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Ultrasonic-assisted extraction and purification of phenolic compounds from sugarcane (Saccharum officinarum L.) rinds
LWT - Food Science and Technology xxx (2014) 1e7
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Ultrasonic-assisted extraction and purification of phenolic compounds from sugarcane (Saccharum officinarum L.) rinds* Simin Feng a, Zisheng Luo a, b, *, Beipei Tao a, Chun Chen a a b
Department of Food Science and Nutrition, Zhejiang University, Hangzhou, 310058, People's Republic of China Fuli Institute of Food Science, Zhejiang University, Hangzhou, 310058, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 September 2013 Received in revised form 1 September 2014 Accepted 13 September 2014 Available online xxx
In order to obtained highest extraction efficiency of phenolic compounds from the sugarcane rinds, a rapid extraction method based on ultrasound-assisted extraction technique using response surface methodology was investigated. Results showed that the optimal conditions were: solvent concentration 52.19 mL/100 mL, solideliquid ratio 14.46 mL/g, ultrasonic temperature 61.54 C and extraction time 31.30 min. The practical phenolic compounds extraction rate was 8.67 g/100 g DW, which was well matched with the predicted value of 8.82 g/100 g DW. Purified by macroporous resins adsorption or solvent extraction method, the total phenolic content of crude phenolic compounds extracts improved from 117.50 ± 13.00 to 302.50 ± 19.50 or 670.00 ± 17.00 mg/g, respectively. Gallic acid, chlorogenic acid and ferulic acid were identified as three main phenolic compounds in the phenolic compounds extract by HPLC analysis. Furthermore, TPC revealed good correlations with antioxidant activities. All of the results indicated that sugarcane rinds could be used as a good source of phenolic compounds with significant antioxidant activities. © 2014 Published by Elsevier Ltd.
Keywords: Sugarcane rinds Phenolic compounds Ultrasonic-assisted extraction Response surface methodology Antioxidant
1. Introduction Sugarcanes (Saccharum officinarum L.) are one of the most important economic plants, and are the most important sources of sugar as 70% of the world's sugars are from sugarcanes. Sugarcanes have high content of phenolic acids, flavonoids and other phenolic compounds (Duarte-Almeida, Salatino, Genovese, & Lajolo, 2011). Five antioxidant compounds, namely syringaresinol, medioresinol, coniferyl alcohol, 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)1-propanone and sinapyl alcohol, were first isolated from a kokuto (a sugarcane product consumed as candies in Japan) extract (Nakasone et al., 1996). More antioxidant compounds, such as chlorogenic acid (Duarte-Almeida et al., 2011) and ferulic acid (Fontaniella, Vicente, de Armas, & Legaz, 2007) were isolated from sugarcane and its products. Sugarcane rinds, as waste by-product of
Abbreviations: TPC, total phenolic content; E1, crude sugarcane rind extract powder; E2, sugarcane rind extract powder purified by MARs; E3, sugarcane rind extract powder purified by solvent extraction. * This manuscript was presented in the international conference of “Food Innova2012” Hangzhou, China, December 12e14, 2012. * Corresponding author. Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, People's Republic of China. Tel.: þ86 571 88982162. E-mail address: [email protected] (Z. Luo).
the sugar industries, are generated in large quantities, about 20% weight of sugarcanes. Recently, response surface methodology (RSM) is widely used for the optimization of the extraction methods. As a type of RSM, BoxeBehnken design (BBD) is an independent quadratic design that does not contain an embedded factorial or fractional factorial design (Zhu & Liu, 2013). BBD was easy to arrange and interpret experiment and more efficient than other response surface designs since they can be used to establish a quadratic response surface (Li et al., 2013). It has been successfully used in the optimization of the extraction of polyphenols from natural sources (Yue, Shao, Yuan, Wang, & Qiang, 2012). However, there are very few reports on the use of BBD in the optimization of extraction of phenolic compounds from sugarcane rinds. Conventional extraction with heating, boiling and refluxing are usually used for covering phenolic compounds. However, the disadvantages are degradation of phenolic compounds during long extraction time. Up to now, several new extraction techniques have been reported for the extraction of bioactive compounds from plants, including microwave-assisted extraction (Bai, Yue, Yuan, & Zhang, 2010), ultrasound-assisted extraction (UAE) (Zou, Xie, Fan, Gu, & Han, 2010), supercritical fluid extraction (Oliveira, Kamimura, & Rabi, 2009). Among these, the UAE is one of simple and efficient extraction techniques (Huang, Xue, Niu, Jia, & Wang,
http://dx.doi.org/10.1016/j.lwt.2014.09.066 0023-6438/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: Feng, S., et al., Ultrasonic-assisted extraction and purification of phenolic compounds from sugarcane (Saccharum officinarum L.) rinds, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.09.066
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S. Feng et al. / LWT - Food Science and Technology xxx (2014) 1e7
2009). The ultrasonic cavitation creates shear forces that can break cell walls mechanically, which allow high diffusion rates across the cell wall (Vinatoru, 2001). Moreover, UAE technique is one of environment friendly techniques that can offer no toxic chemical involvement, less solvent consumption, low temperature and lower energy input. Due to their convenience, low cost, high chemical stability, friendly to environmental protection and easy regeneration, separation method based on macroporous adsorption resins (MARs) was frequently used in the separation and purification of pharmaceutical and natural products (Shi et al., 2002). Some studies show that the MARs method was widely investigated and used for separating bioactive compounds from natural extracts such as red pigments (Zhang, Yin, Kong, & Jiang, 2011), lycopene (Liu, Liu, Chen, Liu, & Di, 2010), phenolics and rosmarinic acid (Lin, Zhao, Dong, Yang, & Zhao, 2012). There is very few references available on the UAE of phenolic compounds from sugarcane rinds. In the present study, UAE technique was used as a rapid extraction process for phenolic compounds from sugarcane rinds for good yields. Several operational parameters that could potentially affect the extraction efficiency were optimized using RSM. Then crude sugarcane rinds extracts were purified by MARs and solvent extraction method. Finally, free radical scavenging activity (FRSA) of sugarcane rinds phenolic compounds were evaluated in vitro study. 2. Materials and methods 2.1. Plant material Sugarcanes (Badila) were collected at commercial maturity from Zhejiang, PR China. The outer layer rinds were obtained by hand peeling and rinds samples were carefully separated from the inner pith with sharp knives. Then the rinds samples were air dried at 40 C, smashed into powder with a pulverizer (Huangchen Machinery Co. Ltd., Zhejiang, China), passed through a 60-mesh (pore size in 0.25 mm) sieve and kept dry at 18 C until use. 2.2. Chemicals FolineCiocalteu reagent, 2, 2-di-phenyl-1-picrylhydrazyl (DPPH) free radical, 4, 6-tripryridyls-triazine (TPTZ), Gallic acid-1hydrate was purchased from Aladdin Industrial Co. (Shanghai, China). Chlorogenic, vanillic, ferulic, gallic, syringic, p-Coumaric and caffeic acids, catechin and quercertin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). MARs D-101 were purchased from Sigma Chemical Co. (St. Louis, MO, USA), X-5, H-103, S8, NKA-9 and AB-8 were purchased from Nankai Hecheng S&T Co. Ltd (Tianjin, China) and their physico chemical properties were listed in Table 1. All the other chemicals used were of analytical grade or HPLC grade.
