Food Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Extraction, chemical composition and antioxidant activity of flavonoids from Cyclocarya paliurus (Batal.) Iljinskaja leaves Jian-Hua Xie a,1, Cai-jun Dong b,1, Shao-Ping Nie a, Feng Li b, Zhi-Jun Wang a, Ming-Yue Shen a, Ming-Yong Xie a,⇑ a b
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China Nantong Agricultural College, Nantong 226007, China
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: Cyclocarya paliurus Microwave-assisted extraction Flavonoids Antioxidant activity Chemical composition Response surface methodology
a b s t r a c t Microwave-assisted extraction of flavonoids from Cyclocarya paliurus (Batal.) Iljinskaja leaves, its chemical composition and antioxidant activity were investigated in this study. The influences of parameters including temperature, extraction time, ratio of material to solvent and solvents on the yield of flavonoids were studied. The optimal conditions were determined and the quadratic response surfaces were drawn from the mathematical models. The maximum extraction yield of 15.64 mg/g was achieved at temperature of 76.8 °C, extraction time of 15 min, alcohol concentration of 63.2% and ratio of solvent to material of 21.4:1. Five main constituents in the extract including quercetin-3-O-b-D-glucuronide, quercetin, kaempferol-3-O-b-D-glucuronide, kaempferol-7-O-a-L-rhamnoside and kaempferol were identified by LC–MS. In vitro antioxidant assays showed that the extract exhibited a strong DPPH radical-scavenging ability with IC50 value of 0.146 mg/mL. Results indicated that MAE was a suitable approach for the selective extraction of flavonoids from C. paliurus (Batal.) Iljinskaja leaves. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus) which belongs to the genus Cyclocarya Iljinskaja (Juglangdaceae), is the sole species in its genus, and mainly found in the mountainous regions in the tropics and subtropics (Xie, Li, Nie, Wang, & Lee, 2006). It grows mainly in the south of China, and is called ‘‘sweet tea tree’’ in Chinese (Xie, Shen et al., 2013). The leaves of C. paliurus are used in folk medicine for the treatment of diabetes mellitus, hypertension, hyperliposis, etc. (Li et al., 2000; Kurihara et al., 2003; Xie, Xie, Nie et al., 2010; Xie, Liu et al., 2013). The leaves of this plant have been widely used in China, which may be related to abundant organic compounds, especially flavonoids. Flavonoids are found to be the main active compounds in C. paliurus (Xie, Wang, Yi, & Wang, 2004), which have many biological activities, such as preventing hyperglycemia, diabetes mellitus, hypertension and coronary heart disease. Many flavonoids were isolated from C. paliurus, such as cyclocarioside, cyclocaric acid, kaempferol, ⇑ Corresponding author. Address: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, Jiangxi, China. Tel./fax: +86 791 83969009. E-mail addresses: [email protected] (J.-H. Xie), [email protected] (M.-Y. Xie). 1 These authors contributed equally to this work.
