Food Anal.

Methods (2010) 3:90–97

DOI 10.1007/s12161-009-9091-2

Analysis of Flavonoids in Portulaca oleracea L. by UV–Vis

Spectrophotometry with Comparative Study on Different
Extraction Technologies
Hongbin Zhu & Yuzhi Wang & Yuxuan Liu & Yalin Xia &
Tian Tang

Received: 25 March 2009 / Accepted: 26 May 2009 / Published online: 12 June 2009
# Springer Science + Business Media, LLC 2009

Abstract Portulaca oleracea L. is a traditional edible and Introduction

medicinal plant in China. Flavonoids are one of the main
active ingredients of this plant. Five extraction technologies Portulaca oleracea L. (P. oleracea L., purslane) is a
of flavonoids from P. oleracea L. were investigated and common, herbaceous succulent annual weed, which is
compared, including microwave-assisted extraction, ultra- distributed extensively in temperate and tropical regions
sonic extraction, reflux extraction, Soxhlet extraction, and worldwide (Radhakrishnan et al. 2001; Garti et al. 1999). It
marinated extraction. The results showed that microwave- has been used as a kind of food and medicinal plant for
assisted extraction was most suitable for the extraction of thousands of years in China. P. oleracea L. is of abundant
flavonoids from P. oleracea L. because of its high effect nutrition with contents of proteins, carbohydrates, Ca, K,
and short extraction time. The found optimum extraction Zn, and Na (Aberoumand 2008). As a kind of Chinese
conditions were that the ethanol concentration was 70% traditional medicine, P. oleracea L. is very important
(v/v), solid–liquid ratio was 1:50, extracting temperature because of its special medical function, and it has been
was 50 °C and irradiation time was 9 min. Quantification used traditionally for the treatment of dysentery with
was performed by means of UV–Vis spectrophotometry bloody stools and externally for boils and sores, eczema,
with chromogenic system of NaNO2–Al (NO3)3–NaOH. erysipelas, and insect and snake bites (Chen et al. 2003;
Under the optimum conditions, the calibration curve for the Zhang et al. 2002; Yazici et al. 2007; Palaniswamya et al.
analyte was linear with the correlation coefficients greater 2004).
than 0.9999. The average recovery was 102.6%, and its Among the effective ingredients of many Chinese
RSD was 1.13%(n=5). Eight types of P. oleracea L. herbal medicines, flavonoids are one of the most
according to different habits were investigated. The total popular compounds in the plant kingdom. Flavonoids
content of flavonoids was 7.16, 7.10, 9.38, 6.82, 6.78, have received much attention because of their richness
11.36, 5.12, and 1.76 mg g−1, respectively. in species numbers and effectiveness in reducing blood
lipid, as an anti-oxidative, in assimilating cholesterol,
Keywords Food Analysis . Portulaca oleracea L. . inhibiting thrombosis, dilating the coronary artery, etc.
Flavonoids . Microwave-Assisted Extraction . (Deng et al. 2008; De Souza et al. 2008; Su et al. 2008;
UV–Vis Spectrophotometry Shao et al. 2007; Volpi and Bergonzini 2006; Zhang et al.
2007; Keith et al. 2007).
Recently, the development of flavonoids in medical
H. Zhu : Y. Wang (*) : Y. Liu : Y. Xia : T. Tang and food fields becomes a hot research. Flavonoids and
State Key Laboratory of Chemo/Biosensing and Chemometrics, polysaccharides are the most abundant effective com-
College of Chemistry and Chemical Engineering, ponents in P. oleracea L. It is reported that seven kinds
Hunan University,
Changsha 410082, People’s Republic of China of flavonoids were found in P. oleracea L., including
e-mail: [email protected] quercetin, kaempferol, myricetin, apigenin, luteolin,
Food Anal. Methods (2010) 3:90–97 91

