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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 medicinal plant in China. Flavonoids are one of the main active ingredients of this plant. Five extraction technologies of flavonoids from P. oleracea L. were investigated and compared, including microwave-assisted extraction, ultra- sonic extraction, reflux extraction, Soxhlet extraction, and marinated extraction. The results showed that microwave- assisted extraction was most suitable for the extraction of flavonoids from P. oleracea L. because of its high effect and short extraction time. The found optimum extraction conditions were that the ethanol concentration was 70% ( v / v ), solid liquid ratio was 1:50, extracting temperature was 50 °C and irradiation time was 9 min. Quantification was performed by means of UV Vis spectrophotometry with chromogenic system of NaNO 2 Al (NO 3 ) 3 NaOH. Under the optimum conditions, the calibration curve for the analyte was linear with the correlation coefficients greater than 0.9999. The average recovery was 102.6%, and its RSD was 1.13%( n =5). Eight types of P. oleracea L. according to different habits were investigated. The total content of flavonoids was 7.16, 7.10, 9.38, 6.82, 6.78, 11.36, 5.12, and 1.76 mg g 1 , respectively.

Keywords Food Analysis . Portulaca oleracea Flavonoids . Microwave-Assisted Extraction . UV Vis Spectrophotometry

H. Zhu : Y. Wang ( * ) : Y. Liu : Y. Xia : T. Tang State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, People s Republic of China e-mail: wyzss@hnu.cn

410082, People ’ s Republic of China e-mail: wyzss@hnu.cn Introduction Portulaca oleracea L. ( P. oleracea

Introduction

Portulaca oleracea L. ( P. oleracea L., purslane) is a common, herbaceous succulent annual weed, which is distributed extensively in temperate and tropical regions worldwide (Radhakrishnan et al. 2001 ; Garti et al. 1999 ). It has been used as a kind of food and medicinal plant for thousands of years in China. P. oleracea L. is of abundant nutrition with contents of proteins, carbohydrates, Ca, K, Zn, and Na (Aberoumand 2008 ). As a kind of Chinese traditional medicine, P. oleracea L. is very important because of its special medical function, and it has been used traditionally for the treatment of dysentery with bloody stools and externally for boils and sores, eczema, erysipelas, and insect and snake bites (Chen et al. 2003 ; Zhang et al. 2002 ; Yazici et al. 2007 ; Palaniswamya et al. 2004 ). Among the effective ingredients of many Chinese herbal medicines, flavonoids are one of the most popular compounds in the plant kingdom. Flavonoids have received much attention because of their richness in species numbers and effectiveness in reducing blood lipid, as an anti-oxidative, in assimilating cholesterol, inhibiting thrombosis, dilat ing the coronary artery, etc. (Deng et al. 2008 ; De Souza et al. 2008 ; Su et al. 2008 ; Shao et al. 2007 ; Volpi and Bergonzini 2006 ; Zhang et al. 2007 ; Keith et al. 2007 ). Recently, the development of flavonoids in medical and food fields becomes a hot research. Flavonoids and polysaccharides are the most abundant effective com- ponents in P. oleracea L. It is reported that seven kinds of flavonoids were found in P. oleracea L., including quercetin, kaempferol, myricetin, apigenin, luteolin,

