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Li Yang1 Jian-Guo Jiang1 Wei-Feng Li1 Jian Chen1 Ding-Yong Wang2 Liang Zhu1
1

Original Paper Optimum extraction Process of polyphenols from the bark of Phyllanthus emblica L. based on the response surface methodology
Phyllanthus emblica L. is an economic plant used in Chinese medicine for the treatment of various diseases. The bark of P. emblica is rich in polyphenols and its extractions have shown strong antioxidative and radical scavenging activity. Response surface methodology (RSM) was used to assess the optimal extraction of polyphenols from P. emblica bark. Various extraction parameters including ethanol concentration, extraction time, temperature, solid liquid ratio, and extraction times were chosen to identify their effects on polyphenols extraction. Among these parameters, extraction times and solvent concentration were found to have significant effect on polyphenols extraction. RSM was applied to obtain the optimal combination of solvent concentration, extraction time, temperature, and extraction time for maximum rate of extraction. The most suitable condition for the extraction of polyphenols was at ethanol concentration 75%, extraction time 25 min, extraction temperature 458C, and extraction times 3. At these optimal extraction parameters, the maximum extraction of polyphenols obtained experimentally was found to be very close to its predicted value. The extraction rate of polyphenols was 19.78% at the optimum conditions. The mathematical model developed was found to fit with the experimental data of polyphenols extraction.
Keywords: Bark extraction rate / Phyllanthus emblica L. / Polyphenols / Response surface methodology / Received: December 19, 2008; revised: January 16, 2009; accepted: January 16, 2009 DOI 10.1002/jssc.200800744

College of Food and Bioengineering, South China University of Technology, Guangzhou, China 2 Department of Natural Drug, Guangdong Phamaceutical University, China

1 Introduction
Phyllanthus emblica, a Euphorbiaceous Phyllanthus plant, is widely distributed in subtropical and tropical areas of China, India, Indonesia, and Malay Peninsula, and used in many traditional medicinal systems such as Chinese herbal medicine, Tibetan medicine, and Ayurvedic medicine [1]. Emblica fruit is reported to have antioxidant [2], hypolipidemic [3], and hypoglycemic activities [4], and acts as an important constituent of many hepatoprotective formulas [5]. It is also used as an antimicrobial agent [6], antitumor [7], or an anti-inflammatory agent [8] and can improve the metal-induced clastogenic effects [9]. Barks of P. emblica are rich in tannin with 21 33% content, and are the main raw materials used for tannin extract production in China. Utilization of resources of P.

Correspondence: Professor Jian-Guo Jiang, College of Food and Bioengineering, South China University of Technology, Guangzhou, 510640, China E-mail: jgjiang@scut.edu.cn Fax: (+86)-20-87113843

emblica has mainly focused on fruit processing. Wang et al. [10] reported that extracts from P. emblica bark had strong antioxidative and radical scavenging activities. Polyphenols are a group of aromatic secondary metabolites mainly existing in the plant's bark, roots, leaves, and fruits. They have been reported to possess multiple biological effects such as antioxidant capacity [11 13] and antimicrobial activity [14], due to which polyphenols have received much attention in the field of pharmaceutical, biochemical, food and cosmetic industry, and so on [15]. The effect of extraction conditions on polyphenols has been a contentious issue, particularly comparing different raw materials [16]. Many factors such as solvent composition, extraction time, extraction temperature [17], and solvent to solid ratio [18] may significantly influence the extraction efficacy and yield [19]. In contrast, microwave-assisted extraction is a fast extraction process where microwave energy is efficiently delivered to materials through molecular interaction with the electromagnetic field and offers a rapid transfer of energy to the extraction solvent and raw plant materials [20, 21].
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The commonly used types of solvent for extracting polyphenols are methanol, ethanol, acetone, and their water mixtures. Thus, alcoholic solvents have been commonly employed to extract phenolics from natural sources: they give quite high yield of total extract even though they are not highly selective for phenols. Particularly, mixtures of alcohols and water have revealed to be more efficient in extracting phenolic constituents than the corresponding mono-component solvent system [22, 23]. RSM had enabled the evaluation of the effects of several process variables and their interactions on response variables. Thus, RSM is a collection of statistical and mathematical techniques that has been successfully used for developing, improving, and optimizing processes [24].

standard curve prepared with gallic acid. The total polyphenols contents were expressed as lg gallic acid per ml.