genistein, and genistin (Chen et al. 2003; Zhang et al. Materials and Methods
2007). The extraction and separation of flavonoids with
high biological activity and high content is very impor- Collection of Samples
tant for the medical and food industry. Therefore, it is of
great significance to study the extraction and In order to investigate the flavonoids content of P. oleracea
the determination technologies of flavonoids from L. samples collected in different months, three different
P. oleracea L. P. oleracea L. samples were used as experimental materials.
In traditional methods, the extraction of flavonoids Two of the three samples are traditional Chinese medicine
from P. oleracea L. is usually carried out by means of (TCM) samples purchased from Traditional Chinese Med-
marinated extraction and reflux extraction. Being a newly icine Co. Ltd (Hunan, China); one of the TCM samples was
arisen extraction method, ultrasonic extraction has also collected in August 2007 and another TCM sample was
been used for the extraction of flavonoids (Jin et al. 2008; collected in July 2007. We collected the third sample (wild
Chen et al. 2008a). Unfortunately, flavonoids could not be P. oleracea L.) from the suburb of Changsha (Hunan,
extracted sufficiently by the mentioned methods due to the China) in June 2008.
high impurity content and the long extraction time, which
restrict the development of the medicine at a certain Samples Preparation
extent. Therefore, it is necessary to develop a more
effective and reliable extraction method. Microwave- Fresh samples were cleaned with water, and some of the
assisted extraction seems promising to fulfill the purpose. fresh samples were split as roots, stems, and leaves for
Microwave-assisted extraction has the advantage of strong study. All of the samples were crushed into powder by a
penetrating force, high selectivity, high heating ability, plant grinder after being dried at 50 °C. The powders of the
less solvent consumption, short extraction time, high samples were kept in an airer.
extraction yield (Ganzler et al. 1990), and as a new kind
of extraction technology it is especially suitable for Chemicals and Instruments
extracting heat-sensitivity components and natural active
constituents (Chen et al. 2008b). In this paper, pressure
self-control microwave decomposition system was used to 1. Chemicals: authentic standard rutin (>97%) was pur-
extract flavonoids from P. oleracea L. The temperature of chased from Guoyao Group of Chemical Reagents Ltd.
the system can be controlled at a certain value. Therefore, (China). All reagents and chemicals used in this work
it is effective to avoid the loss of the components that are were of analytical grade. Triple-distilled water was used
heat sensitive. In the present study, microwave-assisted throughout the study.
extraction, a simple and effective method, has been 2. Instruments: pressure self-control microwave decom-
developed. Meanwhile, in order to demonstrate the position system (MDS-2002AT, Shanghai, China),
advantages of microwave-assisted extraction, the extrac- Auto Science AS-2060B Ultrasonic Cleaner (Tianjin,
tion efficiencies of flavonoids with various methodologies China), and Soxhlet extractor (250 mL, Changsha,
were compared. China) were used for extraction. High-speed centri-
Several methods for the determination of flavonoids fugation (TGL-16C, Shanghai, China) was employed
in medicinal plants have been reported, including to accelerate the phase separation process. A
thin-layer chromatography, high-performance liquid versatile plant pulverizer (FW-100, Beijing, China)
chromatography, capillary electrophoresis, and spectro- was used to make the plant materials into powder.
photometry. Among all these methods, only spectropho- A rotary evaporator (RE52CS, Shanghai, China) was
tometry can be used to determinate the total amount of used to concentrate the sample solution. UV–Vis
the flavonoids. UV–Vis spectrophotometry is the most spectrophotometer V-530 (JASCO, Japan) was used
extensively used method to determine the total content for determination.
of flavonoids in plant. In this technology, direct
mensuration and coloration methods are both common,
but coloration method is better than direct mensuration
because coloration method will not be disturbed by Extraction of Flavonoids
other components such as polysaccharides and saponin.
In this study, we present an easy method that uses UV– Before extraction, P. oleracea L. was crushed into powder
Vis spectrophotometry with chromogenic system of by versatile plant pulverizer. The powders of the samples
NaNO2–Al(NO3)3–NaOH to determine the total amount were degreased by Soxhlet extractor with petroleum ether
of the flavonoids from P. oleracea L. until the color of the eluate became colorless. Flavonoids
92 Food Anal. Methods (2010) 3:90–97