quercetin and isoquercitrin. (Shu, Xu, & Li, 1996; Xie et al., 2004; Zhang et al., 2010). Conventional techniques to obtain flavonoids, such as heating, boiling, or refluxing, usually require several hours or even days for the extraction process and a large volume of solvent, and may result in a loss of flavonoids due to hydrolysis, ionisation and oxidation during extraction (Li, Chen, & Yao, 2005). Microwave irradiation, which has proved to be a clean, efficient and convenient energy source, has been widely utilised in natural products extraction (Jiao et al., 2014; Xie et al., 2012; Zhang et al., 2013). Microwave-assisted extraction (MAE) is known to be a fast and efficient method for the extraction of flavonoids from plants. Compared with the traditional methods, MAE has many advantages, such as higher extraction rate, shorter extraction time, use of less solvent, better productivity and higher quality products (Chen, Xie, & Gong, 2007; Zhang, Yang, & Wang, 2011). Recently, MAE has been applied to extract various bioactive compounds from plants, such as terpenes from caraway seeds (Chemat, Aït-Amar, Lagha, & Esveld, 2005), quercetin from Flos Sophorae (Li et al., 2004) and flavonoids from cultivated Epimedium sagittatum (Zhang et al., 2013). A dynamic MAE system designed by Chen et al. (2008) markedly enhanced extraction yield of flavonoids from Herba Epimedii. To our knowledge, the optimised conditions of
http://dx.doi.org/10.1016/j.foodchem.2014.06.106 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xie, J.-H., et al. Extraction, chemical composition and antioxidant activity of flavonoids from Cyclocarya paliurus (Batal.) Iljinskaja leaves. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.06.106
2
J.-H. Xie et al. / Food Chemistry xxx (2014) xxx–xxx
MAE used in extraction of flavonoids from C. paliurus have not been reported yet. Response surface methodology (RSM), an important tool in process and product improvement, is a collection of experimental design and optimisation techniques that enables the researcher to determine the relationship between the response and the independent variables (Eren & Kaymak-Ertekin, 2007). To the best of our knowledge, there has been no report about the application of RSM in the optimisation of MAE conditions for the extraction of flavonoids from the leaves of C. paliurus. The objective of this study was to optimise the MAE conditions for the extraction of flavonoids from C. paliurus. RSM was employed to study the optimal temperature, extraction time, solid–liquid ratio and concentration of alcohol, which could maximise the yield of flavonoids from C. paliurus. Then, the colorimetric method with AlCl3/methanol system was used to quantify total flavonoids, which has been reported to be a simple, quick and accurate method (Zhao, Xu, & Liu, 2004). In addition, the antioxidant activity of flavonoids from C. paliurus, obtained under optimised MAE extraction conditions was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay. The chemical composition of the MAE extract was also evaluated by liquid chromatography–mass spectrometry (LC–MS) analysis. 2. Materials and methods 2.1. Plant materials The leaves of C. paliurus were collected in Xiushui County, Jiangxi Province, China. A voucher specimen was deposited at the State Key Laboratory of Food Science and Technology, Nanchang University, China. The leaves were air dried and ground into a fine powder (40–60 mesh) in a mill.
cool down to room temperature. Then each extract was filtered and ethanol was added in order to make the total volume of 100 mL (Chen et al., (2007)). 2.4. Experimental design RSM was used to design the MAE of total flavonoids from the leaves of C. paliurus. To explore the effect of independent variables on the response within the range of investigation, a central composite rotate design with four independent variables (X1, temperature, X2, extraction time, X3, solid–liquid ratio and X4, alcohol concentration) at five levels was performed. Each independent variable had coded levels of 2, 1, 0, 1 and 2. The experimental designs of the coded (x) and actual (X) levels of variables are shown in Table 1. The variables were coded according to Eq. (1):
xi ¼
ðX i X 0 Þ DX i
ð1Þ
where xi is the coded value of an independent variable, Xi is the real value of the independent variable, X0 is the real value of an independent variable at the centre point, and DX is the step change value. The yield of flavonoids was considered as the dependent variable or response. For a central composite rotate design with four independent variables at five levels, 31 experimental runs are required. The actual design of experiments is given in Table 1. The experimental results were fitted to a second-order polynomial model, and the regression coefficients were determined. The quadratic model for predicting the optimal point was expressed according to Eq. (2):
Y k ¼ bk0 þ
4 4 4 X X X bki xi þ bkii x2i þ bkij xi xj i¼1
i¼1
ð2Þ
j¼1
where bk0, bki, bkii and bkij are constant regression coefficients of the model, while xi, xj are the independent variables.
