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Ultrasonic impact on viscosity and extraction efficiency of polyethylene glycol: A greener approach for anthocyanins recovery from purple sweet potato
Food Chemistry 283 (2019) 59–67
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Ultrasonic impact on viscosity and extraction efficiency of polyethylene glycol: A greener approach for anthocyanins recovery from purple sweet potato
T
⁎
Hao Huanga, Qin Xua, Tarun Belwala, Li Lia, Halah Aalima, Qiong Wua, Zhenhua Duanb, , ⁎ Xuebing Zhangc, Zisheng Luoa, a
Zhejiang University, College of Biosystems Engineering and Food Science, Key Laboratory of Agro-Products Postharvest Handling of Ministry of Agriculture and Rural Affairs, Zhejiang Key Laboratory for Agri-Food Processing, Hangzhou 310058, People’s Republic of China Institute of Food Science and Engineering, Hezhou University, Hezhou, People’s Republic of China c Hangzhou Wanxiang Polytechnic, Huawu Road 3, Hangzhou 310023, People’s Republic of China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Polyethylene glycol Ultrasound-assisted extraction Anthocyanins Purple sweet potato Viscosity
Purple sweet potatoes are known for its vibrant purple color due to high level of anthocyanins. A polyethylene glycol based ultrasonic-assisted green extraction (PEG-UAE) of anthocyanins from purple sweet potato was proposed. Different types of PEG were tested for anthocyanin extraction along with PEG concentration, liquid-tosolid ratio, ultrasonic temperature and time were investigated for its impact on viscosity and extraction efficiency. The optimum extraction condition, 42 mL/g of ratio, 83% of PEG 200 concentration, 64 °C of ultrasonic temperature and 80 min of sonication time, resulted in better extraction of anthocyanins (83.78 mg CE/100 g DW) and phenolics (994.88 mg GAE/100 g DW). Using UPLC-Triple-TOF/MS, ten anthocyanin and six nonanthocyanin compounds were identified and characterized, with the highest peak area for cyanidin-3-caffeoyl-phydroxybenzoyl sophoroside-5-glucoside (25.9%). Moreover, the anthocyanins and phenolics extraction yield along with antioxidant activity were negatively correlated with PEG viscosity, on which ultrasonication has profound effects.
1. Introduction Purple sweet potato (Ipomoea batatas) is an important staple food mainly cultivated in Asia, especially in China (Zhang, Luo, Zhou, & Zhang, 2018). Although worldwide it is among the seven largest crops (in term of production) after sugar cane, rice, wheat, potatoes, maize, and cassava, it is still underutilized in term of nutraceutical exploration and applications (Esatbeyoglu, Rodríguez-Werner, Schlösser, Winterhalter, & Rimbach, 2017). Previous studies have reported that purple sweet potato contains abundant anthocyanins, including cyanidin and peonidin with acylated p-hydroxybenzoic acid, ferulic acid and caffeic acid (Montilla et al., 2010). Anthocyanins are secondary metabolites, responsible for the developement of purple, blue, red colors in plants (Rommel, Heatherbell, & Wrolstad, 1990). In recent years, there is an increasing interest in anthocyanins research due to their health benefits against cardiovascular diseases, neurodegenerative diseases, cancer and many others (Yousuf, Gul, Wani, & Singh, 2016). Concurrently, Sun, Zhang, Zhu, Lou, and He (2018) also reported that
⁎
anthocyanins of purple sweet potato are attributed to their antioxidant, anticancer, antidiabetic, anti-inflammatory, anti-bacteria, and hepatoprotective activities. Recently, the utilization of agricultural products for extracting value-added bioactive compounds have generated interests among researchers and nutraceutical industry (Azmir et al., 2013), and purple sweet potato could be considered as one of the potential source of bioactive compounds, especially anthocyanins. Anthocyanins are sensitive to high temperature, pH and other extraction conditions, along with the kind of instrument used, which together played an important role in anthocyanin yield and its quality attributes (Belwal et al., 2018). For valorization of purple sweet potato, it is of paramount importance to find novel methodologies to improve the extraction efficiency of anthocyanins. Recently, the special interest on ultrasonic-assisted extraction (UAE) has been drawn, due to its green impacts on extraction process of bioactive compounds, in term of higher yield, shorter processing time and lower maintenance cost (Chemat, Rombaut, & Meullemiestre et al., 2017). The majority of research studies employ UAE focused on the extraction conditions such as type of
Corresponding authors. E-mail addresses: [email protected] (Z. Duan), [email protected] (Z. Luo).