were extracted from P. oleracea L. by five extraction 6 min. Secondly, 0.5 mL of the Al(NO3)3 (10%) solution
methods as follows: was added to the volumetric flask, shaken, and was left to
stand for 6 min. Finally, 3.0 mL of the NaOH (4.3%)
1. Microwave-assisted extraction solution was added to the volumetric flask, followed by
Powders of the samples (0.5 g) were accurately weighed addition of water to the scale, shaken, and left to stand for
and placed in a sealed vessel by adding 25 mL of the 15 min before determination. Using the sample solution
ethanol–water (70:30, v/v) solvent, then the sealed vessel without coloration as reference solution and 500 nm as
was placed into the pressure self-control microwave determination wavelength, the coloration method was used
decomposition system for microwave-assisted extraction. to determine the content of flavonoids in the sample by
The extraction temperature was 50 °C and the irradiation ultraviolet–visible detector.
time was kept at 9 min. After microwave-assisted extrac-
tion, the extract was centrifuged at 11,000 rpm for 30 min; Preparation of Standard Solution
the supernatant was diluted to 25 mL in a volumetric flask
(25 mL) by the ethanol–water (70:30, v/v) solvent. A standard solution (0.16 mg/mL) of rutin was prepared as
follows: rutin (200 mg) was accurately weighed and
2. Ultrasonic Extraction dissolved in ethanol–water (70:30, v/v) solution, and then
Powders of the samples (0.5 g) were accurately weighed the solution was diluted to 100 mL in a volumetric flask
and placed in a sealed vessel by adding 25 mL of the (100 mL) by ethanol–water (70:30, v/v) solvent. Two
ethanol–water (70:30, v/v) solvent, then the sealed vessel milliliters of this solution was removed and diluted to
was placed in the ultrasonic cleaning bath for ultrasonic 25 mL in a volumetric flask (25 mL) by ethanol–water
extraction for an hour at room temperature (25 °C). Then (70:30, v/v) solvent.
the following works were done (as the description in
microwave-assisted extraction). Standard Curve

3. Condensing Reflux Extraction Six portions of the rutin solution were accurately removed
Powders of the sample (0.5 g) were accurately weighed (0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 mL, respectively) in six
and placed in a sealed vessel by adding 25 mL of the volumetric flasks (10 mL), then the following works were
ethanol–water (70:30, v/v) solvent, then the sealed vessel done (as the description in determination of flavonoids).
was placed into the reflux device, followed by the Using the concentration of rutin standard solution as
extraction for 2.5 h. Then the following works were done abscissa and the absorbency as y-coordinate, the linear
(as the description in microwave-assisted extraction). chart was constructed and the standard curve is shown in
Fig. 1, the regression equation was A= −0.00171 +
4. Soxhlet Extraction
11.58482C (r=0.9999).
Powders of the sample (0.5 g) were accurately weighed
and placed in Soxhlet extractor by adding 80 mL of the
ethanol–water (70:30) solvent, followed by the extraction
for 5 h, and then the extract solution was concentrated.
Then the following works were done (as the description in
microwave-assisted extraction).
5. Marinated Extraction
Powders of the sample (0.5 g) were accurately weighed
and placed in a sealed vessel by adding 25 mL of the
ethanol–water (70:30, v/v) solvent, followed by the extrac-
tion for 48 h at room temperature (25 °C). Then the
following works were done (as the description in
microwave-assisted extraction).

Determination of Flavonoids

Firstly, 2 mL of the sample solution was accurately

removed in a volumetric flask (10 mL) by adding 0.6 mL
of NaNO2 (5%) solution, shaken up and then standing for Fig. 1 Standard curve
Food Anal. Methods (2010) 3:90–97 93