2.2. Chemicals and reagents 2.5. Determination of flavonoids content and yield of flavonoids Methanol and formic acid (HPLC grade) were obtained from Merck (Darmstadt, Germany). Ethanol was purchased from Shanghai Chemicals and Reagents Co. (Shanghai, China). Quercetin was supplied by China Institute for Drugs and Biological Products Identification (Beijing, China). Butylated hydroxytoluene, 2,2diphenyl-1-picrylhydrazyl (DPPH), and ascorbic acid (VC) were purchased from Sigma (St. Louis, MO, USA). All the other reagents were of analytical grade. Ultrapure water was prepared by Milli-Q50 (Millipore Corp., Bedford, MA, USA) water purification system. All solvents used for HPLC measurements were filtered (0.45 lm) before being used. 2.3. MAE The MAE method was performed in a closed vessel unit MDS-2002AT (Shanghai Sineo Microwave Chemical Technology Co., Ltd., Shanghai, China). The system was equipped with a temperature sensor for monitoring and regulating the internal temperature of the extraction vessels. Maximum oven power for this system was 800 W. The pressure was pre-established at a safe limit about 1.5 MP which could not go beyond during the extraction process. Dry samples (2 g) were placed into 100 mL PFTE (CEM) extraction vessels and the solvent added. The container was capped, and the system was started-up. The exact temperature was detected by a sensor. When the desired temperature was reached, the heating device would automatically shut down for a while until the temperature dropped. The extraction temperature and time were set at different degrees according to different conditions. After extraction, the vessels were left for several minutes to
The content of flavonoids was determined by spectrophotometry using the aluminium chloride colorimetric method (Amir et al., 2012) with some modifications. Briefly, 0.5 mL diluted solution containing flavonoids was added to a 10 mL test tube, and 0.1 mL of 5% (w/w) NaNO2 and 4 mL of 80% (v/v) ethanol were mixed for 5 min, and then 0.1 mL of 10% AlCl3 (w/w) was added and mixed, 6 min later, 3 mL of 1 mol/L NaOH was added. After 15 min, the absorbance of the solution at 410 nm was measured with a double beam uv/vis spectrophotometer (TU-1900, PGENENAL, Beijing, China) against the same mixture, without the sample as a blank. The calibration curve was prepared by preparing rutin solutions at concentrations from 10 to 100 lg/mL in methanol. The concentration of total flavonoids in extract was expressed as mg of rutin equivalents per gram dry weight of extract. The calibration curve was determined to be as the following: y = 0.0269x + 0.0054, where y is absorbance value of sample, x is sample concentration (10–300 lg/mL) (R2 = 0.9996). In the present work, the percentage extraction of flavonoids was expressed according to Eq. (3):
Percentage extraction ðw=wÞ ¼
Mass of flavonoidsðin extracted solutionÞ 100% Mass of materialðsamplesÞ
ð3Þ
2.6. Determination of antioxidant activity The total flavonoids obtained were subjected to screening for its possible antioxidant activity. The scavenging activity of flavonoids
Please cite this article in press as: Xie, J.-H., et al. Extraction, chemical composition and antioxidant activity of flavonoids from Cyclocarya paliurus (Batal.) Iljinskaja leaves. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.06.106
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J.-H. Xie et al. / Food Chemistry xxx (2014) xxx–xxx
Table 1 Experimental design runs in Statistical Analysis System (SAS version 9.0) and the observed responses value of extraction yield with different combinations of temperature (X1), extraction time (X2), solid–liquid ratio (X3), and alcohol concentration (X4).
a
Experiment number
Coded variables Temperature (°C) x1
Time (min) x2
Solid–liquid ratio (w/v) x3
Ethanol concentration (%) x4
Temperature (°C) X1
Actual variables Time (min) X2
Solid–liquid ratio (w/v) X3
Ethanol concentration (%) X4
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 28 29 30 31
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 0 0 0 0 0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 2 2 0 0 0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 2 2 0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 2 2 0 0 0 0 0 0 0
60 60 60 60 60 60 60 60 80 80 80 80 80 80 80 80 50 90 70 70 70 70 70 70 70 70 70 70 70 70 70
10 10 10 10 20 20 20 20 10 10 10 10 20 20 20 20 15 15 5 25 15 15 15 15 15 15 15 15 15 15 15
1:15 1:15 1:25 1:25 1:15 1:15 1:25 1:25 1:15 1:15 1:25 1:25 1:15 1:15 1:25 1:25 1:20 1:20 1:20 1:20 1:10 1:30 1:20 1:20 1:20 1:20 1:20 1:20 1:20 1:20 1:20
60 80 60 80 60 80 60 80 60 80 60 80 60 80 60 80 70 70 70 70 70 70 50 90 70 70 70 70 70 70 70
Observed yield (mg/g)a
14.84 14.47 14.22 12.70 15.22 13.99 14.36 12.99 15.55 14.47 16.12 14.25 15.33 14.77 15.37 15.17 12.85 14.96 14.52 14.92 14.55 14.55 15.26 12.61 14.73 14.88 14.82 14.77 14.95 14.89 14.89
Mean of duplicate runs.