Food Anal. Methods (2010) 3:90 97

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genistein, and genistin (Chen et al. 2003 ; Zhang et al. 2007 ). The extraction and separation of flavonoids with high biological activity and high content is very impor- tant for the medical and food industry. Therefore, it is of great significance to study the extraction and the determination technologies of flavonoids from P. oleracea L. In traditional methods, the extraction of flavonoids from P. oleracea L. is usually carried out by means of marinated extraction and reflux extraction. Being a newly arisen extraction method, ult rasonic extraction has also been used for the extraction of flavonoids (Jin et al. 2008 ; Chen et al. 2008a ). Unfortunately, flavonoids could not be extracted sufficiently by the mentioned methods due to the high impurity content and the long extraction time, which restrict the development of the medicine at a certain extent. Therefore, it is necessary to develop a more effective and reliable extraction method. Microwave- assisted extraction seems pro mising to fulfill the purpose. Microwave-assisted extracti on has the advantage of strong penetrating force, high selectivity, high heating ability, less solvent consumption, sh ort extraction time, high extraction yield (Ganzler et al. 1990 ), and as a new kind of extraction technology it is especially suitable for extracting heat-sensitivity components and natural active constituents (Chen et al. 2008b ). In this paper, pressure self-control microwave decomposition system was used to extract flavonoids from P. oleracea L. The temperature of the system can be controlled at a certain value. Therefore, it is effective to avoid the loss of the components that are heat sensitive. In the present study, microwave-assisted extraction, a simple and effective method, has been developed. Meanwhile, in order to demonstrate the advantages of microwave-assi sted extraction, the extrac- tion efficiencies of flavonoids with various methodologies were compared. Several methods for the determination of flavonoids in medicinal plants have been reported, including thin-layer chromatography, high-performance liquid chromatography, capillary electrophoresis, and spectro- photometry. Among all these methods, only spectropho- tometry can be used to determinate the total amount of the flavonoids. UV Vis spectrophotometry is the most extensively used method to determine the total content 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 other components such as polysaccharides and saponin. In this study, we present a n easy method that uses UV Vis spectrophotometry with chromogenic system of NaNO 2 Al(NO 3 ) 3 NaOH to determine the total amount of the flavonoids from P. oleracea L.

Materials and Methods

Collection of Samples

In order to investigate the flavonoids content of P. oleracea L. samples collected in different months, three different P. oleracea L. samples were used as experimental materials. Two of the three samples are traditional Chinese medicine (TCM) samples purchased from Traditional Chinese Med- icine Co. Ltd (Hunan, China); one of the TCM samples was collected in August 2007 and another TCM sample was collected in July 2007. We collected the third sample (wild P. oleracea L.) from the suburb of Changsha (Hunan, China) in June 2008.

Samples Preparation

Fresh samples were cleaned with water, and some of the fresh samples were split as roots, stems, and leaves for study. All of the samples were crushed into powder by a plant grinder after being dried at 50 °C. The powders of the samples were kept in an airer.

Chemicals and Instruments

1. Chemicals: authentic standard rutin (>97%) was pur- chased from Guoyao Group of Chemical Reagents Ltd. (China). All reagents and chemicals used in this work were of analytical grade. Triple-distilled water was used throughout the study.

2. Instruments: pressure se lf-control microwave decom- position system (MDS-2002AT, Shanghai, China), Auto Science AS-2060B Ultrasonic Cleaner (Tianjin, China), and Soxhlet extractor (250 mL, Changsha, China) were used for extraction. High-speed centri- fugation (TGL-16C, Shanghai, China) was employed to accelerate the phase separation process. A versatile plant pulverizer (FW-100, Beijing, China) was used to make the plant materials into powder. A rotary evaporator (RE52CS, Shanghai, China) was used to concentrate the sample solution. UV Vis spectrophotometer V-530 (JASCO, Japan) was used for determination.

Extraction of Flavonoids

Before extraction, P. oleracea L. was crushed into powder by versatile plant pulverizer. The powders of the samples were degreased by Soxhlet extractor with petroleum ether until the color of the eluate became colorless. Flavonoids

were degreased by Soxhlet extractor with petroleum ether until the color of the eluate became colorless.

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Food Anal. Methods (2010) 3:90 97

were extracted from P. oleracea L. by five extraction methods as follows:

1. Microwave-assisted extraction

Powders of the samples (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, then the sealed vessel was placed into the pressure self-control microwave decomposition system for microwave-assisted extraction. The extraction temperature was 50 °C and the irradiation time was kept at 9 min. After microwave-assisted extrac- tion, the extract was centrifuged at 11,000 rpm for 30 min; the supernatant was diluted to 25 mL in a volumetric flask (25 mL) by the ethanolwater (70:30, v / v ) solvent.

2. Ultrasonic Extraction

Powders of the samples (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, then the sealed vessel was placed in the ultrasonic cleaning bath for ultrasonic extraction for an hour at room temperature (25 °C). Then the following works were done (as the description in microwave-assisted extraction).

3. Condensing Reflux 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, then the sealed vessel was placed into the reflux device, followed by the extraction for 2.5 h. Then the following works were done (as the description in microwave-assisted extraction).