Table 1 Physicochemical properties of the macroporous adsorption resins. Type
Polarity
Surface area (m2/g)
Particle diameter (mm)
Average pore diameter (Å)
S-8 NKA-9 AB-8 X-5 H-103 D-101
Polar Polar Weak-polar Nonpolar Nonpolar Nonpolar
10e120 250e290 480e520 500e600 1000e1100 500e550
0.3e1.25 0.3e1.25 0.3e1.25 0.3e1.25 0.3e1.25 0.3e1.25
28e30 15e16.5 13e14 29e30 8.5e9.5 9e10
2.3. UAE of phenolic compounds UAE was performed with an ultrasonic apparatus (Ningshang Ultrasonic Instrument Co. Ltd., Shanghai, China). Pre-weighed amounts of sugarcane rinds powder were placed into a volumetric flask (100 mL), soaked with ethanol solvent (varying ethanol concentration from 30 to 80 mL/100 mL; varying liquid to solid ratio from 5:1 to 25:1, mL/g) and then placed in ultrasonic cleaning bath at 40 kHz for certain time (varying extraction time from 5 to 50 min) at a constant temperature (varying extraction temperature from 30 to 80 C). After filtration through filter paper, anhydrous ethanol was added to bring the final volume of the extract to 50 mL. The phenolic concentration of sugarcane rinds extract was measured according to the method described in Section 2.5. Phenolic compounds extraction rate (Y) was expressed as g phenolic compounds/100 g sample. All results were expressed by dry weight (DW). Then bulk solvents were removed from the extract in a rotary vacuum evaporator (IKAWerke-GmbH & Co., Staufen, Germany) at 40 C. At last, the crude sugarcane rinds extract powder (E1) was freeze-dried and kept sealed in the dark. 2.4. Design of experiment Box-Behnken (BB) design was used to determine the optimal conditions of the UAE. According to the principle of BB design, solvent concentration (mL/100 mL) (X1), solideliquid ratio (mL/g) (X2), ultrasonic temperature ( C) (X3) and extraction time (min) (X4), were taken as independent variables tested in a 29-run experiment. As we can see from Table 2, the four independent variables were prescribed into 3 levels, coded 1, 0 and þ1 for low, intermediate and high value, respectively. Phenolic compounds extraction rate (Y) was taken as response of the design experiments. The experimental design used in the study is showed in Table 2. A second-degree polynomial regression model was used to Table 2 BB design and the responses values for Phenolic compounds extraction rate. Run Solvent Solideliquid Ultrasonic Extraction Y(g/100 g DW) concentration ratio temperature time X4(min) X1(mL/100 mL) X2(mL/g) X3( C) 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
45(1) 50(0) 50(0) 50(0) 45(1) 50(0) 50(0) 50(0) 50(0) 50(0) 50(0) 50(0) 55(1) 55(1) 55(1) 50(0) 50(0) 50(0) 55(1) 45(1) 55(1) 45(1) 45(1) 45(1) 50(0) 55(1) 50(0) 50(0) 50(0)
15(0) 10(1) 15(0) 15(0) 15(0) 15(0) 20(1) 15(0) 15(0) 10(1) 10(1) 20(1) 15(0) 15(0) 20(1) 15(0) 15(0) 15(0) 10(1) 10(1) 15(0) 15(0) 20(1) 15(0) 20(1) 15(0) 10(1) 20(1) 15(0)
55(1) 55(1) 55(1) 60(0) 60(0) 60(0) 60(0) 65(1) 60(0) 60(0) 60(0) 55(1) 60(0) 55(1) 60(0) 65(1) 55(1) 60(0) 60(0) 60(0) 60(0) 65(1) 60(0) 60(0) 60(0) 65(1) 65(1) 65(1) 60(0)
30(0) 30(0) 20(1) 30(0) 20(1) 30(0) 20(1) 20(1) 30(0) 20(1) 40(1) 30(0) 20(1) 30(0) 30(0) 40(1) 40(1) 30(0) 30(0) 30(0) 40(1) 30(0) 30(0) 40(1) 40(1) 30(0) 30(0) 30(0) 30(0)
6.17 6.42 6.06 8.63 6.53 8.64 6.88 6.92 8.61 6.71 6.88 6.79 7.12 6.38 7.56 7.09 6.57 8.65 8.44 6.34 7.78 6.68 6.94 7.37 8.11 8.22 7.92 7.34 8.63
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S. Feng et al. / LWT - Food Science and Technology xxx (2014) 1e7
correlate the relationship between independent variables and response (phenolic compounds extraction rate), and the equation was.
Y4 ¼ a0 þ
4 X i¼1
ai Xi þ
4 X
aii Xi2 þ
i¼1
3 4 X X
aij Xi Xj
(1)
i¼1 j¼iþ1
where Y4 is the response variables. a0, ai, aii and aij are the intercept, linear, quadratic and interactive coefficients, respectively. Xi and Xj are the levels of the independent variables (isj). 2.5. Total phenolic content (TPC) The total phenol content was determined according to the FolineCiocalteu method (Zhang et al., 2013) with minor modifications. Gallic acid was used as standard, and the results were expressed as mg gallic acid/mL or g of sample. 0.2 mL of the extract (2 mg/mL) was mixed with 1 mL of the Folin Ciocalteu's reagent and 2 mL of 15 (g/100 mL) Na2CO3. The 10 mL volumetric flasks were shaken and allowed to stand for 2 h at room temperature. The absorbance was measured with a spectrophotometer (Shimadzu Co., Tokyo, Japan) at 765 nm. All assays were run in three replicates. 2.6. Purification of phenolic compounds by the macroporous adsorption resins (MARs) 2.6.1. Static adsorption of MARs and static desorption tests MARs X-5, D-101, H-103, S-8, NKA-9 and AB-8 were washed with abundant distilled water to remove salts and impurities. Before the adsorption experiment, pre-weighed amounts of adsorbents were washed with ethanol and subsequently the ethanol was thoroughly substituted with deionized water (Zhang et al., 2011). Experiments on the adsorption/desorption behaviors of 6 different MARs were carried out to select suitable resin for the purification of phenolic compounds. Static adsorption tests of extractions were performed as follows: pre-weighed amounts of hydrated MARs (1.000 g) were introduced into a 250 mL air-tight conical flask. Then each 25 mL of different concentrations of sugarcane rinds extracts (E1) was added into each flask. The flasks were shaken (100 rpm) in a constant temperature water-bath shaker for 24 h at 25 C. The phenolic concentration of the solution at different time was measured according to the method described in Section 2.5. Static desorption tests were carried out as follows: After adsorption experiment, each MARs was first filtered, washed by distilled water. Then resins were desorbed with 25 mL ethanolewater solution (30, 40, 50, 60, 70, 80 and 90 mL/100 mL) in the 250 mL air-tight conical flask. The flasks were shaken (100 rpm) in a constant temperature water-bath shaker for 24 h at 25 C. The phenolic concentration of supernatant was measured according to the method described in Section 2.5. Adsorption capacity was calculated by using the following equations (Lin et al., 2012; Liu et al., 2010):
Acðmg=gÞ ¼ ðC0 C1 Þ V=M
(2)
Adsorption rate was calculated by using the following equation:
Arð%Þ ¼ 100% ðC0 C1 Þ=C0
(3)
Desorption rate was calculated by using the following equation:
Drð%Þ ¼ 100% Cd Vd =ððC0 C1 Þ VÞ
(4)
where Ac is the adsorption capacity (mg/g). C0 is the phenolics concentration of the sample solution before adsorption (mg/mL). C1
3
is the phenolics concentration of the sample solution after adsorption (mg/mL). M is the weight of the hydrated resin. V is the volume of phenolics solutions used in the study (mL). Ar is the adsorption rate of the sample (%). Dr is the desorption rate (%). Cd is the phenolics concentration in the desorption solution (mg/mL). Vd is the volume of the desorption solution (mL). 2.6.2. Purification of phenolic compounds 300 mL of crude sugarcane rinds extract was first processed by resin X-5 adsorption, and then the effluent was concentrated in a rotary vacuum evaporator (Yarong biochemical Co. Ltd., Shanghai, China) at 40 C after desorption. At last, the sugarcane rinds extract powder purified by MARs (E2) was freeze-dried and kept sealed in the dark. 2.7. Purification of phenolic compounds by solvent extraction method A certain amount of sodium hydroxide standard solution was first added to 300 mL of crude sugarcane rinds extract to adjust the pH value to 6.5. Then ethyl acetate was used as liquid extractant to purify the sugarcane rinds extract for three times. After the extraction, the liquid extractant was concentrated in a rotary vacuum evaporator at 40 C. At last, the sugarcane rinds extract powder purified by solvent extraction (E3) was freeze-dried and kept sealed in the dark. 2.8. High performance liquid chromatography (HPLC) analysis Identification and quantification of phenolic substances were carried out using an LC-2010 (Japan AT) system with an ultravioletvisible detector. 0.2 g of samples (E1, E2 and E3) was dissolved in 2 mL of methanol and filtered through 0.5 mm Fluorpore membranes (Millipore). The injection volume was 10 ml. Separation was performed on a Luna 5m C-18 column (250mm 4.6 mmi.d., Phenomenex Ltd.). The mobile phase with a flow rate of 0.8 mL/min consisted of two solvents: A, 5 mL/100 mL acetic acid solution and B, acetonitrile. The solvent gradient in volume ratios was as follows: 0e5 min: 0e5% B; 5e22 min: 5e11.5% B; 22e32 min: 11.5e11.7% B; 32e62 min: 11.7e71.7% B; 62e65 min: 71.7 to 5% B; 65e72 min: 5 to 0% B. All the analyses were performed at temperature of 40 C, and the wavelength used for detection was 280 nm. Identification followed comparison of retention times and UV spectra with authentic standards. Standards used for phenylpropanoid analyses were chlorogenic, vanillic, catechin, ferulic, gallic, syringic, p-coumaric, quercertin and caffeic acids. All the standards were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Quantification was based on external calibration and the final concentrations were expressed as mg/g of sample. 2.9. Evaluation of free radical scavenging activity (FRSA) The FRSA of the extracts was evaluated according to the method of (Wang, Li, Zeng, & Liu, 2008), with slight modifications. Ascorbic acid was used as a positive control. Three samples (E1, E2 and E3) were dissolved in 50 mL/100 mL aqueous ethanol with different concentrations: 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/mL, respectively. The samples of 2.0 mL were added to 4.0 mL of 0.1 mmol DPPH. The mixtures were shaken and reacted in the dark at 25 C for 30 min. The FRSA (%) of the tested samples, were evaluated by comparison with a control (4.0 mL DPPH solution and 2 mL of 50 mL/100 mL ethanol solution). Scavenging capacity was measured by monitoring the absorbance at 517 nm.