from C. paliurus towards DPPH-radical was measured according to the method by Xie, Xie, Nie et al. (2010) with some modifications. Briefly, 0.2 mmol/L solution of DPPH in ethanol was prepared daily before measurements, 2 mL of various concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mg/g) of the sample solutions were thoroughly mixed with 2 mL of freshly prepared DPPH and 1 mL ethanol. The mixture was shaken vigorously and allowed to stand for 30 min in the dark, and the absorbance at 517 nm was then measured against a blank with a double beam uv/vis spectrophotometer (TU-1900, PGENENAL, Beijing, China). A lower absorbance value of the reaction mixture indicates a higher free radical scavenging activity. Ethanol was used as the blank control, ascorbic acid and BHT were used as positive controls. All tests were carried out in triplicate. The capability to scavenge the free radical DPPH in percentage of sample (I%) was calculated according to the following equation:
I% ¼
A0 ðA2 A1 Þ 100 A0
ð4Þ
where A0 is the absorbance of the incubation DPPH solution without addition of the sample or positive controls, A1 is the absorbance of the sample without DPPH solution and A2 is the absorbance of the incubation mixture containing both the test sample and DPPH solution. The concentration of sample producing a 50% reduction of the radical absorbance (IC50) was used as an index to compare the antioxidant activity.
2.7. Liquid chromatography–mass spectrometry (LC–MS) analysis Chemical composition of flavonoids extracted from the leaves of C. paliurus was analysed by LC–MS method (Coppin et al., 2013). Methanol and 0.1% (v/v) formic acid were used as the mobile phase. The elution program was as follows: 0–40 min (30–60% methanol), 40–60 min (60–80% methanol), 60–70 min (80–30% methanol). The flow rate was 0.8 mL/min and the injection volume was 5 lL. The UV detector was set at the wavelength of 270 nm and the column temperature was maintained at 30 °C. In order to reduce the contamination flow into the ion source, the eluent was discharged to waste for the first 2 min. The ESI interface operated in the negative mode was used. The eluent was monitored by electrospray ion mass spectrometer (ESI-MS) under positive ion mode and scanned from m/z 100 to 600. ESI was conducted by using a needle voltage of 3.5 kV under optimum collision energy level of 60%. Pure nitrogen (99.999%) was used as a dry gas and at a flow rate of 250 L/h and capillary temperature at 350 °C. Nitrogen was used as nebulizer at 60 psi. Drying nitrogen was heated to 150 °C. The chemical composition of flavonoids extracted from the leaves of C. paliurus were separated and identified by the retention time in HPLC and mass spectral data. The ion spray voltage was held at 3000 V in positive-ion mode. 2.8. Statistical analysis All data collected from MAE extraction experiments were centred by using three parallel measurements of mean ± SD. The
Please cite this article in press as: Xie, J.-H., et al. Extraction, chemical composition and antioxidant activity of flavonoids from Cyclocarya paliurus (Batal.) Iljinskaja leaves. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.06.106