https://doi.org/10.1016/j.foodchem.2019.01.017 Received 23 October 2018; Received in revised form 10 December 2018; Accepted 4 January 2019 Available online 14 January 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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for 80 min in an ultrasonic bath (SB-4200D, Scientz Biotechnology Co., Ningbo, China). Calorimetric measurements were applied to assess actual ultrasound power (Both, Chemat, & Strube, 2014; Sicaire et al., 2016), calculated by Eq. (1).
solvents, its concentration, liquid-to-solid ratio, sonication temperature and time (Belwal et al., 2018; Chemat, Rombaut, & Sicaire et al., 2017). However, for green extraction of bioactive compounds, the selection of non-toxic, high efficiency, low cost and environmental friendly solvent is of paramount importance (Chemat, Rombaut, & Meullemiestre et al., 2017). Polyethylene glycol (PEG) is one of such kind of green solvent which is non-toxic, low cost and safe to the environment (Zhang & Wang, 2016). Moreover, a number of bioactive compounds have been successfully extracted using PEG and the yield of many of these are higher as compared to other solvents. For instance, Zhou, Liu, Ma, and Zhang (2014) reported PEG offered higher extraction yield of polysaccharides than that of the water-based extraction. It has also been reported that PEG had higher efficiency for the extraction of flavonoid compounds, compared with ethanol (Liu, Liu, Zhang, & Zhang, 2012) and methanol (Zhou, Xiao, Li, & Cai, 2011). PEG has also been applied in food (Harris, 2013) and pharmaceutical products (Fishburn, 2008), due to its non-toxic nature. However, no study has been reported for its use in the extraction of anthocyanins from purple sweet potato. Moreover, the relationship study between extraction efficiency and various PEG associated factors including viscosity is still lacking. In the present study, for efficient extraction of anthocyanins from purple sweet potatoes, we investigated the effect of different PEG at various concentrations and other extraction conditions to develop a polyethylene glycol-based ultrasonic-assisted green extraction method (PEG-based-UAE). The relationship between anthocyanin yield and PEG viscosity was also be established during the investigation. For the identification and characterization of anthocyanin compounds, a highend UPLC-Triple-TOF/MS analytical method was also employed. Although, many researches have been conducted on anthocyanins extraction from purple sweet potatoes, however the current proposed research work is in continuation of developing greener extraction method for anthocyanins, which can further be utilized for its multiindustrial applications.
P(W) = m × Cp ×
dT dt
(1)
where Cp is the heat capacity of the solvent at constant pressure (J/g/ °C), m is the mass of solvent (g) and dT/dt is temperature rise per second (°C/s). The actual ultrasound power in present study was around 100 W. The total anthocyanin yield (YTA) was determined to select the best type of PEG and viscosity of each was also estimated. 2.3.2. Yield of total anthocyanin (YTA) YTA was determined according to pH differential method (Zhu et al., 2017) with some modifications. An aliquot (1 mL each) of the extract was mixed with 0.025 M potassium chloride buffer (pH 1.0, 3 mL) and 0.4 M sodium acetate buffer (pH 4.5, 3 mL), separately. The absorbance of the mixture was measured at 510 and 700 nm by UV–Vis spectrophotometer (UV-1750, Shimadzu Co., Ltd., Japan). YTA was calculated as cyanidin-3-glucoside equivalents according to Eq. (2).