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2.10. Statistical analysis Analysis of variance (ANOVA) was used to determine the significant difference in phenolic compounds yield extracted from sugarcane rinds under different conditions. A second-order polynomial regressed equation was established on the basis of analysis of BB experimental data, and the optimal conditions for extraction were found using the software of Design Expert version 6.0.10 (Stat-Ease, Inc.). 3. Results and discussion 3.1. Optimization of UAE condition of phenolic compounds by RSM 3.1.1. Extraction model The BB design was employed to study the interactions among four individual parameters. The independent variables and the corresponding response values obtained in different experimental combination are listed in Table 2. The phenolic compounds extraction rate ranged from 6.06 to 8.65 g/100 g DW. Multiple regression analysis was used to analyze the experimental data and thus a quadratic polynomial equation of the response surfaces obtained is as follow:
Y ¼ 215:865 þ 2:245X1 þ 1:857X2 þ 4:65X3 þ 0:616X4 0:015X1 X2 þ 0:013X1 X3 9 103 X1 X4 9:5 103 X2 X3 þ 5:3 103 X2 X4 0:027X12 0:023X22 0:042X32 8:643 103 X42
(5)
The statistical significance of Eq. (5) was checked by F-test, and ANOVA results of experiment model are showed in Table 3. The model F-value of 16.27 obtained by ANOVA indicated that the model was very significant (P < 0.01). It indicated that the quadratic model was in good agreement with the experimental results. The R2 value closer to one suggested a better correlation between the observed and predicted values (Gangadharan, Sivaramakrishnan, Nampoothiri, Sukumaran, & Pandey, 2008). The determination coefficient (R2) was 0.9421 implying that the regression model could explain 94.21% of the result in the case of phenolic compounds extraction rate. Moreover, a low value of coefficient of variation (CV ¼ 3.98%) suggested that the model was reproducible (Wanasundara & Shahidi, 1996). The results indicated that the model could work well for the prediction of phenolic compounds
Table 3 ANOVA results of experiment model. Source
Sum of squares
df
Mean square
F value
P-value
Modle X1 X2 X3 X4 X21 X1X2 X1X3 X1X4 X22 X2X3 X2X4 X23 X3X4 X24 Residual Cor total
19.34 2.49 0.069 2.78 1.07 2.96 0.55 0.44 0.0081 2.15 0.23 0.28 7.11 0.029 4.85 1.19 20.53
14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 28
1.38 2.49 0.069 2.78 1.07 2.96 0.55 0.44 0.0081 2.15 0.23 0.28 7.11 0.029 4.85 0.085
16.27 29.38 0.81 32.8 12.58 34.88 6.45 5.21 0.095 25.32 2.66 3.31 83.75 0.34 57.1
<0.0001*** <0.0001*** 0.3825 <0.0001*** 0.0032** <0.0001*** 0.0236* 0.0386* 0.7619 0.0002*** 0.1253 0.0903 <0.0001*** 0.5688 <0.0001***
R2 ¼ 0.9421 R2Adj ¼ 0.8842 *P < 0.05 **P < 0.01 ***P < 0.001.
extract from sugarcane rinds. The P-values were used to check the significance of each factor on phenolic compounds extraction rate. As shown in Table 3, the independent variables (X1, X3 and X4) and quadratic terms (X21, X1X2, X1X3, X22, X23 and X24) significantly affected the phenolic compounds extraction rate (P < 0.05). The results showed that solvent concentration and ultrasonic temperature were the two most significant single factors, which affected the phenolic compounds extraction rate followed by extraction time and solideliquid ratio. 3.1.2. Optimization of extraction conditions and verification of the model In order to obtain desirable response goals multiple response optimizations were carried out to determine optimum levels of independent variables. Six independent response surface plots are showed in Fig. 1. Each pair of parameters within the experimental rang was depicted in 3-D surface plots, while the two other parameters were kept constant at zero level. The shape of the 3-D surface plots indicated a moderate interaction between these tested variables (Pan, Yu, Zhu, & Qiao, 2012) and facilitated the identification of the optimum experimental conditions (Chen, Wang, Zhang, & Huang, 2012). Fig. 1(a, d and f) show the effects of solvent concentration with each of the three other variables on phenolic compounds extraction rate. As can be seen from Fig. 1(a, d and f), the phenolic compounds extraction rate increased with increasing solvent concentration at first. However, the trend was reversed when the phenolic compounds extraction rate reached a certain value. According to theory of similarity and intermiscibility, the phenolic compounds are easily dissolved from plant cells, when polarities of solvent are similar to phenolic compounds (Gribova, Filippenko, Nikolaevskii, Belaya, & Tsybulenko, 2008) When solvent concentration amount exceeded the certain value, polarities of solvent changed again and the phenolic compounds extraction rate decreased. Fig. 1(a, c and e) show the effects of solideliquid ratio with each of the three other variables on phenolic compounds extraction rate. As shown, the increase in the solideliquid ratio led to a gradual increase in phenolic compounds extraction rate and extraction rate reached a maximum when the solideliquid ratio was up to a certain value, with no significantly further improvement thereafter. The effects of ultrasonic temperature interaction with each of the three other factors on the phenolic compounds extraction rate are displayed in Fig. 1(b, c and e). As shown, the phenolic compounds extraction rate increased readily with increasing ultrasonic temperature up to 60 C and followed by a slight decrease thereafter. With suitable temperature, more phenolic compounds would be extracted into the solvent, as heating might weaken the cell wall integrity, soften the plant tissue, hydrolyze the bonds of bound phenolic compounds (phenoleprotein or phenolepolysaccharide) (Spigno, Trarnelli, & De Faveri, 2007; Tabaraki & Nateghi, 2011). However, high temperature promoted degradation such as the oxidation of phenolic compounds, resulting in decreased extraction yield when temperatures is above 60 C, and it might also cause higher furfural (a carcinogenic compound) contents in the phenolic extract (Rodrigues & Pinto, 2007). Fig. 1(b, c and e) show the effects of extraction time with each of the three other variables on phenolic compounds extraction rate. In all situations, the phenolic compounds extraction rate increased with increasing extraction time from 20 to 30 min, and then fell above 30 min, which indicated that the extraction time has a remarkable effect on phenolic compounds extraction rate. The optimal conditions were listed as follows: solvent concentration 52.19 mL/100 mL, solideliquid ratio 14.46 mL/g, ultrasonic temperature 61.54 C and extraction time 31.30 min. However,
Please cite this article in press as: Feng, S., et al., Ultrasonic-assisted extraction and purification of phenolic compounds from sugarcane (Saccharum officinarum L.) rinds, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.09.066
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Fig. 1. Response surface graph showing effects of the extraction parameters on phenolic compounds extraction rate (a) at varying solvent concentration and solideliquid ratio, (b) at varying ultrasonic temperature and extraction time, (c) at varying ultrasonic temperature and solideliquid ratio, (d) at varying ultrasonic temperature and solvent concentration, (e) at varying extraction time and solideliquid ratio, and (f) at varying extraction time and solvent concentration.
considering the operability in actual production, optimal conditions can be modified as follows: solvent concentration 52%, solideliquid ratio 14 mL/g, ultrasonic temperature 62 C and extraction time 31 min. The predicted phenolic compounds extraction rate was 8.82 g/100 g DW, which was consistent with the practical phenolic compounds extraction rate of 8.67 g/100 g DW. These data indicated that the model designed in this study was valid.