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data of RSM were analysed using Statistical Analysis System (SAS version 9.0, SAS Institute Inc., Cary, North Carolina, USA) and used to design central composite rotatable design and analyse the experimental data. Data from the quadratic general rotary design were analysed by multiple regressions. Fischer’s test was used to determine the type of model equation, while the student’s t-test was performed for the determination of statistical significance of regression coefficients. P value
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Extraction, chemical composition and antioxidant activity of flavonoids from Cyclocarya paliurus (Batal.) Iljinskaja leaves Jian-Hua Xie a,1, Cai-jun Dong b,1, Shao-Ping Nie a, Feng Li b, Zhi-Jun Wang a, Ming-Yue Shen a, Ming-Yong Xie a,⇑ a b
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China Nantong Agricultural College, Nantong 226007, China
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: Cyclocarya paliurus Microwave-assisted extraction Flavonoids Antioxidant activity Chemical composition Response surface methodology
a b s t r a c t Microwave-assisted extraction of flavonoids from Cyclocarya paliurus (Batal.) Iljinskaja leaves, its chemical composition and antioxidant activity were investigated in this study. The influences of parameters including temperature, extraction time, ratio of material to solvent and solvents on the yield of flavonoids were studied. The optimal conditions were determined and the quadratic response surfaces were drawn from the mathematical models. The maximum extraction yield of 15.64 mg/g was achieved at temperature of 76.8 °C, extraction time of 15 min, alcohol concentration of 63.2% and ratio of solvent to material of 21.4:1. Five main constituents in the extract including quercetin-3-O-b-D-glucuronide, quercetin, kaempferol-3-O-b-D-glucuronide, kaempferol-7-O-a-L-rhamnoside and kaempferol were identified by LC–MS. In vitro antioxidant assays showed that the extract exhibited a strong DPPH radical-scavenging ability with IC50 value of 0.146 mg/mL. Results indicated that MAE was a suitable approach for the selective extraction of flavonoids from C. paliurus (Batal.) Iljinskaja leaves. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus) which belongs to the genus Cyclocarya Iljinskaja (Juglangdaceae), is the sole species in its genus, and mainly found in the mountainous regions in the tropics and subtropics (Xie, Li, Nie, Wang, & Lee, 2006). It grows mainly in the south of China, and is called ‘‘sweet tea tree’’ in Chinese (Xie, Shen et al., 2013). The leaves of C. paliurus are used in folk medicine for the treatment of diabetes mellitus, hypertension, hyperliposis, etc. (Li et al., 2000; Kurihara et al., 2003; Xie, Xie, Nie et al., 2010; Xie, Liu et al., 2013). The leaves of this plant have been widely used in China, which may be related to abundant organic compounds, especially flavonoids. Flavonoids are found to be the main active compounds in C. paliurus (Xie, Wang, Yi, & Wang, 2004), which have many biological activities, such as preventing hyperglycemia, diabetes mellitus, hypertension and coronary heart disease. Many flavonoids were isolated from C. paliurus, such as cyclocarioside, cyclocaric acid, kaempferol, ⇑ Corresponding author. Address: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, Jiangxi, China. Tel./fax: +86 791 83969009. E-mail addresses: [email protected] (J.-H. Xie), [email protected] (M.-Y. Xie). 1 These authors contributed equally to this work.
