reagents
Standard quercetin was obtained from National Institute for
the Control of Pharmaceutical and Biological Products
(Batch number: 100081-200406) Methanol used was of
HPLC-specific grade (Yu Wang-Chemical Industry
Com-pany, Shan Dong; China)
Sample Preparation
The pulverized plant material (about 60 mesh; 2 g) was
reflux-extracted twice, using 50 ml of methanol for 1 h
each time in a water bath at 80°C The extracts were
filtered through filter paper (Whatman filter paper,
1003090/grade No 3) and collected in a flask The pooled
extract was evaporated under vacuum to dryness The
residue was dissolved in 30 ml of distilled water and
extracted six times using 30 ml of petroleum ether each
time The petroleum ether extraction liquid was discarded
and 10 ml of HCL (10%) was added to water liquid extract
which was extracted for 30 min in a water bath at 90°C
After rapid cool off, the liquid was extracted five times
using 30 ml of ethyl acetate each time The ethyl acetate
extraction was collected, evaporated to dryness,
re-dis-solved in methanol, transferred to a 25-ml measuring flask
and filtered with 0.45-lm Millipore membrane filter before use
RP-HPLC Quercetin contents in Euphorbia helioscopia L were ana-lyzed by using RP-HPLC At first, three different buffers including methanol buffer (buffer A), acetonitrile buffer (buffer B), and 0.4% phosphoric acid buffer (buffer C), were mixed at three different ratios such as 60:20:20; 65:20:15; and 70:15:15 It was found that the whole separation course required almost 21 min and the peak of quercetin was obtained at the 15th minute and was asymmetric Thus, we opted to use 0.2% phosphoric acid buffer (buffer D) and mixed buffer A and buffer D at the ratios of 70:30; 60:40; and 65:35 The quercetin peak time now shifted to the fifth minute point and the whole separation course required 9 min while the peak obtained was symmetric
Since quercetin is reported to have the maximum absorption at 250 and 360 nm wavelengths, we, therefore, compared the shapes of quercetin peaks at these wave-lengths and found that the shape of the peak was more symmetric and higher at the detection wavelength of
360 nm We used Agilent 1120 system with TC-C18 col-umn (250 9 4.6 mm, 5 lm) The mobile phase was methanol-0.2% phosphoric acid (65:35) solution The flow rate was 1.0 ml min-1 The column temperature was 30°C and the detection wavelength was 360 nm The quercetin peaks were identified and quantified against the external reference standards Quercetin standard was purchased from the National Institute for the Control of Pharmaceu-tical and Biological Products, China The solvents used were of spectra analytical grade and were filtered through 0.45 lm filters before use
Statistical Analysis The data were acquired and analyzed by Agilent 1120 HPLC software The standard curve of quercetin was obtained by plotting concentrations against peak area and the regression equation and correlation coefficient (r2) were determined The sample analyses were performed in trip-licate and the mean values were calculated RSD \ 3% was considered as significant for the precision (repeat-ability) and the accuracy (recovery) of the method used
Results RP-HPLC Optimization Conditions The best mobile phase was methanol-0.2% phosphoric acid (65:35) solution, the flow rate was 1.0 ml min-1, the
Trang 3column temperature was 30°C, and the detection
wave-length was 360 nm The standard (reference) and sample
peaks of quercetin are shown in Figs.1and2, respectively
Validation
Six samples from the same source were reflux-extracted,
processed in parallel, analyzed by RP-HPLC, and
com-pared The relative standard deviation (RSD) was 1.77%
Recovery Rate
By adding different concentrations of standard quercetin (e.g., 4.05, 2.70, and 1.35 mg g-1) into samples with known (2.69 mg g-1) quercetin content, the recovery rate
of the method was determined at the reaction conditions used As shown in Table 1, the recovery rate ranged from 97.50 to 103.30% (average 100.40%; RSD 2.28%;
n = 6)
Fig 1 Standard peak of quercetin Quercetin content in Euphorbia
helioscopia L was determined by RP-HPLC using Agilent 1120
system with TC-C 18 column (250 9 4.6 mm, 5 lm) and UV detector
system The assay was optimized using the mobile phase of
methanol-0.2% phosphoric acid (65:35) solution and a flow rate of
1.0 ml min -1 at the column temperature of 30°C and the detection
wavelength of 360 nm The standard solutions of quercetin were prepared at the concentrations of 0.025, 0.050, 0.075, 0.100, 0.125 and 0.150 mg ml -1 The least-square regression analysis of quercetin calibration graph was: y = 6.00460 9 108 x ? 41710.9 (r = 0.9995) where, y represents peak area, x represents concentration in mg ml -1
and r represents correlation coefficient
Fig 2 Sample peak of quercetin Quercetin content in Euphorbia
helioscopia L were determined by RP-HPLC as described before.