Fig. 2 The color reaction of

flavonoids and chromogenic
system

Results and Discussion Selection of Detective Wavelength

The Color Reaction of Flavonoids and Chromogenic Ultraviolet scanning (presented in Fig. 3) was performed to
System determine the detective wavelength. The solution of rutin
and the solution of sample were prepared (as the description
The basic structure of flavonoids was presented in Fig. 2a, in determination of flavonoids). From Fig. 3, we can see
and most of the flavonoids in P. oleracea L. have 3′,4′- that both rutin and sample showed maximum absorption at
dihydroxy-substituted structure (as shown in Fig. 2b). the wavelength of 500 nm in visible range, so the
Flavonoids with 3′,4′-dihydroxy-substituted structure can wavelength of 500 nm can be selected for detection of
show special color by reacting with the system of NaNO2– target analyte.
Al(NO3)3–NaOH. The color reaction of flavonoids and
chromogenic system is presented in Fig. 2c. As shown in Comparison of Extraction Efficiency with Different
Fig. 2, this method is based on the reaction of aluminum Solvents
ion with flavonoid at alkaline medium forming red chelates.
By measuring the absorption of such red chelates, it is The extraction of the three mentioned solvents was
possible to determine the flavonoids. investigated with microwave-assisted extraction, including

Fig. 3 The UV–Vis spectra of rutin and sample solution Fig. 4 Effect of the solvents on extracting efficiency
94 Food Anal. Methods (2010) 3:90–97

methanol–water, ethanol–water, and acetone–water to tion effects of the optimum levels of each factor may not be
check their efficiency for extraction of flavonoids from P. the optimum extraction conditions. The orthogonal test is a
oleracea L., and the results are shown in Fig. 4. From scientific method of arranging multi-factors. Based on a
Fig. 4, we can see that extraction of flavonoids from P. single factor test, the optimal extraction conditions can be
oleracea L. with ethanol–water gave the highest efficiency. worked out by orthogonal test. In this paper, the optimal
extraction conditions of microwave-assisted extraction were
Selection of Extraction Method worked out through single factor test and orthogonal test.

Five different extraction methods were investigated, includ- 1. Single Factor Test
ing microwave-assisted extraction, marinated extraction, There are many factors affecting the extraction yield,
Soxhlet extraction, reflux extraction, and ultrasonic extrac- among which the extraction solvent, solid–liquid ratio,
tion, and the flavonoids of these samples were determined radiating time of microwave, and extracting temperature are
by UV–Vis spectrophotometry. The results are shown in the main factors. Single factor test was performed by one
Table 1. factor varied with different levels while other factors fixed.
From the results shown in Table 1, we can find that
microwave-assisted extraction had the highest extraction (a) Selection of Extraction Solvent
efficiency. Compared with Soxhlet extraction, the extrac- The extraction by three mentioned solvents of different
tion time of microwave-assisted extraction was shortened strengths was investigated; other conditions of microwave
by 97%, and compared with reflux extraction, 94% of radiation was kept the same (ratio of material to solution
extraction time was shortened. was 1:30, extracting temperature was 35 °C, and extraction
Microwave-assisted extraction is a rapid, efficient, and time was 5 min). As shown in Fig. 4, the absorbance was
economic method for extracting flavonoids from P. oler- the highest when extraction solvent was ethanol of 70%
acea L. Unlike classical conductive heating methods, (v/v), so ethanol–water of 70% (v/v) was chosen in the
microwave heats the whole sample simultaneously because following experiments.
microwave rays are very penetrating. The microwave rays
travel through the microwave-transparent extraction medi- (b) Selection of Solid–liquid Ratio
um and reach the inner vascular bundles and cell systems of The extraction by solid–liquid ratio of 1:10 (g/mL), 1:15,
the plant material. The result is a sudden rise in temperature 1:20, 1:30, 1:40, and 1:50 was investigated, other con-
inside the material; the internal pressure exceeds the ditions of microwave radiation was kept same (ethanol–
capacity of expansion of the cell walls, so the cell walls water of 70%, extracting temperature was 35 °C, and
cracked and the substances in the cells are then free to flow extraction time was 5 min). As shown in Fig. 5, the
out of the cells into the extract solvent. Therefore, extraction efficiency increased when the ratio increased
microwave-assisted extraction had the highest extraction from 1:15 to 1:40 and then tended to remain constant in the
efficiency and it was most suitable for extracting flavonoids ratio range of 1:40–1:50. Hence, the solid–liquid ratio of
from P. oleracea L. 1:40 (g/mL) was chosen in the following experiments.
(c) Selection of Extraction Time
Optimization of Microwave-Assisted Extraction Conditions