3.2. Screening of optimum MARs adsorption method
Fig. 2. Adsorption/desorption properties of phenolic compounds on six macroporous adsorption resins: AB-8, D-101, H-103, NKA-9, S-8, and X-5. Results were means ± SD of three independent experiments (n ¼ 3) followed by the different letters, which indicated significantly different at P < 0.05. adsorption rate; desorption rate.
As illustrated in Fig. 2, adsorption/desorption properties of six MARs for purifying phenolic compounds were studied in the static adsorption/desorption tests. It could be seen that the adsorption rates were calculated as 50.89% and 60.11% for X-5 and H-103 resins, respectively, and they were considerably higher than those of other resins. Our results were in accord with the results of Sun, Guo, Fu, Li, & Li, 2013, who found that X-5 resin was verified to offer the best adsorption capacity and desorption ratio for total polyphenols. Desorption rate of phenolic compounds was the highest for X-5. As can be seen from Fig. 3a, adsorption capacity (for phenolic compounds absorbed on X-5 resins) reached the saturation plateau when the initial concentration of phenolic compounds
Please cite this article in press as: Feng, S., et al., Ultrasonic-assisted extraction and purification of phenolic compounds from sugarcane (Saccharum officinarum L.) rinds, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.09.066
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Fig. 3. Effect of initial concentration of the crude sugarcane rinds extract E1 on adsorption capacity and effect of ethanol concentration on desorption of resin X-5. Results are means ± SD of three independent experiments (n ¼ 3).
was 6.001 mg/mL. Thus, the concentration of phenolic compounds in the feed solution was selected at 6.0 mg/mL. In order to find the most suitable desorption solution of X-5, different concentrations of ethanol solutions were used to perform desorption tests. Desorption rates (for phenolic compounds absorbed on X-5 resins) in different concentrations of ethanol solution are presented in Fig. 3b. At a range of ethanol concentration from 30 to 70 mL/ 100 mL, the desorption rate (for phenolic compounds absorbed on X-5 resins) increased. However, when the ethanol concentration surpassed 70 mL/100 mL, the desorption rate mildly decreased. Therefore, optimal ethanol concentration for desorption was 70 mL/100 mL. 3.3. Phenolic composition of sugarcane rinds extracts Phenolic substances are suggested to be important antioxidants in sugarcane products (Duarte-Almeida et al., 2011). After 300 mL crude sugarcane rinds extract (6.0 mg/mL) were purified by MAR X-
5 and solvent extraction method, 680 ± 22 mg E2 and 302 ± 16 mg E3 was collected. The TPC of three samples (E1, E2 and E3) determined by Folin-Ciocalteau procedure, were relatively high, of 117.50 ± 13.00, 302.50 ± 19.50 and 670.00 ± 17.00 mg/g for E1, E2 and E3, respectively. The result indicated that solvent extraction method was more appropriate for the purification of phenolic compounds. Phenolic compounds peaks in the samples (E1, E2 and E3) studied here were identified by comparing the HPLC retention times and UV spectra with authentic standards. As shown in Fig. 4, three main phenolic compounds were identified in three samples: gallic acid (retention time 5.800 min), chlorogenic acid (retention time 19.983 min) and ferulic acid (retention time 44.575 min). Among the phenolic compounds detected (Table 4), Gallic acid was the highest concentration with 74.53 ± 9.50, 91.47 ± 11.20 and 125.11 ± 14.12 mg/g for E1, E2 and E3, respectively.
3.4. Free radical scavenging activity The DPPH free radical has been widely used as a tool for estimating the free radical-scavenging activity of antioxidant (Nagai, Inoue, Inoue, & Suzuki, 2003). The results of DPPH free radicalscavenging ability of ascorbic acid and three samples (E1, E2 and E3) are showed in Fig. 5. As shown in Fig. 5, three samples (E1, E2 and E3) show a good DPPH scavenging activity (approximately 96.36% of that of the ascorbic acid at the same concentration of 0.6 mg/mL). Good correlations between FRSA and concentration of sample were found in certain concentration (y ¼ 129.58xþ7.072, 0.1e0.6 mg/mL, R2 ¼ 0.993 for E1; y ¼ 161.39x þ2.353, 0.1e0.5 mg/mL, R2 ¼ 0.997 for E2; and y ¼ 192.07xþ14.155, 0.1e0.4 mg/mL, R2 ¼ 0.949 for E3). The increasing DPPH scavenging activity will slow down when the dosage of the sample E2, E3 exceeded 0.5 and 0.4 mg/mL, respectively. The increase in FRSA observed with the increase in Table 4 Content of the main phenolic compounds (mg/g) in sample (mean ± SD, n ¼ 3). Values followed by different letters are significantly different at P < 0.05. Peak
Identification
Phenolic compounds content (mg phenolic compounds/g sample) Sample E11
1 2 3 Fig. 4. HPLC chromatogram at 280 nm of three extract samples: the crude sugarcane rinds extract powder E1 (A), the sugarcane rinds extract powder purified by macroporous adsorption resins E2 (B) and the sugarcane rinds extract powder purified by solvent extraction E3 (C).
Gallic acid Chlorogenic acid Ferulic acid
Sample E22 b
74.53 ± 9.50 1.49 ± 0.28b
91.47 ± 11.20 3.16 ± 1.20b
2.10 ± 0.38b
1.62 ± 0.18b
Sample E33 b
125.11 ± 14.12a 11.14 ± 2.40a 9.13 ± 2.50a
1
Sample E1 is the crude sugarcane rind extract powder. Sample E2 is the sugarcane rind extract powder purified by macroporous adsorption resins. 3 Sample E3 is the sugarcane rind extract powder purified by solvent extraction. 2
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S. Feng et al. / LWT - Food Science and Technology xxx (2014) 1e7
Fig. 5. Scavenging effects of ascorbic acid and three samples: the crude the sugarcane rinds extract powder purified sugarcane rinds extract powder (E1), by macroporous adsorption resins (E2) and the sugarcane rinds extract powder purified by solvent extraction (E3) on DPPH free radical. Results were means ± SD of three independent experiments (n ¼ 3) followed by the different letters, which indicated significantly different at P < 0.05.