2.4 Extraction rate of polyphenols

On the basis of the polyphenols content of prepared samples measured by the standard curve, the extraction rate of polyphenols was calculated by the equation D% C6V 2 6100% M6V1 61 000 000

Where D represents the extraction rate of polyphenols (%), V2 is the total volume of prepared sample (mL), C is the polyphenols content of the prepared sample (lg/mL), M is the quality of raw material (g), and V1 is the testing volume of prepared sample (mL).

2 Experimental section
2.1 Plant materials
The bark of P. emblica was collected from Baise, Guangxi province of China, where the samples are used to exclusively produce tannin extract. Collected samples were air-dried in an oven at 458C, and then ground in a cutting mill to pass through 100 mesh sieves to obtain fine power.

2.5 Design of statistical experiments

The effects of extraction condition on polyphenols yield including ethanol concentration, extraction time, temperature, solid liquid ratio, and times were investigated by the single factor method. On the basis of the single factor experimental results, major influence factors were confirmed, and then a response surface methodology was conducted to design experimental project. DPS (Design Expert 7.0) software package was used to establish a mathematical model and obtain the optimum conditions of technological progress. The variables were coded according to the equation [26] xi Xi X0 DX 1

2.2 Polyphenols extraction

The bark power (3 g) was extracted for polyphenols by microwave-assisted extraction with different concentrations (30, 50, 60, 70, 90%) with a ratio of solid liquid (g/ml) (ranging from 1:10 to 1:50) for a given time (extraction time ranging from 10 to 50 min), while the temperature of the water bath ranged from 20 to 608C and was kept steady (within l 1.08C). After filtering of the extract through 0.45 lm filter paper, filtrates were combined and concentrated using a rotary evaporator at 458C, and then freeze-dried in vacuum. The obtained dry power was dissolved by ethanol (60%) to a defined volume (100 ml). The experiment was repeated three times.

where xi is the (dimensionless) coded value of the variable Xi, X0 is the value of Xi at the center point, and (DX is the step change. Table 1 shows the actual design of experiments. The behavior of the system was explained by the following second degree polynomial equation Y X A0
4 X i1

A i Xi

4 X i1

Aii X2 i

3 X 4 X i1 ji1

Aij Xi Xj

2.3 Determination of polyphenols content

Prussion blue method [25] was employed to measure the total content of polyphenols extracted from the bark of P. emblica. One milliliter of prepared sample was added to a 25 ml volumetric flask. Then ferric chloride solution (0.5 ml, 0.10 mol/L), potassium ferricyanide solution (0.5 ml, 0.008 mol/L), and hydrochloric acid solution (0.5 ml, 0.10 mol/L) were added, respectively. The mixture was finally diluted with ethanol (60%) to a defined volume (25 mL). The absorbance of the mixture at 695 nm was measured immediately in comparison to a

3 Results and discussion

3.1 Single factor analysis method 3.1.1 Effect of different concentrations of ethanol on extraction rate of polyphenols
Soluble phenolic compounds are mainly distributed in the cell vacuoles, while most lignin, flavonoids, and insoluble polyphenols deposit in the cell wall to combine the hydrogen bond, hydrophobic bond with proteins and polysaccharides [27]. Water and low concentration
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Figure 1. Effect of different concentrations of alcohol (a), time (b), temperature (c), and solid-to-liquid ratio (d) on the extraction rate of polyphenols.

of ethanol can access to cells, but high concentration of ethanol can cause protein denaturation, preventing the dissolution of polyphenols and then influencing the extraction rate. Different concentrations of solvent have an effect on the polarity of the solvent, so it is very important to find a proper concentration in order to get a higher extraction rate [16]. To study the effects of different concentrations of ethanol on the extraction rate of polyphenols, different ethanol concentrations of 30, 50, 60, 70, and 90% were used, accompanied by a ratio of solid/liquid (g/mL) (3:40), extraction time 40 min, the temperature of the water bath 508C by microwaveassisted. Figure 1a shows that the extraction rate of polyphenols increases when the concentration of ethanol increased from 30 to 70%, and then decreases when the concentration of ethanol was more than 70%. The reason was that more uncertain liposoluble materials were extracted over high concentration of ethanol, which led to the increase of interference factors, making the purification progress more difficult. Therefore, the ethanol concentration of 70% was considered to be optimal in the present experiment.