YTA (mg CE/100g DW) = [(A510 - A700)pH1.0 - (A510 - A700)pH4.5] × V× n×M × 100 ε × L×m
(2)
where, A510 and A700 represent the absorbance at 510 nm and 700 nm, respectively. V as volume of extract (mL), n is the dilution factor, M is the molecular weight of cyanidin-3-glucoside (449.2 g/mol), ε is the extinction coefficient of cyanidin-3-glucoside (26,900 L/mol/cm), L is the width of the cuvette (1 cm), and m is the weight of sample (1 g). 2.3.3. Yield of total polyphenol (YTP) YTP was determined according to the Folin-Ciocalteu procedure (Feng, Luo, Tao, & Chen, 2015) with some modifications. Briefly, 0.125 mL of extract was mixed with 0.5 mL of Folin-Ciocalteu reagent and 2.125 mL of distilled water. Thereafter, 1.250 mL of 70 mg/mL sodium carbonate was added. After 90 min of incubation in dark, the absorbance was measured at 760 nm by a spectrophotometer (UV-1750, Shimadzu Co., Japan). YTP was expressed as milligrams of gallic acid equivalents per 100 g dry weight (mg GAE/100 g DW) of sample.
2. Materials and methods 2.1. Chemicals and reagents Folin-Ciocalteu reagent was purchased from Dingguo Changsheng Biotechnology Co. (Beijing, China). PEG (200, 300, 400, 600), potassium chloride, sodium acetate, sodium carbonate, ascorbic acid, 2,2diphenyl-1-picrylhydrazyl (DPPH) and standard of gallic acid were purchased from Aladdin Industrial Co. (Shanghai, China). Hydrochloric acid and ethanol were provided by Sinopharm Chemical Reagent Co. (Shanghai, China). All solvents and standards used for chromatographic analyses were of HPLC grade.
2.3.4. Antioxidant activity (YAA) The antioxidant activity was determined by DPPH assay (Pandey, Belwal, Sekar, Bhatt, & Rawal, 2018). Briefly, 2 mL of extract was mixed with 4 mL of 0.1 mM DPPH solution and kept in dark for 30 min at room temperature. The absorbance was measured at 517 nm using UV–Vis spectrophotometer (UV-1750, Shimadzu Co., Japan). The antioxidant activity was expressed as ascorbic acid equivalent per gram dry weight (mg AAE/100 g DW).
2.2. Materials and preparation Purple sweet potatoes cv. Yamakawamurasaki were collected from a commercial vegetable market of Hangzhou City, Zhejiang province, China. The purple sweet potatoes were peeled and cut into slices of 0.3–0.6 cm. The slices were then dried in an oven at 45 °C for 24 h. The dried samples were grounded into powder with a pulveriser (Huangchen HC-280T, Zhejiang, China) and passed through a 60-mesh sieve and stored at 4 °C until use.
2.3.5. Viscosity The viscosity of different PEG solvents and model experimental runs were measured using a rheometer (Haake Co., Coesfeld, Germany) according to the protocol of Marták and Schlosser (2017). A cone and plate system was applied at 25 °C with shear rates of 0.1 s−1. The correlation of viscosity with the YTA, YTP and YAA was calculated using SPSS 20.0 software.
2.3. Polyethylene glycol-based ultrasonic-assisted extraction (PEG-UAE) 2.3.6. Design of experiments (DoE) 2.3.6.1. Single-factor experiments. Single-factor experiments were firstly conducted to determine the optimal level of individual factors i.e., concentration of PEG, liquid-to-solid ratio, sonication temperature and sonication time on YTA (Table S1). Briefly, about 1.0 g of purple sweet potato powder was extracted in 50-mL centrifuge tube with different concentration of PEG and varying liquid-to-solid ratio. The ultrasonication was carried out in an ultrasonic bath at different
2.3.1. Selection of PEG PEG was considered to show various extraction efficiency depends on its composition and molecular weight. Thus, different types of PEG such as PEG 200, PEG 300, PEG 400 and PEG 600 were tested in a preliminary experiment for selecting the most efficient type. About 1.0 g of purple sweet potato powder was mixed with 30 mL of PEG (60%, v/v) and ultrasonicated (maximum up to 400 W, 40 KHz) at 50 °C 60