concentration of sample confirms the usual correlation between TPC and antioxidant activity (Anagnostopoulou, Kefalas, Papageorgiou, Assimopoulou, & Boskou, 2006). 4. Conclusions Due to some advantages including reduction of solvents, low temperature, less time water consumption for extraction, UAE is an environment-friendly and effective method for extraction of phenolic compounds from sugarcane rinds. Results from BB design indicated solvent concentration and ultrasonic temperature as the two most significant factors followed by extraction time and solideliquid ratio. Sugarcane rinds could be a good commercial source of gallic acid, chlorogenic acid and ferulic acid with high antioxidant ability. Acknowledgments The research was financially supported by the Zhejiang Provincial Natural Science Foundation of China (LR13C200001) and the National Natural Science Foundation of China (31371856). References Anagnostopoulou, M. A., Kefalas, P., Papageorgiou, V. P., Assimopoulou, A. N., & Boskou, D. (2006). Radical scavenging activity of various extracts and fractions of sweet orange-peel (Citrus sinensis). Food Chemistry, 94(1), 19e25. http:// dx.doi.org/10.1016/j.foodchem.2004.09.047. Bai, X. L., Yue, T. L., Yuan, Y. H., & Zhang, H. W. (2010). Optimization of microwaveassisted extraction of polyphenols from apple pomace using response surface methodology and HPLC analysis. Journal of Separation Science, 33(23e24), 3751e3758. http://dx.doi.org/10.1002/jssc.201000430. Chen, W., Wang, W. P., Zhang, H. S., & Huang, Q. (2012). Optimization of ultrasonicassisted extraction of water-soluble polysaccharides from Boletus edulis mycelia using response surface methodology. Carbohydrate Polymers, 87(1), 614e619. http://dx.doi.org/10.1016/j.carbpol.2011.08.029. Duarte-Almeida, J. M., Salatino, A., Genovese, M. I., & Lajolo, F. M. (2011). Phenolic composition and antioxidant activity of culms and sugarcane (Saccharum officinarum L.) products. Food Chemistry, 125(2), 660e664. http://dx.doi.org/ 10.1016/j.foodchem.2010.09.059. Fontaniella, B., Vicente, C., de Armas, R., & Legaz, M. E. (2007). Effect of leaf scald (Xanthomonas albilineans) on polyamine and phenolic acid metabolism of two sugarcane cultivars. European Journal of Plant Pathology, 119(4), 401e409. http:// dx.doi.org/10.1007/S10658-007-9172-2.
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Ultrasonic-assisted extraction and purification of phenolic compounds from sugarcane (Saccharum officinarum L.) rinds* Simin Feng a, Zisheng Luo a, b, *, Beipei Tao a, Chun Chen a a b
Department of Food Science and Nutrition, Zhejiang University, Hangzhou, 310058, People's Republic of China Fuli Institute of Food Science, Zhejiang University, Hangzhou, 310058, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 September 2013 Received in revised form 1 September 2014 Accepted 13 September 2014 Available online xxx
In order to obtained highest extraction efficiency of phenolic compounds from the sugarcane rinds, a rapid extraction method based on ultrasound-assisted extraction technique using response surface methodology was investigated. Results showed that the optimal conditions were: solvent concentration 52.19 mL/100 mL, solideliquid ratio 14.46 mL/g, ultrasonic temperature 61.54 C and extraction time 31.30 min. The practical phenolic compounds extraction rate was 8.67 g/100 g DW, which was well matched with the predicted value of 8.82 g/100 g DW. Purified by macroporous resins adsorption or solvent extraction method, the total phenolic content of crude phenolic compounds extracts improved from 117.50 ± 13.00 to 302.50 ± 19.50 or 670.00 ± 17.00 mg/g, respectively. Gallic acid, chlorogenic acid and ferulic acid were identified as three main phenolic compounds in the phenolic compounds extract by HPLC analysis. Furthermore, TPC revealed good correlations with antioxidant activities. All of the results indicated that sugarcane rinds could be used as a good source of phenolic compounds with significant antioxidant activities. © 2014 Published by Elsevier Ltd.
Keywords: Sugarcane rinds Phenolic compounds Ultrasonic-assisted extraction Response surface methodology Antioxidant
1. Introduction Sugarcanes (Saccharum officinarum L.) are one of the most important economic plants, and are the most important sources of sugar as 70% of the world's sugars are from sugarcanes. Sugarcanes have high content of phenolic acids, flavonoids and other phenolic compounds (Duarte-Almeida, Salatino, Genovese, & Lajolo, 2011). Five antioxidant compounds, namely syringaresinol, medioresinol, coniferyl alcohol, 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)1-propanone and sinapyl alcohol, were first isolated from a kokuto (a sugarcane product consumed as candies in Japan) extract (Nakasone et al., 1996). More antioxidant compounds, such as chlorogenic acid (Duarte-Almeida et al., 2011) and ferulic acid (Fontaniella, Vicente, de Armas, & Legaz, 2007) were isolated from sugarcane and its products. Sugarcane rinds, as waste by-product of
Abbreviations: TPC, total phenolic content; E1, crude sugarcane rind extract powder; E2, sugarcane rind extract powder purified by MARs; E3, sugarcane rind extract powder purified by solvent extraction. * This manuscript was presented in the international conference of “Food Innova2012” Hangzhou, China, December 12e14, 2012. * Corresponding author. Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, People's Republic of China. Tel.: þ86 571 88982162. E-mail address: [email protected] (Z. Luo).
the sugar industries, are generated in large quantities, about 20% weight of sugarcanes. Recently, response surface methodology (RSM) is widely used for the optimization of the extraction methods. As a type of RSM, BoxeBehnken design (BBD) is an independent quadratic design that does not contain an embedded factorial or fractional factorial design (Zhu & Liu, 2013). BBD was easy to arrange and interpret experiment and more efficient than other response surface designs since they can be used to establish a quadratic response surface (Li et al., 2013). It has been successfully used in the optimization of the extraction of polyphenols from natural sources (Yue, Shao, Yuan, Wang, & Qiang, 2012). However, there are very few reports on the use of BBD in the optimization of extraction of phenolic compounds from sugarcane rinds. Conventional extraction with heating, boiling and refluxing are usually used for covering phenolic compounds. However, the disadvantages are degradation of phenolic compounds during long extraction time. Up to now, several new extraction techniques have been reported for the extraction of bioactive compounds from plants, including microwave-assisted extraction (Bai, Yue, Yuan, & Zhang, 2010), ultrasound-assisted extraction (UAE) (Zou, Xie, Fan, Gu, & Han, 2010), supercritical fluid extraction (Oliveira, Kamimura, & Rabi, 2009). Among these, the UAE is one of simple and efficient extraction techniques (Huang, Xue, Niu, Jia, & Wang,
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S. Feng et al. / LWT - Food Science and Technology xxx (2014) 1e7
2009). The ultrasonic cavitation creates shear forces that can break cell walls mechanically, which allow high diffusion rates across the cell wall (Vinatoru, 2001). Moreover, UAE technique is one of environment friendly techniques that can offer no toxic chemical involvement, less solvent consumption, low temperature and lower energy input. Due to their convenience, low cost, high chemical stability, friendly to environmental protection and easy regeneration, separation method based on macroporous adsorption resins (MARs) was frequently used in the separation and purification of pharmaceutical and natural products (Shi et al., 2002). Some studies show that the MARs method was widely investigated and used for separating bioactive compounds from natural extracts such as red pigments (Zhang, Yin, Kong, & Jiang, 2011), lycopene (Liu, Liu, Chen, Liu, & Di, 2010), phenolics and rosmarinic acid (Lin, Zhao, Dong, Yang, & Zhao, 2012). There is very few references available on the UAE of phenolic compounds from sugarcane rinds. In the present study, UAE technique was used as a rapid extraction process for phenolic compounds from sugarcane rinds for good yields. Several operational parameters that could potentially affect the extraction efficiency were optimized using RSM. Then crude sugarcane rinds extracts were purified by MARs and solvent extraction method. Finally, free radical scavenging activity (FRSA) of sugarcane rinds phenolic compounds were evaluated in vitro study. 2. Materials and methods 2.1. Plant material Sugarcanes (Badila) were collected at commercial maturity from Zhejiang, PR China. The outer layer rinds were obtained by hand peeling and rinds samples were carefully separated from the inner pith with sharp knives. Then the rinds samples were air dried at 40 C, smashed into powder with a pulverizer (Huangchen Machinery Co. Ltd., Zhejiang, China), passed through a 60-mesh (pore size in 0.25 mm) sieve and kept dry at 18 C until use. 2.2. Chemicals FolineCiocalteu reagent, 2, 2-di-phenyl-1-picrylhydrazyl (DPPH) free radical, 4, 6-tripryridyls-triazine (TPTZ), Gallic acid-1hydrate was purchased from Aladdin Industrial Co. (Shanghai, China). Chlorogenic, vanillic, ferulic, gallic, syringic, p-Coumaric and caffeic acids, catechin and quercertin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). MARs D-101 were purchased from Sigma Chemical Co. (St. Louis, MO, USA), X-5, H-103, S8, NKA-9 and AB-8 were purchased from Nankai Hecheng S&T Co. Ltd (Tianjin, China) and their physico chemical properties were listed in Table 1. All the other chemicals used were of analytical grade or HPLC grade.