quercetin and isoquercitrin. (Shu, Xu, & Li, 1996; Xie et al., 2004; Zhang et al., 2010). Conventional techniques to obtain flavonoids, such as heating, boiling, or refluxing, usually require several hours or even days for the extraction process and a large volume of solvent, and may result in a loss of flavonoids due to hydrolysis, ionisation and oxidation during extraction (Li, Chen, & Yao, 2005). Microwave irradiation, which has proved to be a clean, efficient and convenient energy source, has been widely utilised in natural products extraction (Jiao et al., 2014; Xie et al., 2012; Zhang et al., 2013). Microwave-assisted extraction (MAE) is known to be a fast and efficient method for the extraction of flavonoids from plants. Compared with the traditional methods, MAE has many advantages, such as higher extraction rate, shorter extraction time, use of less solvent, better productivity and higher quality products (Chen, Xie, & Gong, 2007; Zhang, Yang, & Wang, 2011). Recently, MAE has been applied to extract various bioactive compounds from plants, such as terpenes from caraway seeds (Chemat, Aït-Amar, Lagha, & Esveld, 2005), quercetin from Flos Sophorae (Li et al., 2004) and flavonoids from cultivated Epimedium sagittatum (Zhang et al., 2013). A dynamic MAE system designed by Chen et al. (2008) markedly enhanced extraction yield of flavonoids from Herba Epimedii. To our knowledge, the optimised conditions of
http://dx.doi.org/10.1016/j.foodchem.2014.06.106 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xie, J.-H., et al. Extraction, chemical composition and antioxidant activity of flavonoids from Cyclocarya paliurus (Batal.) Iljinskaja leaves. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.06.106
2
J.-H. Xie et al. / Food Chemistry xxx (2014) xxx–xxx
MAE used in extraction of flavonoids from C. paliurus have not been reported yet. Response surface methodology (RSM), an important tool in process and product improvement, is a collection of experimental design and optimisation techniques that enables the researcher to determine the relationship between the response and the independent variables (Eren & Kaymak-Ertekin, 2007). To the best of our knowledge, there has been no report about the application of RSM in the optimisation of MAE conditions for the extraction of flavonoids from the leaves of C. paliurus. The objective of this study was to optimise the MAE conditions for the extraction of flavonoids from C. paliurus. RSM was employed to study the optimal temperature, extraction time, solid–liquid ratio and concentration of alcohol, which could maximise the yield of flavonoids from C. paliurus. Then, the colorimetric method with AlCl3/methanol system was used to quantify total flavonoids, which has been reported to be a simple, quick and accurate method (Zhao, Xu, & Liu, 2004). In addition, the antioxidant activity of flavonoids from C. paliurus, obtained under optimised MAE extraction conditions was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay. The chemical composition of the MAE extract was also evaluated by liquid chromatography–mass spectrometry (LC–MS) analysis. 2. Materials and methods 2.1. Plant materials The leaves of C. paliurus were collected in Xiushui County, Jiangxi Province, China. A voucher specimen was deposited at the State Key Laboratory of Food Science and Technology, Nanchang University, China. The leaves were air dried and ground into a fine powder (40–60 mesh) in a mill.
cool down to room temperature. Then each extract was filtered and ethanol was added in order to make the total volume of 100 mL (Chen et al., (2007)). 2.4. Experimental design RSM was used to design the MAE of total flavonoids from the leaves of C. paliurus. To explore the effect of independent variables on the response within the range of investigation, a central composite rotate design with four independent variables (X1, temperature, X2, extraction time, X3, solid–liquid ratio and X4, alcohol concentration) at five levels was performed. Each independent variable had coded levels of 2, 1, 0, 1 and 2. The experimental designs of the coded (x) and actual (X) levels of variables are shown in Table 1. The variables were coded according to Eq. (1):
xi ¼
ðX i X 0 Þ DX i
ð1Þ
where xi is the coded value of an independent variable, Xi is the real value of the independent variable, X0 is the real value of an independent variable at the centre point, and DX is the step change value. The yield of flavonoids was considered as the dependent variable or response. For a central composite rotate design with four independent variables at five levels, 31 experimental runs are required. The actual design of experiments is given in Table 1. The experimental results were fitted to a second-order polynomial model, and the regression coefficients were determined. The quadratic model for predicting the optimal point was expressed according to Eq. (2):
Y k ¼ bk0 þ
4 4 4 X X X bki xi þ bkii x2i þ bkij xi xj i¼1
i¼1
ð2Þ
j¼1
where bk0, bki, bkii and bkij are constant regression coefficients of the model, while xi, xj are the independent variables.