tion time increases the chance of oxidation of phenolics unless reducing agents are added to the solvent system [28]. Extraction process was carried out under the condition of different extraction times from 10 to 50 min, while other extraction condition was as follows: a ratio of solid/liquid (g/mL) (3:40), ethanol concentrations 70%, the temperature of the water bath 508C by microwaveassisted. As shown in Fig. 1b, when the extraction time varied from 10 to 20 min, the variation on the extraction rate was relative plateau, then steadily rose from 20 to 40 min, and then reached the peak at 40 min. After extraction time exceeded 40 min, the variation on the extraction rate dropped sharply. The reason was that it may induce more chemical reaction on the longer extraction time and then cause oxidative conversion of polyphenols.

3.1.3 Effect of different temperature on extraction rate of polyphenols

Extraction temperature is one of the important factors affecting the extraction rate of polyphenols. At higher temperature, a higher yield of polyphenols extracted from fruit can be obtained. High-temperature solvent will promote polysaccharides on cell wall to distribute to solvent, and to weaken or undermine the integrity of the cell wall so that more solvent can be contacted with polyphenols [29]. To study the effect of different temperatures
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3.1.2 Effect of different time on extraction rate of polyphenols

Reasonably, a long extraction time causes the enhancement of extraction efficiency. However, longer extrac-

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on the extraction rate of polyphenols, the extraction process was carried out under the condition of different extraction temperature of the water bath from 20 to 608C by microwave-assisted, while other extraction condition was as follows: a ratio of solid liquid (g/mL) (3:40), ethanol concentrations 70%, extraction time 40 min. Figure 1c shows the effect of different temperatures on the extraction rate of polyphenols from the bark of P. emblica. Fig. 1c shows that when the temperature increased from 20 to 508C, the total yield of polyphenols rose steadily, and then reached the peak at 508C, and finally dropped from 50 to 608C. This phenomenon could be explained that, with the increase in temperature, solvent viscidity declined and the movement of molecular accelerated, which led to the dissolution of polyphenols for the enlargement of diffusion coefficient and the increase of solubility. However, much higher temperature will cause the loss of solvent, resulting in a lower yield. In addition, high temperature promoted the oxidation of polyphenols. Therefore, the optimal extraction temperature was considered to be 508C in the present experiment.

Figure 2. Effect of different times of extraction on the extraction rate of polyphenols. Table 1. Factors and levels of response surface methodology A Ethanol concentration (%) 1 0 +1 40 60 80 B (min) 20 30 40 C Tempera- D Times ture (8C) (time) 30 40 50 1 2 3

3.1.4 Effect of different solid liquid ratio on the extraction rate of polyphenols
With the solid liquid ratio increase, the extraction rate of polyphenols also increases [16]. To study the different solid liquid ratio on the extraction rate of polyphenols, the extraction process was carried out using the different ratio of solid liquid (g/mL) from 1:10 to 1:50, while other extraction condition was as follows: ethanol concentrations 70%, extraction time 40 min, the temperature of the water bath 508C by microwave-assisted. The effect of solid liquid ratio on the extraction rate of polyphenols was not very obvious (Fig. 1d). From the lower-cost point of view, the ratio of solid liquid (1:30) was chosen in the present experiment.

3.2 Optimization of the extraction progress 3.2.1 Mathematical model and optimization of extraction conditions
According to the method of Central Composite designed experiment and the levels of independent variables were chosen based on the values obtained in the single factor experiment, ethanol concentration (A,%, V/V), extraction time (B, min), temperature (C,8C) and times (D, time), which have a great effect on the extraction rate of polyphenols, were selected as design variables in the RSM, the extraction rate of polyphenols (Y,%) was employed as a response value, and the four factors and three levels' RSM test were designed (Tables 1, 2). Zero experiment was carried out in duplicate. Through performing in the form of analysis of ANOVA for the quadratic model, it was required to test the significance and adequacy of the model. Table 3 shows that the model of Prob A F (0.0071), correlation coefficient (R2 A 0.8), and the dispersion coefficient was not very large (1.7000), which meant that it was a high precision and applicable model. All the values of Prob A F of B, D, BD, and D2 were lower than 0.05, indicating that the interaction between extraction time and times had an significant influence on the extraction rate of polyphenols. The model established by regression equation can replace the experimental real point to explain response results. The regression equation was
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3.1.5 Effect of different times of extraction on extraction rate of polyphenols