There are many factors affecting the extraction of flavo- The radiating time of microwave of 5, 6, 7, 8, 9, and
noids from P. oleracea L. We worked out the optimum 10 min were investigated; other conditions of microwave
levels of each factor by the single factor test. However, radiation was kept the same (ethanol–water of 70%, solid–
because of the interaction among the factors, the combina- liquid ratio was 1:40, and extracting temperature was

Table 1 Comparison of different extraction methods (n=5)

Extraction methods Extraction time Numbers of Absorbance of Average extraction RSD % (n=5)
(min) the samples each sample efficiency (mg/g)

Microwave-assisted extraction 9 5 0.390, 0.393, 0.391, 0.387, 0.388 7.1 0.29

Ultrasonic extraction 60 5 0.371, 0.373, 0.373, 0.376, 0.373 6.7 0.48
Soxhlet extraction 300 5 0.391, 0.390, 0.389, 0.390, 0.388 7.0 0.29
Condensing reflux extraction 150 5 0.378, 0.374, 0.375, 0.373, 0.374 6.8 0.51
Marinated extraction 2880 5 0.313, 0.316, 0.312, 0.317, 0.313 5.6 0.70
Food Anal. Methods (2010) 3:90–97 95

Table 3 Results of L9 (34) orthogonal test

A B C D Abs

1 60% 1:30 7 40 0.289

2 60% 1:40 8 45 0.295
3 60% 1:50 9 50 0.368
4 70% 1:30 8 50 0.373
5 70% 1:40 9 40 0.387
6 70% 1:50 7 45 0.373
7 80% 1:30 9 45 0.310
8 80% 1:40 7 50 0.347
9 80% 1:50 8 40 0.307
K1 0.952 0.972 1.009 0.983 T=3.049
K2 1.133 1.029 0.975 0.978
K3 0.964 1.048 1.065 1.088
Fig. 5 Effect of microwave-assisted extraction conditions: solid–
liquid ratio (1:10, 1:15, 1:20, 1:30, 1:40, and 1:50), radiation time (5, k1 =K1/3 0.317 0.324 0.336 0.328
6, 7, 8, 9, and 10 min), and extraction temperature (30, 35, 40, 45, 50, k2 =K2/3 0.378 0.343 0.325 0.326
and 55 °C) k3 =K3/3 0.321 0.349 0.355 0.363
R 0.061 0.025 0.030 0.037
35 °C). As shown in Fig. 5, the extraction efficiency
increased when microwave treatment time increased from 4
to 8 min and then tended to remain constant from 8 to extraction. The experimental design and the results are
10 min. When other factors are fixed, the extraction shown in Tables 2 and 3.
efficiency increased when extraction time increased, and The K and R values were calculated and are listed in
then it reached equilibrium in a certain time. Hence, the Table 3. K value is the average extracting efficiency of
radiating time of microwave of 8 min was chosen in the every factor at each level. R value is the range of K value.
following experiments. Range analysis results of the orthogonal experiment
indicated that ethanol concentration is the most important
(d) Selection of Extraction Temperature
factor, followed by extraction temperature, radiating time of
Microwave conditions of ethanol–water of 70%, solid– microwave, and solid–liquid ratio. We also can see that K2
liquid ratio of 1:40, and the radiating time of 8 min were has the highest K value in factor A, which means that,
fixed, and extraction temperature varied with different comparing with other levels of factor A, level two has the
levels (30, 35, 40, 45, 50, and 55 °C) were investigated. highest extracting efficiency and it is the most suitable level
As shown in Fig. 5, the extraction efficiency was the for extraction. Based on the same principle, level three of
highest when the extraction temperature was 45 °C. factor B, level three of factor C, and level three of factor D
However, when the temperature was higher, the extraction were chosen for extraction. In other words, the maximum
efficiency decreased. It could speculate that some of the yield of the active component was obtained at the following
heat-sensitive components in P. oleracea L. decomposed in conditions: 70% ethanol solution, 1:50 ratio of solid to
higher temperature. Therefore, the extraction temperature of liquid, 9 min of extraction time, and 50°C of extraction
45 °C was chosen as the best extraction temperature. temperature, respectively. In order to validate the optimum
2. Orthogonal Design Experiment conditions, a sample made under these optimum conditions
was determined, and the result indicated that the extraction
Orthogonal experiment of four factors and three levels efficiency was higher than any group in the orthogonal
was adopted to optimize the microwave conditions of the experiment table (the absorbance is 0.390, n=5).