2.2. Chemicals and reagents 2.5. Determination of flavonoids content and yield of flavonoids Methanol and formic acid (HPLC grade) were obtained from Merck (Darmstadt, Germany). Ethanol was purchased from Shanghai Chemicals and Reagents Co. (Shanghai, China). Quercetin was supplied by China Institute for Drugs and Biological Products Identification (Beijing, China). Butylated hydroxytoluene, 2,2diphenyl-1-picrylhydrazyl (DPPH), and ascorbic acid (VC) were purchased from Sigma (St. Louis, MO, USA). All the other reagents were of analytical grade. Ultrapure water was prepared by Milli-Q50 (Millipore Corp., Bedford, MA, USA) water purification system. All solvents used for HPLC measurements were filtered (0.45 lm) before being used. 2.3. MAE The MAE method was performed in a closed vessel unit MDS-2002AT (Shanghai Sineo Microwave Chemical Technology Co., Ltd., Shanghai, China). The system was equipped with a temperature sensor for monitoring and regulating the internal temperature of the extraction vessels. Maximum oven power for this system was 800 W. The pressure was pre-established at a safe limit about 1.5 MP which could not go beyond during the extraction process. Dry samples (2 g) were placed into 100 mL PFTE (CEM) extraction vessels and the solvent added. The container was capped, and the system was started-up. The exact temperature was detected by a sensor. When the desired temperature was reached, the heating device would automatically shut down for a while until the temperature dropped. The extraction temperature and time were set at different degrees according to different conditions. After extraction, the vessels were left for several minutes to
The content of flavonoids was determined by spectrophotometry using the aluminium chloride colorimetric method (Amir et al., 2012) with some modifications. Briefly, 0.5 mL diluted solution containing flavonoids was added to a 10 mL test tube, and 0.1 mL of 5% (w/w) NaNO2 and 4 mL of 80% (v/v) ethanol were mixed for 5 min, and then 0.1 mL of 10% AlCl3 (w/w) was added and mixed, 6 min later, 3 mL of 1 mol/L NaOH was added. After 15 min, the absorbance of the solution at 410 nm was measured with a double beam uv/vis spectrophotometer (TU-1900, PGENENAL, Beijing, China) against the same mixture, without the sample as a blank. The calibration curve was prepared by preparing rutin solutions at concentrations from 10 to 100 lg/mL in methanol. The concentration of total flavonoids in extract was expressed as mg of rutin equivalents per gram dry weight of extract. The calibration curve was determined to be as the following: y = 0.0269x + 0.0054, where y is absorbance value of sample, x is sample concentration (10–300 lg/mL) (R2 = 0.9996). In the present work, the percentage extraction of flavonoids was expressed according to Eq. (3):
Percentage extraction ðw=wÞ ¼
Mass of flavonoidsðin extracted solutionÞ 100% Mass of materialðsamplesÞ
ð3Þ
2.6. Determination of antioxidant activity The total flavonoids obtained were subjected to screening for its possible antioxidant activity. The scavenging activity of flavonoids
Please cite this article in press as: Xie, J.-H., et al. Extraction, chemical composition and antioxidant activity of flavonoids from Cyclocarya paliurus (Batal.) Iljinskaja leaves. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.06.106
3
J.-H. Xie et al. / Food Chemistry xxx (2014) xxx–xxx
Table 1 Experimental design runs in Statistical Analysis System (SAS version 9.0) and the observed responses value of extraction yield with different combinations of temperature (X1), extraction time (X2), solid–liquid ratio (X3), and alcohol concentration (X4).
a
Experiment number
Coded variables Temperature (°C) x1
Time (min) x2
Solid–liquid ratio (w/v) x3
Ethanol concentration (%) x4
Temperature (°C) X1
Actual variables Time (min) X2
Solid–liquid ratio (w/v) X3
Ethanol concentration (%) X4
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 28 29 30 31
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 0 0 0 0 0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 2 2 0 0 0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 2 2 0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 2 2 0 0 0 0 0 0 0
60 60 60 60 60 60 60 60 80 80 80 80 80 80 80 80 50 90 70 70 70 70 70 70 70 70 70 70 70 70 70
10 10 10 10 20 20 20 20 10 10 10 10 20 20 20 20 15 15 5 25 15 15 15 15 15 15 15 15 15 15 15
1:15 1:15 1:25 1:25 1:15 1:15 1:25 1:25 1:15 1:15 1:25 1:25 1:15 1:15 1:25 1:25 1:20 1:20 1:20 1:20 1:10 1:30 1:20 1:20 1:20 1:20 1:20 1:20 1:20 1:20 1:20
60 80 60 80 60 80 60 80 60 80 60 80 60 80 60 80 70 70 70 70 70 70 50 90 70 70 70 70 70 70 70
Observed yield (mg/g)a
14.84 14.47 14.22 12.70 15.22 13.99 14.36 12.99 15.55 14.47 16.12 14.25 15.33 14.77 15.37 15.17 12.85 14.96 14.52 14.92 14.55 14.55 15.26 12.61 14.73 14.88 14.82 14.77 14.95 14.89 14.89
Mean of duplicate runs.