Multiple-step extraction is an important method to improve the extraction rate of polyphenols from the bark of P. emblica. To study the effect of different times of extraction on the extraction rate of polyphenols, the extraction process was carried out using the different times of extraction, and then the filtrates were combined, while other extraction conditions were as follows: ethanol concentration 70%, extraction time 40 min, the temperature of the water bath 508C by microwaveassisted. We can see from the Fig. 2 that the yield of polyphenols by three times of extraction was significantly higher than that by one time. Compared to the yield and cost, extraction three times can be appropriate in the present research.

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Table 2. The plan and results for response surface methodology Run A Ethanol B concen- (min) tration (%) 60 40 60 60 40 80 60 80 60 60 40 60 60 80 60 60 60 60 60 60 80 40 40 60 40 80 80 20 40 30 40 30 30 20 20 20 40 30 30 40 30 30 30 30 20 30 30 30 30 20 40 30 40 30 C Tem- D Times Y Extraction perature (time) rate of poly(8C) phenols (%) 40 40 40 40 40 50 30 40 50 30 40 30 50 40 50 40 30 40 50 40 40 50 40 40 30 40 30 3 2 2 3 3 2 2 2 2 2 1 3 2 1 1 2 1 1 3 2 3 2 2 1 2 2 2 19.9013 18.7205 19.7631 19.1476 19.6249 19.5495 19.4993 19.0219 19.4742 18.8712 18.6325 19.2481 18.6451 18.1929 18.2682 19.6501 18.6325 17.8662 19.4114 19.5370 19.6877 19.1224 18.8084 18.9340 18.8461 18.4315 18.6953

conditions were: ethanol concentration 74.63%, extraction time 23.16 min, extraction temperature 45.998C, extraction times 2.95. Under the optimal progress, the predictive value of extraction rate of polyphenols was 20.02%.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

3.2.2 The interaction between the variables

The graph of RSM was a 3D response surface plot consisting of response values of experimental variables (presented in Fig. 3). They can reflect the interaction between the variables (ethanol concentration, extraction time, temperature and times). From Fig. 3A, it can be seen that the effect of ethanol concentration on the extraction rate of polyphenols was not very obvious at a certain extraction time. With the increase in ethanol concentration from 40 to 80%, the extraction rate of polyphenols increased at the beginning, and then fell. When the ethanol concentration was at a certain value, the extraction rate of polyphenols also increased and then decreased as the extraction time prolonged. These data were consistent with the conclusion of the single factor test. Therefore, both extraction time and ethanol concentration would have independent optimum condition parameters. Figure 3B shows that with the ethanol concentration and extraction temperature increasing in the beginning, the extraction rate of polyphenols increased. However, when they were at a certain value, the extraction rate of polyphenols dropped. The best point of balance should be sought for the maximum extraction rate of polyphenols between ethanol concentration and extraction temperature. When ethanol concentration was at a certain value, the extraction rate of polyphenols increased with the extraction times extended (Fig. 3C). However, when extraction times were unchanged, the extraction rate of polyphenols rose and then declined as the ethanol concentration increased. It was not significant that the increase of ethanol concentration affected the extraction rate of polyphenols at certain extraction times. When the extraction time was at a certain value, the extraction rate of polyphenols rose, and then dropped as the extraction temperature gradually rose. Their trends of changes were consistent with the interaction between ethanol concentration and temperature on the extraction rate of polyphenols (Fig. 3D). It can be seen from Fig. 3 E that when extraction time was at a certain value, the extraction rate of polyphenols obviously increased with the extraction times added from one to three. In addition, when the extraction time was one, the extraction rate of polyphenols increased as the extraction time extended. However, when the extraction times were three, the extraction rate of polyphenols did not increase and slightly fell as the extraction time
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Y = +19.65 0.085* A-0.24*B + 0.057*C+0.53*D + 0.12*A*B + 0.14*A*C + 0.16*A*D 0.050*B*C 0.46* B*D + 0.13*C*D 0.31*A2 0.29*B2 0.30*C2 0.40*D2 (2-2) The negative coefficients of A and B variables indicated that their negative changes can bring about reduction in response value; the positive coefficients of C and D variables revealed that their positive changes can cause increase in the response value; negative quadratic coefficient concluded that the opening of equation paraboloid was downward, which illuminated that it included maximum point and could carry out optimal analysis. By observing linear and quadratic coefficients, we conclude that the order of factors influencing the response value of the extraction rate of polyphenols was as follows: extraction times A extraction time A solvent concentration A extraction temperature. The effects of extraction times and time to response values were significant (P a 0.05). By carrying out parameter optimization on the basis of the built mathematical model, obtained experimental