Quercetin sample peaks were identified and quantified against the
external reference standards The assay was performed in triplicate and average quercetin content (mg g-1) was calculated
Trang 4The linearity of the method was found to be from 0.025 to
0.150 mg ml-1 The standard solutions of quercetin were
prepared at the concentrations of 0.025, 0.050, 0.075, 0.100,
0.125 and 0.150 mg ml-1 The least-square regression
analysis of quercetin calibration graph was: y = 6.00460 9
108 x ? 41710.9; r = 0.9995 where y represents peak area,
x represents concentration in mg ml-1, and r represents
correlation coefficient
Quercetin Contents of Whole Plant at Different Times
The average quercetin contents in the whole plant
were 3.57 mg g-1 (RSD = 1.22%), 2.69 mg g-1(RSD =
1.49%), and 1.48 mg g-1(RSD = 1.17%) in June, July, and
August, respectively The results are shown in Table2
Quercetin Contents of Leaves, Stems and Roots
The average quercetin contents of leaves, stems, and roots
were 1.42 mg g-1 (RSD = 1.38%), 0.02 mg g-1(RSD =
1.42%) and 0.04 mg g-1 (RSD = 1.87%), respectively
The results are shown in Table3
Discussion HPLC method is gaining importance for the analysis of plant extracts Although TLC is also a simple method, it sometimes produces doubtful results RP-HPLC has been previously used for the analysis of flavonoids in plants e.g
to distinguish species based on quantitative variation of flavonoids [28] and for quantitative analysis of flavonoid aglycones [29] The analysis of quercetin content in Euphorbia helioscopia L using RP-HPLC has not been previously reported, and in this study, we standardized the RP-HPLC method for the detection of quercetin in Euphorbia helioscopia L
During optimization of RP-HPLC, we initially used different mobile phases comprising three different buffers mixed at three different ratios Since the quercetin peak obtained was asymmetric, we re-adjusted the mobile phase and found that methanol buffer mixed with 0.2% phos-phoric acid buffer at the ratio of 65:35 yielded a highly symmetric quercetin peak at the flow rate of 1.0 ml min-1
at 30°C Since the previous studies [30,31] indicate that quercetin has the maximum absorption at 250 and 360 nm,
we tested and found that the shape of the peak was more symmetric at the detection UV wavelength of 360 nm using Agilent 1120 system with TC-C18column (250 9 4.6 mm,
Table 1 Recovery rates (n = 6)
Background (mg g-1) Added (mg g-1) Detected (mg g-1) Recovery rate (%) Average recovery rate (%) RSD (%)
RSD represents relative standard deviation
Table 2 Quercetin contents of whole plant at different times
Month No Quercetin contents
(mg g-1)
Average (mg g-1)
RSD (%)
2 3.62
3 3.55
2 2.65
3 2.69
2 1.49
3 1.49
RSD represents relative standard deviation
Table 3 Quercetin contents of roots, stems and leaves Parts No Quercetin contents
(mg g-1)
Average (mg g-1)
RSD (%)
2 1.4308
3 1.4274
2 0.0208
3 0.0214
2 0.0428
3 0.0443 RSD represents relative standard deviation
Trang 55 lm) The quercetin peaks were identified and quantified
against the external reference standards
For validation of analytical methods, the guidelines from
the International Conference on the Harmonization of
Tech-nical Requirements for the Human Use (ICH) [32] and USP 24
[33] recommend that the tests of linearity, sensitivity,
preci-sion, specificity, and accuracy of the method be performed
The type of method and its use determine the parameters to be
evaluated, particularly when the samples are complex
bio-logic matrices such as extractive solutions from plants In this
study, the linearity of the HPLC method for quercetin was
found to be in the range 0.025–0.150 mg ml-1 at six
con-centration levels and the representative linear equation for
quercetin was: y = 6.00460 9 108 x ? 41710.9 (n = 6;
r = 0.9995) The method was found to be highly sensitive as