from C. paliurus towards DPPH-radical was measured according to the method by Xie, Xie, Nie et al. (2010) with some modifications. Briefly, 0.2 mmol/L solution of DPPH in ethanol was prepared daily before measurements, 2 mL of various concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mg/g) of the sample solutions were thoroughly mixed with 2 mL of freshly prepared DPPH and 1 mL ethanol. The mixture was shaken vigorously and allowed to stand for 30 min in the dark, and the absorbance at 517 nm was then measured against a blank with a double beam uv/vis spectrophotometer (TU-1900, PGENENAL, Beijing, China). A lower absorbance value of the reaction mixture indicates a higher free radical scavenging activity. Ethanol was used as the blank control, ascorbic acid and BHT were used as positive controls. All tests were carried out in triplicate. The capability to scavenge the free radical DPPH in percentage of sample (I%) was calculated according to the following equation:
I% ¼
A0 ðA2 A1 Þ 100 A0
ð4Þ
where A0 is the absorbance of the incubation DPPH solution without addition of the sample or positive controls, A1 is the absorbance of the sample without DPPH solution and A2 is the absorbance of the incubation mixture containing both the test sample and DPPH solution. The concentration of sample producing a 50% reduction of the radical absorbance (IC50) was used as an index to compare the antioxidant activity.
2.7. Liquid chromatography–mass spectrometry (LC–MS) analysis Chemical composition of flavonoids extracted from the leaves of C. paliurus was analysed by LC–MS method (Coppin et al., 2013). Methanol and 0.1% (v/v) formic acid were used as the mobile phase. The elution program was as follows: 0–40 min (30–60% methanol), 40–60 min (60–80% methanol), 60–70 min (80–30% methanol). The flow rate was 0.8 mL/min and the injection volume was 5 lL. The UV detector was set at the wavelength of 270 nm and the column temperature was maintained at 30 °C. In order to reduce the contamination flow into the ion source, the eluent was discharged to waste for the first 2 min. The ESI interface operated in the negative mode was used. The eluent was monitored by electrospray ion mass spectrometer (ESI-MS) under positive ion mode and scanned from m/z 100 to 600. ESI was conducted by using a needle voltage of 3.5 kV under optimum collision energy level of 60%. Pure nitrogen (99.999%) was used as a dry gas and at a flow rate of 250 L/h and capillary temperature at 350 °C. Nitrogen was used as nebulizer at 60 psi. Drying nitrogen was heated to 150 °C. The chemical composition of flavonoids extracted from the leaves of C. paliurus were separated and identified by the retention time in HPLC and mass spectral data. The ion spray voltage was held at 3000 V in positive-ion mode. 2.8. Statistical analysis All data collected from MAE extraction experiments were centred by using three parallel measurements of mean ± SD. The
Please cite this article in press as: Xie, J.-H., et al. Extraction, chemical composition and antioxidant activity of flavonoids from Cyclocarya paliurus (Batal.) Iljinskaja leaves. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.06.106
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data of RSM were analysed using Statistical Analysis System (SAS version 9.0, SAS Institute Inc., Cary, North Carolina, USA) and used to design central composite rotatable design and analyse the experimental data. Data from the quadratic general rotary design were analysed by multiple regressions. Fischer’s test was used to determine the type of model equation, while the student’s t-test was performed for the determination of statistical significance of regression coefficients. P value