4. Soxhlet Extraction

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 NaNO 2 (5%) solution, shaken up and then standing for

of NaNO 2 (5%) solution, shaken up and then standing for 6 min. Secondly, 0.5 mL

6 min. Secondly, 0.5 mL of the Al(NO 3 ) 3 (10%) solution was added to the volumetric flask, shaken, and was left to stand for 6 min. Finally, 3.0 mL of the NaOH (4.3%)

solution was added to the volumetric flask, followed by addition of water to the scale, shaken, and left to stand for 15 min before determination. Using the sample solution without coloration as reference solution and 500 nm as determination wavelength, the coloration method was used

to determine the content of flavonoids in the sample by

ultravioletvisible detector.

Preparation of Standard Solution

A standard solution (0.16 mg/mL) of rutin was prepared as

follows: rutin (200 mg) was accurately weighed and dissolved in ethanol water (70:30, v / v ) solution, and then the solution was diluted to 100 mL in a volumetric flask (100 mL) by ethanol water (70:30, v / v ) solvent. Two milliliters of this solution was removed and diluted to 25 mL in a volumetric flask (25 mL) by ethanol water (70:30, v /v ) solvent.

Standard Curve

Six portions of the rutin solution were accurately removed (0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 mL, respectively) in six volumetric flasks (10 mL), then the following works were done (as the description in determination of flavonoids). Using the concentration of rutin standard solution as abscissa and the absorbency as y -coordinate, the linear chart was constructed and the standard curve is shown in Fig. 1 , the regression equation was A = 0.00171+ 11.58482 C ( r =0.9999).

shown in Fig. 1 , the regression equation was A = − 0.00171+ 11.58482 C (

Fig. 1 Standard curve

Food Anal. Methods (2010) 3:90 97

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Fig. 2 The color reaction of flavonoids and chromogenic system

2 The color reaction of flavonoids and chromogenic system Results and Discussion The Color Reaction of

Results and Discussion

The Color Reaction of Flavonoids and Chromogenic System

The basic structure of flavonoids was presented in Fig. 2a , and most of the flavonoids in P. oleracea L. have 3 ,4 - dihydroxy-substituted structure (as shown in Fig. 2b ). Flavonoids with 3 ,4 -dihydroxy-substituted structure can show special color by reacting with the system of NaNO 2 Al(NO 3 ) 3 NaOH. The color reaction of flavonoids and chromogenic system is presented in Fig. 2c . As shown in Fig. 2 , this method is based on the reaction of aluminum ion with flavonoid at alkaline medium forming red chelates. By measuring the absorption of such red chelates, it is possible to determine the flavonoids.

Selection of Detective Wavelength

Ultraviolet scanning (presented in Fig. 3 ) was performed to determine the detective wavelength. The solution of rutin and the solution of sample were prepared (as the description in determination of flavonoids). From Fig. 3 , we can see that both rutin and sample showed maximum absorption at the wavelength of 500 nm in visible range, so the wavelength of 500 nm can be selected for detection of target analyte.

Comparison of Extraction Efficiency with Different Solvents

The extraction of the three mentioned solvents was investigated with microwave-assisted extraction, including

investigated with microwave-assisted extraction, including Fig. 3 The UV – Vis spectra of rutin and sample

Fig. 3 The UV Vis spectra of rutin and sample solution

Fig. 3 The UV – Vis spectra of rutin and sample solution Fig. 4 Effect of

Fig. 4 Effect of the solvents on extracting efficiency

Fig. 3 The UV – Vis spectra of rutin and sample solution Fig. 4 Effect of

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Food Anal. Methods (2010) 3:90 97

methanol water, ethanol water, and acetone water to check their efficiency for extraction of flavonoids from P. oleracea L., and the results are shown in Fig. 4 . From Fig. 4 , we can see that extraction of flavonoids from P. oleracea L. with ethanolwater gave the highest efficiency.