Table 1 Physicochemical properties of the macroporous adsorption resins. Type
Polarity
Surface area (m2/g)
Particle diameter (mm)
Average pore diameter (Å)
S-8 NKA-9 AB-8 X-5 H-103 D-101
Polar Polar Weak-polar Nonpolar Nonpolar Nonpolar
10e120 250e290 480e520 500e600 1000e1100 500e550
0.3e1.25 0.3e1.25 0.3e1.25 0.3e1.25 0.3e1.25 0.3e1.25
28e30 15e16.5 13e14 29e30 8.5e9.5 9e10
2.3. UAE of phenolic compounds UAE was performed with an ultrasonic apparatus (Ningshang Ultrasonic Instrument Co. Ltd., Shanghai, China). Pre-weighed amounts of sugarcane rinds powder were placed into a volumetric flask (100 mL), soaked with ethanol solvent (varying ethanol concentration from 30 to 80 mL/100 mL; varying liquid to solid ratio from 5:1 to 25:1, mL/g) and then placed in ultrasonic cleaning bath at 40 kHz for certain time (varying extraction time from 5 to 50 min) at a constant temperature (varying extraction temperature from 30 to 80 C). After filtration through filter paper, anhydrous ethanol was added to bring the final volume of the extract to 50 mL. The phenolic concentration of sugarcane rinds extract was measured according to the method described in Section 2.5. Phenolic compounds extraction rate (Y) was expressed as g phenolic compounds/100 g sample. All results were expressed by dry weight (DW). Then bulk solvents were removed from the extract in a rotary vacuum evaporator (IKAWerke-GmbH & Co., Staufen, Germany) at 40 C. At last, the crude sugarcane rinds extract powder (E1) was freeze-dried and kept sealed in the dark. 2.4. Design of experiment Box-Behnken (BB) design was used to determine the optimal conditions of the UAE. According to the principle of BB design, solvent concentration (mL/100 mL) (X1), solideliquid ratio (mL/g) (X2), ultrasonic temperature ( C) (X3) and extraction time (min) (X4), were taken as independent variables tested in a 29-run experiment. As we can see from Table 2, the four independent variables were prescribed into 3 levels, coded 1, 0 and þ1 for low, intermediate and high value, respectively. Phenolic compounds extraction rate (Y) was taken as response of the design experiments. The experimental design used in the study is showed in Table 2. A second-degree polynomial regression model was used to Table 2 BB design and the responses values for Phenolic compounds extraction rate. Run Solvent Solideliquid Ultrasonic Extraction Y(g/100 g DW) concentration ratio temperature time X4(min) X1(mL/100 mL) X2(mL/g) X3( C) 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
45(1) 50(0) 50(0) 50(0) 45(1) 50(0) 50(0) 50(0) 50(0) 50(0) 50(0) 50(0) 55(1) 55(1) 55(1) 50(0) 50(0) 50(0) 55(1) 45(1) 55(1) 45(1) 45(1) 45(1) 50(0) 55(1) 50(0) 50(0) 50(0)
15(0) 10(1) 15(0) 15(0) 15(0) 15(0) 20(1) 15(0) 15(0) 10(1) 10(1) 20(1) 15(0) 15(0) 20(1) 15(0) 15(0) 15(0) 10(1) 10(1) 15(0) 15(0) 20(1) 15(0) 20(1) 15(0) 10(1) 20(1) 15(0)
55(1) 55(1) 55(1) 60(0) 60(0) 60(0) 60(0) 65(1) 60(0) 60(0) 60(0) 55(1) 60(0) 55(1) 60(0) 65(1) 55(1) 60(0) 60(0) 60(0) 60(0) 65(1) 60(0) 60(0) 60(0) 65(1) 65(1) 65(1) 60(0)
30(0) 30(0) 20(1) 30(0) 20(1) 30(0) 20(1) 20(1) 30(0) 20(1) 40(1) 30(0) 20(1) 30(0) 30(0) 40(1) 40(1) 30(0) 30(0) 30(0) 40(1) 30(0) 30(0) 40(1) 40(1) 30(0) 30(0) 30(0) 30(0)
6.17 6.42 6.06 8.63 6.53 8.64 6.88 6.92 8.61 6.71 6.88 6.79 7.12 6.38 7.56 7.09 6.57 8.65 8.44 6.34 7.78 6.68 6.94 7.37 8.11 8.22 7.92 7.34 8.63
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S. Feng et al. / LWT - Food Science and Technology xxx (2014) 1e7
correlate the relationship between independent variables and response (phenolic compounds extraction rate), and the equation was.
Y4 ¼ a0 þ
4 X i¼1
ai Xi þ
4 X
aii Xi2 þ
i¼1
3 4 X X
aij Xi Xj
(1)
i¼1 j¼iþ1
where Y4 is the response variables. a0, ai, aii and aij are the intercept, linear, quadratic and interactive coefficients, respectively. Xi and Xj are the levels of the independent variables (isj). 2.5. Total phenolic content (TPC) The total phenol content was determined according to the FolineCiocalteu method (Zhang et al., 2013) with minor modifications. Gallic acid was used as standard, and the results were expressed as mg gallic acid/mL or g of sample. 0.2 mL of the extract (2 mg/mL) was mixed with 1 mL of the Folin Ciocalteu's reagent and 2 mL of 15 (g/100 mL) Na2CO3. The 10 mL volumetric flasks were shaken and allowed to stand for 2 h at room temperature. The absorbance was measured with a spectrophotometer (Shimadzu Co., Tokyo, Japan) at 765 nm. All assays were run in three replicates. 2.6. Purification of phenolic compounds by the macroporous adsorption resins (MARs) 2.6.1. Static adsorption of MARs and static desorption tests MARs X-5, D-101, H-103, S-8, NKA-9 and AB-8 were washed with abundant distilled water to remove salts and impurities. Before the adsorption experiment, pre-weighed amounts of adsorbents were washed with ethanol and subsequently the ethanol was thoroughly substituted with deionized water (Zhang et al., 2011). Experiments on the adsorption/desorption behaviors of 6 different MARs were carried out to select suitable resin for the purification of phenolic compounds. Static adsorption tests of extractions were performed as follows: pre-weighed amounts of hydrated MARs (1.000 g) were introduced into a 250 mL air-tight conical flask. Then each 25 mL of different concentrations of sugarcane rinds extracts (E1) was added into each flask. The flasks were shaken (100 rpm) in a constant temperature water-bath shaker for 24 h at 25 C. The phenolic concentration of the solution at different time was measured according to the method described in Section 2.5. Static desorption tests were carried out as follows: After adsorption experiment, each MARs was first filtered, washed by distilled water. Then resins were desorbed with 25 mL ethanolewater solution (30, 40, 50, 60, 70, 80 and 90 mL/100 mL) in the 250 mL air-tight conical flask. The flasks were shaken (100 rpm) in a constant temperature water-bath shaker for 24 h at 25 C. The phenolic concentration of supernatant was measured according to the method described in Section 2.5. Adsorption capacity was calculated by using the following equations (Lin et al., 2012; Liu et al., 2010):
Acðmg=gÞ ¼ ðC0 C1 Þ V=M
(2)
Adsorption rate was calculated by using the following equation:
Arð%Þ ¼ 100% ðC0 C1 Þ=C0
(3)
Desorption rate was calculated by using the following equation:
Drð%Þ ¼ 100% Cd Vd =ððC0 C1 Þ VÞ
(4)
where Ac is the adsorption capacity (mg/g). C0 is the phenolics concentration of the sample solution before adsorption (mg/mL). C1
3
is the phenolics concentration of the sample solution after adsorption (mg/mL). M is the weight of the hydrated resin. V is the volume of phenolics solutions used in the study (mL). Ar is the adsorption rate of the sample (%). Dr is the desorption rate (%). Cd is the phenolics concentration in the desorption solution (mg/mL). Vd is the volume of the desorption solution (mL). 2.6.2. Purification of phenolic compounds 300 mL of crude sugarcane rinds extract was first processed by resin X-5 adsorption, and then the effluent was concentrated in a rotary vacuum evaporator (Yarong biochemical Co. Ltd., Shanghai, China) at 40 C after desorption. At last, the sugarcane rinds extract powder purified by MARs (E2) was freeze-dried and kept sealed in the dark. 2.7. Purification of phenolic compounds by solvent extraction method A certain amount of sodium hydroxide standard solution was first added to 300 mL of crude sugarcane rinds extract to adjust the pH value to 6.5. Then ethyl acetate was used as liquid extractant to purify the sugarcane rinds extract for three times. After the extraction, the liquid extractant was concentrated in a rotary vacuum evaporator at 40 C. At last, the sugarcane rinds extract powder purified by solvent extraction (E3) was freeze-dried and kept sealed in the dark. 2.8. High performance liquid chromatography (HPLC) analysis Identification and quantification of phenolic substances were carried out using an LC-2010 (Japan AT) system with an ultravioletvisible detector. 