the limit of detection (LOD) values were within the range
0.025–0.150 mg ml-1 Since the target component was
baseline separated during the period of merely 9 min, the
analytical method also proved to be appreciably rapid
For further validation, we also determined precision
(repeatability) and accuracy (recovery) of the method The
repeatability of RP-HPLC analysis was demonstrated with
an RSD of 1.77% (n = 6) The accuracy of HPLC method for
quercetin analysis, based on percent recovery, was
deter-mined by adding different concentrations of standard
quer-cetin (e.g., 4.05, 2.70, 1.35 mg g-1) into samples with
known quercetin content (i.e., the background of
2.69 mg g-1) The recovery rate for quercetin was found to
be 97.50–103.30% (average 100.40%; RSD = 2.28%),
indicating that the analyses performed were highly accurate
We found that quercetin content in the plant were the
highest with an average of 3.57 mg g-1 dry weight in the
month of June, followed by July, and August samples with
the average contents of 2.69 and 1.48 mg g-1dry weight,
respectively This indicates that June is the best harvest
time for optimal quercetin yield from Euphorbia
helios-copia L More importantly, leaves had the highest
quer-cetin content with an average of 1.42 mg g-1 dry weight
while stems and roots had lower quercetin contents (0.02
and 0.04 mg g-1, respectively) Thus, we conclude that
quercetin is mainly distributed in leaves of Euphorbia
helioscopia L
HPLC with diode array detector (DAD) and electrospray
ionization mass spectrometry (ESI–MS) was previously
used to detect 13 bioactive compounds in Flos Lonicerae
In this study [31], the optimal chromatographic conditions
were obtained on a C18 column (250 9 4.6 mm; 5 lm) at
30°C The mobile phase comprises acetic acid aqueous
(0.4% v/v) and acetonitrile with a gradient elution at the
flow rate of 1 ml min-1 at 360 nm The method provided
satisfactory precision and accuracy In another study [34], a
capillary zone electrophoresis method was established for
analysis of aglycone quercetin in mulberry (Morus alba L.)
leaves Baseline separation of all compounds was obtained within 16.5 min under the following conditions: 150 mM boric acid (pH 10.0) using a fused-silica capillary with an effective length of 42.5 cm (50 lm inner diameter) at 32°C and the voltage of 15 kV
Although all the above methods provide satisfactory pre-cision and accuracy in determination of flavonoids, there are also some caveats involved As for example, the capillary zone electrophoresis results are influenced by too many factors such
as electrolyte concentrations and pH, surfactant concentra-tions, organic solvents, temperature, and the voltage used We used HPLC for determination of quercetin content because it
is a relatively simple method as compared with HPLC com-bined with ESI–MS whereas the efficiency of the latter approach may be slightly better In this study, however, we have determined only one constituent of Euphorbia helios-copia L; therefore, more studies will be required to further evaluate sensitivity, precision and accuracy of this method for simultaneous determination of different flavonoids in the plant samples collected preferably from different geographic regions in China and/or elsewhere
In conclusion, an RP-HPLC method was successfully developed in this study for the determination of quercetin contents in Euphorbia helioscopia L The data show that RP-HPLC is a powerful analytical technique for this pur-pose which has the potential benefits of high sensitivity, accuracy, reproducibility, and time-saving
Acknowledgments We thank Lanzhou City Science Foundation (Grant # 06-1-04) and Gansu Provincial Science and Technology Council (Grant # QS061-C33-40) for financial support.
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