Selection of Extraction Method

Five different extraction methods were investigated, includ- ing microwave-assisted extraction, marinated extraction, Soxhlet extraction, reflux extraction, and ultrasonic extrac- tion, and the flavonoids of these samples were determined by UV Vis spectrophotometry. The results are shown in Table 1 . From the results shown in Table 1 , we can find that microwave-assisted extraction had the highest extraction efficiency. Compared with Soxhlet extraction, the extrac- tion time of microwave-assisted extraction was shortened by 97%, and compared with reflux extraction, 94% of extraction time was shortened. Microwave-assisted extraction is a rapid, efficient, and economic method for extracting flavonoids from P. oler- acea L. Unlike classical cond uctive heating methods, microwave heats the whole sample simultaneously because microwave rays are very penetrating. The microwave rays travel through the microwave-transparent extraction medi- um and reach the inner vascular bundles and cell systems of the plant material. The result is a sudden rise in temperature inside the material; the internal pressure exceeds the capacity of expansion of the cell walls, so the cell walls cracked and the substances in the cells are then free to flow out of the cells into the extract solvent. Therefore, microwave-assisted extraction had the highest extraction efficiency and it was most suitable for extracting flavonoids from P. oleracea L.

Optimization of Microwave-Assisted Extraction Conditions

There are many factors affecting the extraction of flavo- noids from P. oleracea L. We worked out the optimum levels of each factor by the single factor test. However, because of the interaction among the factors, the combina-

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

tion effects of the optimum levels of each factor may not be the optimum extraction conditions. The orthogonal test is a scientific method of arranging multi-factors. Based on a single factor test, the optimal extraction conditions can be worked out by orthogonal test. In this paper, the optimal extraction conditions of microwave-assisted extraction were worked out through single factor test and orthogonal test.

1. Single Factor Test

There are many factors affecting the extraction yield, among which the extraction solvent, solid liquid ratio, radiating time of microwave, and extracting temperature are the main factors. Single factor test was performed by one factor varied with different levels while other factors fixed.

(a) Selection of Extraction Solvent

The extraction by three mentioned solvents of different strengths was investigated; other conditions of microwave radiation was kept the same (ratio of material to solution was 1:30, extracting temperature was 35 °C, and extraction time was 5 min). As shown in Fig. 4 , the absorbance was the highest when extraction solvent was ethanol of 70% ( v / v ), so ethanol water of 70% ( v / v ) was chosen in the following experiments.

(b) Selection of Solid liquid Ratio

The extraction by solid liquid ratio of 1:10 (g/mL), 1:15, 1:20, 1:30, 1:40, and 1:50 was investigated, other con- ditions of microwave radiation was kept same (ethanol water of 70%, extracting temperature was 35 °C, and extraction time was 5 min). As shown in Fig. 5 , the extraction efficiency increased when the ratio increased from 1:15 to 1:40 and then tended to remain constant in the ratio range of 1:401:50. Hence, the solid liquid ratio of 1:40 (g/mL) was chosen in the following experiments.

(c) Selection of Extraction Time

The radiating time of microwave of 5, 6, 7, 8, 9, and 10 min were investigated; other conditions of microwave radiation was kept the same (ethanol water of 70%, solid liquid ratio was 1:40, and extracting temperature was

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

Food Anal. Methods (2010) 3:90 – 97 95 Fig. 5 Effect of microwave-assisted extraction conditions: solid

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, 6, 7, 8, 9, and 10 min), and extraction temperature (30, 35, 40, 45, 50, and 55 °C)

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

10 min. When other factors are fixed, the extraction

efficiency increased when extraction time increased, and then it reached equilibrium in a certain time. Hence, the

radiating time of microwave of 8 min was chosen in the following experiments.

(d) Selection of Extraction Temperature

Microwave conditions of ethanolwater of 70%, solid liquid ratio of 1:40, and the radiating time of 8 min were fixed, and extraction temperature varied with different levels (30, 35, 40, 45, 50, and 55 °C) were investigated. As shown in Fig. 5 , the extraction efficiency was the highest when the extracti on temperature was 45 °C. However, when the temperature was higher, the extraction efficiency decreased. It could speculate that some of the heat-sensitive components in P. oleracea L. decomposed in higher temperature. Therefore, the extraction temperature of 45 °C was chosen as the best extraction temperature.