0.2 g of samples (E1, E2 and E3) was dissolved in 2 mL of methanol and filtered through 0.5 mm Fluorpore membranes (Millipore). The injection volume was 10 ml. Separation was performed on a Luna 5m C-18 column (250mm 4.6 mmi.d., Phenomenex Ltd.). The mobile phase with a flow rate of 0.8 mL/min consisted of two solvents: A, 5 mL/100 mL acetic acid solution and B, acetonitrile. The solvent gradient in volume ratios was as follows: 0e5 min: 0e5% B; 5e22 min: 5e11.5% B; 22e32 min: 11.5e11.7% B; 32e62 min: 11.7e71.7% B; 62e65 min: 71.7 to 5% B; 65e72 min: 5 to 0% B. All the analyses were performed at temperature of 40 C, and the wavelength used for detection was 280 nm. Identification followed comparison of retention times and UV spectra with authentic standards. Standards used for phenylpropanoid analyses were chlorogenic, vanillic, catechin, ferulic, gallic, syringic, p-coumaric, quercertin and caffeic acids. All the standards were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Quantification was based on external calibration and the final concentrations were expressed as mg/g of sample. 2.9. Evaluation of free radical scavenging activity (FRSA) The FRSA of the extracts was evaluated according to the method of (Wang, Li, Zeng, & Liu, 2008), with slight modifications. Ascorbic acid was used as a positive control. Three samples (E1, E2 and E3) were dissolved in 50 mL/100 mL aqueous ethanol with different concentrations: 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/mL, respectively. The samples of 2.0 mL were added to 4.0 mL of 0.1 mmol DPPH. The mixtures were shaken and reacted in the dark at 25 C for 30 min. The FRSA (%) of the tested samples, were evaluated by comparison with a control (4.0 mL DPPH solution and 2 mL of 50 mL/100 mL ethanol solution). Scavenging capacity was measured by monitoring the absorbance at 517 nm.
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2.10. Statistical analysis Analysis of variance (ANOVA) was used to determine the significant difference in phenolic compounds yield extracted from sugarcane rinds under different conditions. A second-order polynomial regressed equation was established on the basis of analysis of BB experimental data, and the optimal conditions for extraction were found using the software of Design Expert version 6.0.10 (Stat-Ease, Inc.). 3. Results and discussion 3.1. Optimization of UAE condition of phenolic compounds by RSM 3.1.1. Extraction model The BB design was employed to study the interactions among four individual parameters. The independent variables and the corresponding response values obtained in different experimental combination are listed in Table 2. The phenolic compounds extraction rate ranged from 6.06 to 8.65 g/100 g DW. Multiple regression analysis was used to analyze the experimental data and thus a quadratic polynomial equation of the response surfaces obtained is as follow:
Y ¼ 215:865 þ 2:245X1 þ 1:857X2 þ 4:65X3 þ 0:616X4 0:015X1 X2 þ 0:013X1 X3 9 103 X1 X4 9:5 103 X2 X3 þ 5:3 103 X2 X4 0:027X12 0:023X22 0:042X32 8:643 103 X42
(5)
The statistical significance of Eq. (5) was checked by F-test, and ANOVA results of experiment model are showed in Table 3. The model F-value of 16.27 obtained by ANOVA indicated that the model was very significant (P < 0.01). It indicated that the quadratic model was in good agreement with the experimental results. The R2 value closer to one suggested a better correlation between the observed and predicted values (Gangadharan, Sivaramakrishnan, Nampoothiri, Sukumaran, & Pandey, 2008). The determination coefficient (R2) was 0.9421 implying that the regression model could explain 94.21% of the result in the case of phenolic compounds extraction rate. Moreover, a low value of coefficient of variation (CV ¼ 3.98%) suggested that the model was reproducible (Wanasundara & Shahidi, 1996). The results indicated that the model could work well for the prediction of phenolic compounds
Table 3 ANOVA results of experiment model. Source
Sum of squares
df
Mean square
F value
P-value
Modle X1 X2 X3 X4 X21 X1X2 X1X3 X1X4 X22 X2X3 X2X4 X23 X3X4 X24 Residual Cor total
19.34 2.49 0.069 2.78 1.07 2.96 0.55 0.44 0.0081 2.15 0.23 0.28 7.11 0.029 4.85 1.19 20.53
14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 28
1.38 2.49 0.069 2.78 1.07 2.96 0.55 0.44 0.0081 2.15 0.23 0.28 7.11 0.029 4.85 0.085
16.27 29.38 0.81 32.8 12.58 34.88 6.45 5.21 0.095 25.32 2.66 3.31 83.75 0.34 57.1
<0.0001*** <0.0001*** 0.3825 <0.0001*** 0.0032** <0.0001*** 0.0236* 0.0386* 0.7619 0.0002*** 0.1253 0.0903 <0.0001*** 0.5688 <0.0001***
R2 ¼ 0.9421 R2Adj ¼ 0.8842 *P < 0.05 **P < 0.01 ***P < 0.001.
extract from sugarcane rinds. The P-values were used to check the significance of each factor on phenolic compounds extraction rate. As shown in Table 3, the independent variables (X1, X3 and X4) and quadratic terms (X21, X1X2, X1X3, X22, X23 and X24) significantly affected the phenolic compounds extraction rate (P < 0.05). The results showed that solvent concentration and ultrasonic temperature were the two most significant single factors, which affected the phenolic compounds extraction rate followed by extraction time and solideliquid ratio. 3.1.2. Optimization of extraction conditions and verification of the model In order to obtain desirable response goals multiple response optimizations were carried out to determine optimum levels of independent variables. Six independent response surface plots are showed in Fig. 1. Each pair of parameters within the experimental rang was depicted in 3-D surface plots, while the two other parameters were kept constant at zero level. The shape of the 3-D surface plots indicated a moderate interaction between these tested variables (Pan, Yu, Zhu, & Qiao, 2012) and facilitated the identification of the optimum experimental conditions (Chen, Wang, Zhang, & Huang, 2012). Fig. 1(a, d and f) show the effects of solvent concentration with each of the three other variables on phenolic compounds extraction rate. As can be seen from Fig. 1(a, d and f), the phenolic compounds extraction rate increased with increasing solvent concentration at first. However, the trend was reversed when the phenolic compounds extraction rate reached a certain value. According to theory of similarity and intermiscibility, the phenolic compounds are easily dissolved from plant cells, when polarities of solvent are similar to phenolic compounds (Gribova, Filippenko, Nikolaevskii, Belaya, & Tsybulenko, 2008) When solvent concentration amount exceeded the certain value, polarities of solvent changed again and the phenolic compounds extraction rate decreased. Fig. 1(a, c and e) show the effects of solideliquid ratio with each of the three other variables on phenolic compounds extraction rate. As shown, the increase in the solideliquid ratio led to a gradual increase in phenolic compounds extraction rate and extraction rate reached a maximum when the solideliquid ratio was up to a certain value, with no significantly further improvement thereafter. The effects of ultrasonic temperature interaction with each of the three other factors on the phenolic compounds extraction rate are displayed in Fig. 1(b, c and e). As shown, the phenolic compounds extraction rate increased readily with increasing ultrasonic temperature up to 60 C and followed by a slight decrease thereafter. With suitable temperature, more phenolic compounds would be extracted into the solvent, as heating might weaken the cell wall integrity, soften the plant tissue, hydrolyze the bonds of bound phenolic compounds (phenoleprotein or phenolepolysaccharide) (Spigno, Trarnelli, & De Faveri, 2007; Tabaraki & Nateghi, 2011). However, high temperature promoted degradation such as the oxidation of phenolic compounds, resulting in decreased extraction yield when temperatures is above 60 C, and it might also cause higher furfural (a carcinogenic compound) contents in the phenolic extract (Rodrigues & Pinto, 2007). Fig. 1(b, c and e) show the effects of extraction time with each of the three other variables on phenolic compounds extraction rate. In all situations, the phenolic compounds extraction rate increased with increasing extraction time from 20 to 30 min, and then fell above 30 min, which indicated that the extraction time has a remarkable effect on phenolic compounds extraction rate. The optimal conditions were listed as follows: solvent concentration 52.19 mL/100 mL, solideliquid ratio 14.46 mL/g, ultrasonic temperature 61.54 C and extraction time 31.30 min. However,
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Fig. 1. Response surface graph showing effects of the extraction parameters on phenolic compounds extraction rate (a) at varying solvent concentration and solideliquid ratio, (b) at varying ultrasonic temperature and extraction time, (c) at varying ultrasonic temperature and solideliquid ratio, (d) at varying ultrasonic temperature and solvent concentration, (e) at varying extraction time and solideliquid ratio, and (f) at varying extraction time and solvent concentration.