Table 2 Factors and levels of orthogonal test Recovery Studies

1 2 3 Recovery studies were performed by adding 5.0 mg of rutin

A Ethanol concentration (v/v) 60% 70% 80%
to 0.5 g of powders of the sample, determined by
B The solid–liquid ratio (g mL−1) 1:30 1:40 1:50
ultraviolet–visible detector after microwave-assisted extrac-
C Radiation time (min) 7 8 9
tion. The results are shown in Table 4. We can see from the
table that the mean recovery for flavonoids from P.
D Extraction temperature (°C) 40 45 50
oleracea L. extracted by microwave-assisted extraction
96 Food Anal. Methods (2010) 3:90–97

Table 4 The results of recovery studies

No. Sample (g) Content (mg) Added (mg) Found (mg) Recovery (%) Mean recovery (%) RSD (%)

1 0.5008 4.6975 5.0 9.8791 103.6 102.6 1.13

2 0.5002 4.6919 5.0 9.8255 102.7
3 0.5007 4.6966 5.1 9.8887 103.8
4 0.5007 4.6966 5.0 9.8881 101.8
5 0.5005 4.6947 5.2 9.9516 101.1

and determined by UV–Vis spectrophotometry was 102.6% Sample Analysis

(RSD=1.13%), which indicates good effectiveness of the
method. Flavonoids from different kinds of P. oleracea L. extracted
by optimized microwave-assisted extraction conditions
were determined by means of UV–Vis spectrophotometry,
Methodology Studies and the results are shown in Table 5.
We could conclude from Table 5 that the extraction
Precision Test of the Apparatus efficiency of flavonoids in P. oleracea L. were different
from different batches, and the flavonoids content in
Apparatus precision was evaluated by the analysis of a different parts of P. oleracea L. (root, stem, and leaf)
solution of the sample for ten consecutive times by indicated that the root was rich in flavonoids, followed by
ultraviolet–visible detector. RSD obtained was 0.22%, stem and leaf. A comparison of flavonoids content from
which showed that the precision of the apparatus was P. oleracea L. between degreased and non-degreased
satisfactory. samples was also investigated (seen in Table 5); from the
table, we can see that lipid material had influence on the
Repeatability of Analytical Method detection results, so the samples used were all degreased in
our study.
To assess the repeatability of the method, five solutions
made by microwave extraction were determined. RSD
obtained was 0.57%, which showed acceptable Concluding Remarks
repeatability.
In the present study, the microwave-assisted extraction has
Stability Test of the Solution been established to extract flavonoids from P. oleracea L.
and quantification was performed by means of UV–Vis
Stability was evaluated by the analysis a solution of the spectrophotometry. The extraction yield of flavonoids by
sample extracted by microwave every 15 min. RSD microwave-assisted extraction is 0.71%. It is higher than
obtained was 0.32%, which indicated that the stability of other extractions, which confirms that microwave-assisted
the solution had excellent stability in 2 h. extraction is very suitable for the sample preparation of

Table 5 The analytical results of flavonoids in different samples (n=5)

Samples Degreased or not Flavonoids contents mg/g RSD %

P. oleracea L. (TCM, July) No 7.16 0.29

P. oleracea L. (TCM, July) Yes 7.10 0.61
P. oleracea L. (TCM, August) Yes 9.38 0.57
Wild P. oleracea L. (Yuelu Mountains, Changsha) No 6.82 0.39
Wild P. oleracea L. (Yuelu Mountains, Changsha) Yes 6.78 0.30
Root of wild P. oleracea L. Yes 11.36 0.32
Stem of wild P. oleracea L. Yes 5.12 0.81
Leaf of wild P. oleracea L. Yes 1.76 1.54
Food Anal. Methods (2010) 3:90–97 97

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