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Figure 3. Correlative effects of ethanol solution concentration and time (A), ethanol solution concentration and temperature (B), ethanol solution concentration and times (C), time and temperature (D), time and times (E) and temperature and times (F) on phenolics productivity.

prolonged. It was concluded that extraction times affected significantly on extraction rate of polyphenols. From Fig. 3F, when extraction temperature was at a certain value the extraction rate of polyphenols increased as the extraction times prolonged. When extraction times did not vary, extraction rate of polyphenols increased and then decreased with the extraction temperature. It showed that while extraction times were unchanged, it was not obvious that the improvement of

extraction temperature affected the extraction rate of polyphenols.

3.2.3 Verification of predictive model

To further test the reliability of the experimental method, the extraction experiment was carried out by adopting the program of optimal analytical model. Adjusted extraction conditions are shown in Table 4. The result showed that the experimental values were not
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Table 3. Analysis of mean square deviation of regress equation Source Model A-ethanol concentration B-time C-temperature D-times AB AC AD BC BD CD A2 B2 C2 D2 Residual Lack of Fit Pure Error Cor Total Sum of squares 6.4897 0.0876 0.6634 0.0383 3.3539 0.0619 0.0835 0.1066 0.0101 0.8295 0.0696 0.4980 0.4432 0.4658 0.8699 1.2657 1.2401 0.0256 7.7554 Degree of freedom Mean square 14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12 10 2 26 0.4636 0.0876 0.6634 0.0383 3.3539 0.0619 0.0835 0.1066 0.0101 0.8295 0.0696 0.4980 0.4432 0.4658 0.8699 0.1055 0.1241 0.0128 F-value 4.3950 0.8306 6.2895 0.3635 31.7989 0.5867 0.7916 1.0110 0.0958 7.8644 0.6598 4.7216 4.2025 4.4161 8.2475 9.7032 Prob A F 0.0071 0.3800 0.0275 0.5578 0.0001 0.4585 0.3911 0.3345 0.7623 0.0159 0.4324 0.0505 0.0629 0.0574 0.0140 0.0970 Not significant Significant Significant

Table 4. The result of experimental verification Term Predict Experiment Ethanol concentration (%) 74.63 75.00 Extraction time (min) 23.16 25.00 Extraction temperature (8C) 45.99 45.00 Extraction times (time) 2.95 3.00 Extraction rate of polyphenols (%) 20.02 19.78

only consistent with the predictive values, but were also better than any single factor experiments. Therefore, the extraction conditions obtained by response surface methodology were not only accurate and reliable, but also with practical value reflecting the expected optimization.

polyphenols 19.78%, which was better than any single factor test. Research on extraction, separation and purification plant polyphenols from bark of P. emblica will help improve the added value of P. emblica resource. This project was supported by National Key Technology R&D Program 2006BAD27B04. Authors declare no conflict of interest

4 Conclusions
The bark of P. emblica is rich in polyphenols. The performance of the extraction of polyphenols from bark of P. emblica was studied using response surface methodology. The conclusions can be drawn that among the four parameters studied, extraction times had the most significant effect on the extraction rate of polyphenols; according to the mathematical model parameters optimization analysis, the optimal conditions were: ethanol concentration 74.63%, extraction time 23.16 min, extraction temperature 45.998C, extraction times 2.95 and the extraction rate of polyphenols 20.02%; in order to facilitate the operation of verification experiment, the extraction conditions were adjusted as follows: ethanol concentration 75%, extraction time 25 min, extraction temperature 458C, extraction three-times and the extraction rate of

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