2. Orthogonal Design Experiment

Orthogonal experiment of four factors and three levels was adopted to optimize the microwave conditions of the

Table 2 Factors and levels of orthogonal test

123

A Ethanol concentration (v /v)

60%

70%

80%

B The solid liquid ratio (g mL 1 )

1:30

1:40

1:50

C Radiation time (min)

7

8

9

D Extraction temperature (°C)

40

45

50

Table 3 Results of L 9 (3 4 ) 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

K 1

0.952

0.972

1.009

0.983

T =3.049

K 2

1.133

1.029

0.975

0.978

K 3 k 1 = K 1 /3

0.964

1.048

1.065

1.088

0.317

0.324

0.336

0.328

k 2 = K 2 /3

0.378

0.343

0.325

0.326

k 3 = K 3 /3

0.321

0.349

0.355

0.363

R

0.061

0.025

0.030

0.037

extraction. The experimental design and the results are shown in Tables 2 and 3 . The K and R values were calculated and are listed in Table 3 . K value is the average extracting efficiency of every factor at each level. R value is the range of K value. Range analysis results of the orthogonal experiment indicated that ethanol concentration is the most important factor, followed by extraction temperature, radiating time of microwave, and solid liquid ratio. We also can see that K 2 has the highest K value in factor A, which means that, comparing with other levels of factor A, level two has the highest extracting efficiency and it is the most suitable level for extraction. Based on the same principle, level three of factor B, level three of factor C, and level three of factor D were chosen for extraction. In other words, the maximum yield of the active component was obtained at the following conditions: 70% ethanol solution, 1:50 ratio of solid to liquid, 9 min of extraction time, and 50°C of extraction temperature, respectively. In order to validate the optimum conditions, a sample made under these optimum conditions was determined, and the result indicated that the extraction efficiency was higher than any group in the orthogonal experiment table (the absorbance is 0.390, n =5).

Recovery Studies

Recovery studies were performed by adding 5.0 mg of rutin to 0.5 g of powders of the sample, determined by ultravioletvisible detector after microwave-assisted extrac- tion. The results are shown in Table 4 . We can see from the table that the mean recovery for flavonoids from P. oleracea L. extracted by microwave-assisted extraction

see from the table that the mean recovery for flavonoids from P. oleracea L. extracted by

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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% (RSD=1.13%), which indicates good effectiveness of the method.

Methodology Studies

Precision Test of the Apparatus

Apparatus precision was evaluated by the analysis of a solution of the sample for ten consecutive times by ultraviolet visible detector. RSD obtained was 0.22%, which showed that the precision of the apparatus was satisfactory.

Repeatability of Analytical Method

To assess the repeatability of the method, five solutions made by microwave extraction were determined. RSD obtained was 0.57%, which showed acceptable repeatability.

Stability Test of the Solution

Stability was evaluated by the analysis a solution of the sample extracted by microwave every 15 min. RSD obtained was 0.32%, which indicated that the stability of the solution had excellent stability in 2 h.

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

Sample Analysis

Flavonoids from different kinds of P. oleracea L. extracted by optimized microwave-ass isted extraction conditions were determined by means of UV Vis spectrophotometry, and the results are shown in Table 5 . We could conclude from Table 5 that the extraction efficiency of flavonoids in P. oleracea L. were different from different batches, and the flavonoids content in different parts of P. oleracea L. (root, stem, and leaf) indicated that the root was rich in flavonoids, followed by stem and leaf. A comparison of flavonoids content from P. oleracea L. between degreased and non-degreased samples was also investigated (seen in Table 5 ); from the table, we can see that lipid material had influence on the detection results, so the samples used were all degreased in our study.

Concluding Remarks

In the present study, the microwave-assisted extraction has been established to extract flavonoids from P. oleracea L. and quantification was performed by means of UV Vis spectrophotometry. The extraction yield of flavonoids by microwave-assisted extraction is 0.71%. It is higher than other extractions, which confirms that microwave-assisted extraction is very suitable for the sample preparation of

 

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

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flavonoids from P. oleracea L., and the analytical method performed in this article shows it was convenient, rapid, reliable, and useful in the determination of flavonoids. The effective components in P. oleracea L. are compli- cated and varied. However, according to our knowledge, there is no special component in P. oleracea L. that has ever been reported. Flavonoids are major effective components of P. oleracea L., and flavonoids from P. oleracea L. have many special roles in medical and health function. Therefore, the rapid and practical method established in our work to extract and determine flavonoids from P. oleracea L. has great significance for studying the active ingredients of P. oleracea L. and for its quality control.

Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 20875025, No. J0830415) and the Hunan Provincial Natural Science Foundation (No.

07JJ3018).

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