considering the operability in actual production, optimal conditions can be modified as follows: solvent concentration 52%, solideliquid ratio 14 mL/g, ultrasonic temperature 62 C and extraction time 31 min. The predicted phenolic compounds extraction rate was 8.82 g/100 g DW, which was consistent with the practical phenolic compounds extraction rate of 8.67 g/100 g DW. These data indicated that the model designed in this study was valid.
3.2. Screening of optimum MARs adsorption method
Fig. 2. Adsorption/desorption properties of phenolic compounds on six macroporous adsorption resins: AB-8, D-101, H-103, NKA-9, S-8, and X-5. Results were means ± SD of three independent experiments (n ¼ 3) followed by the different letters, which indicated significantly different at P < 0.05. adsorption rate; desorption rate.
As illustrated in Fig. 2, adsorption/desorption properties of six MARs for purifying phenolic compounds were studied in the static adsorption/desorption tests. It could be seen that the adsorption rates were calculated as 50.89% and 60.11% for X-5 and H-103 resins, respectively, and they were considerably higher than those of other resins. Our results were in accord with the results of Sun, Guo, Fu, Li, & Li, 2013, who found that X-5 resin was verified to offer the best adsorption capacity and desorption ratio for total polyphenols. Desorption rate of phenolic compounds was the highest for X-5. As can be seen from Fig. 3a, adsorption capacity (for phenolic compounds absorbed on X-5 resins) reached the saturation plateau when the initial concentration of phenolic compounds
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Fig. 3. Effect of initial concentration of the crude sugarcane rinds extract E1 on adsorption capacity and effect of ethanol concentration on desorption of resin X-5. Results are means ± SD of three independent experiments (n ¼ 3).
was 6.001 mg/mL. Thus, the concentration of phenolic compounds in the feed solution was selected at 6.0 mg/mL. In order to find the most suitable desorption solution of X-5, different concentrations of ethanol solutions were used to perform desorption tests. Desorption rates (for phenolic compounds absorbed on X-5 resins) in different concentrations of ethanol solution are presented in Fig. 3b. At a range of ethanol concentration from 30 to 70 mL/ 100 mL, the desorption rate (for phenolic compounds absorbed on X-5 resins) increased. However, when the ethanol concentration surpassed 70 mL/100 mL, the desorption rate mildly decreased. Therefore, optimal ethanol concentration for desorption was 70 mL/100 mL. 3.3. Phenolic composition of sugarcane rinds extracts Phenolic substances are suggested to be important antioxidants in sugarcane products (Duarte-Almeida et al., 2011). After 300 mL crude sugarcane rinds extract (6.0 mg/mL) were purified by MAR X-
5 and solvent extraction method, 680 ± 22 mg E2 and 302 ± 16 mg E3 was collected. The TPC of three samples (E1, E2 and E3) determined by Folin-Ciocalteau procedure, were relatively high, of 117.50 ± 13.00, 302.50 ± 19.50 and 670.00 ± 17.00 mg/g for E1, E2 and E3, respectively. The result indicated that solvent extraction method was more appropriate for the purification of phenolic compounds. Phenolic compounds peaks in the samples (E1, E2 and E3) studied here were identified by comparing the HPLC retention times and UV spectra with authentic standards. As shown in Fig. 4, three main phenolic compounds were identified in three samples: gallic acid (retention time 5.800 min), chlorogenic acid (retention time 19.983 min) and ferulic acid (retention time 44.575 min). Among the phenolic compounds detected (Table 4), Gallic acid was the highest concentration with 74.53 ± 9.50, 91.47 ± 11.20 and 125.11 ± 14.12 mg/g for E1, E2 and E3, respectively.
3.4. Free radical scavenging activity The DPPH free radical has been widely used as a tool for estimating the free radical-scavenging activity of antioxidant (Nagai, Inoue, Inoue, & Suzuki, 2003). The results of DPPH free radicalscavenging ability of ascorbic acid and three samples (E1, E2 and E3) are showed in Fig. 5. As shown in Fig. 5, three samples (E1, E2 and E3) show a good DPPH scavenging activity (approximately 96.36% of that of the ascorbic acid at the same concentration of 0.6 mg/mL). Good correlations between FRSA and concentration of sample were found in certain concentration (y ¼ 129.58xþ7.072, 0.1e0.6 mg/mL, R2 ¼ 0.993 for E1; y ¼ 161.39x þ2.353, 0.1e0.5 mg/mL, R2 ¼ 0.997 for E2; and y ¼ 192.07xþ14.155, 0.1e0.4 mg/mL, R2 ¼ 0.949 for E3). The increasing DPPH scavenging activity will slow down when the dosage of the sample E2, E3 exceeded 0.5 and 0.4 mg/mL, respectively. The increase in FRSA observed with the increase in Table 4 Content of the main phenolic compounds (mg/g) in sample (mean ± SD, n ¼ 3). Values followed by different letters are significantly different at P < 0.05. Peak
Identification
Phenolic compounds content (mg phenolic compounds/g sample) Sample E11
1 2 3 Fig. 4. HPLC chromatogram at 280 nm of three extract samples: the crude sugarcane rinds extract powder E1 (A), the sugarcane rinds extract powder purified by macroporous adsorption resins E2 (B) and the sugarcane rinds extract powder purified by solvent extraction E3 (C).
Gallic acid Chlorogenic acid Ferulic acid
Sample E22 b
74.53 ± 9.50 1.49 ± 0.28b
91.47 ± 11.20 3.16 ± 1.20b
2.10 ± 0.38b
1.62 ± 0.18b
Sample E33 b
125.11 ± 14.12a 11.14 ± 2.40a 9.13 ± 2.50a
1
Sample E1 is the crude sugarcane rind extract powder. Sample E2 is the sugarcane rind extract powder purified by macroporous adsorption resins. 3 Sample E3 is the sugarcane rind extract powder purified by solvent extraction. 2
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S. Feng et al. / LWT - Food Science and Technology xxx (2014) 1e7
Fig. 5. Scavenging effects of ascorbic acid and three samples: the crude the sugarcane rinds extract powder purified sugarcane rinds extract powder (E1), by macroporous adsorption resins (E2) and the sugarcane rinds extract powder purified by solvent extraction (E3) on DPPH free radical. Results were means ± SD of three independent experiments (n ¼ 3) followed by the different letters, which indicated significantly different at P < 0.05.
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