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temperatures with varying time. Thereafter, the extract was centrifuged at 12,000 rpm for 15 min and the supernatant was collected and stored at 4 °C for further analysis. 2.3.6.2. Multi-factor experiments. According to single-factor experiments, the optimal level of each factor was chosen as center point for the multi-factorial experimental design to determine the linear, quadratic and interactive effects between the variables. The response surface methodology (RSM) was used to optimize the extraction process parameters. A four-factor (X1, X2, X3 and X4) and three levels (−1, 0 and 1) Box–Behnken design (BBD) was applied. X1, X2, X3 and X4 represented concentration of PEG (%, v/v), liquid-to-solid ratio (mL/g), sonication temperature (℃) and sonication time (min), respectively. The responses i.e., YTA, YTP and YAA along with viscosity of each experimental run were presented in Table S2. Design Expert software (V. 8.0.6) was used for the experimental design, regression and graphical analysis. Analysis of variance (ANOVA) was used to determine the significant difference of linear, quadratic and interactive variables, statistical significance of the model and the regression coefficients. 2.3.7. Validation of the model For model validation, the optimized condition which showed highest desirability was selected using Design Expert software (V. 8.0.6) and tested for its validation. The model predicted value was compared with the experimental values. 2.4. UPLC-triple-TOF/MS analysis The ultra-high pressure liquid chromatography (Waters Co., Milford, USA) combined with triple-TOF mass spectrophotometry (UPLC-Triple-TOF/MS) 5600+ System (AB SCIEX Co., Framingham, USA) for anthocyanins were conducted according to Cai’s protocol with minor modifications (Cai et al., 2016). The PEG extract was filtered through 0.5 μm fluoropore membrane filter (Millipore Co., Shanghai, China). The injection volume was 5 μL and the separation was carried out using a ZORBAX-SB C18, 100 mm × 4.6 mm, 1.8 µm column (Agilent Technologies Co., Santa Clara, USA), and the temperature was maintained at 30 °C. The UV detector was set at 530 nm. The mobile phase was selected as solvent A (formic acid: water = 1:1000) and solvent B (formic acid: acetonitrile = 1:1000). The elution gradient was as follows: 0–2 min, 5% B; 2–25 min, 5% to 50% B; 25–35 min, 50% to 95% B; 35–37 min, 95% B, 37–40 min 95% to 5% B and the flow rate was set at 0.8 mL min−1. The MS condition was set at the positive ion mode as follows: scan range m/z 200–2000, capillary voltage 3.0 kV, sampling cone 35 V, collision energy 4 eV, source temperature 100 °C, desolvation temperature 300 °C, desolvation gas 500 L/h. 2.5. Statistical analysis All experiments were performed in triplicates and the data were statistically analyzed by the SAS 9.4 program and presented as means ± SD. Significant differences were calculated using ANOVA and p < 0.05 was considered as significant. 3. Results and discussion 3.1. Selection of PEG Among the tested PEG, PEG 200 was found to be the most effective solvent for the extraction of anthocyanins (YTA) (Fig. 1A). With increasing molecular weight of PEG from 200 to 600 Da, the extraction efficiency was significantly decreased. It was also recorded that the viscosity of PEG increased with increasing molecular weight, which played significant role in lower down extraction efficiency. During ultrasonication the cavitation bubbles were formed which disrupted the (caption on next page) 61
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Fig. 1. Effect of different PEG on YTA and viscosity (A), effects of PEG 200 concentration (B), liquid-to-solid ratio (C), sonication temperature (D) and sonication time (E) on YTA. Values are presented as means ± SD (n = 3). Different letters above the bar showed significant difference at p < 0.05.
Table 1 Regression coefficient (β) and analysis of the variance (ANOVA) of the response surface quadratic models for the investigated parameters. YTA
cells and increase the release of bioactive compounds. However, the viscous medium prevent the distribution of ultrasonic waves and formation of cavitation bubbles (Challis & Pinfield, 2014), thus decreases the mass transfer and extraction yield, as seen during the present study. Some previous studies were also support this phenomenon while extracting different bioactive compounds using PEG (Zhang & Wang, 2016). 3.2. Single-factor experiments The effect of various PEG-based-UAE factors i.e., PEG concentration, liquid-to-solid ratio, sonication temperature and time on YTA were estimated. All these factors showed significant differences in YTA under different tested levels (Fig. 1). For instance, at lower concentration of PEG 200, the YTA was significantly lower than that at higher concentration of 80%, however, when concentration increased to 100%, a significant decrease in YTA has been seen (Fig. 1B). Thus, 80% was considered as the optimum concentration of PEG 200. In case of liquidto-solid ratio and sonication temperature, 40 mL of PEG 200 for 1 g of powder sample and 60 °C sonication temperature was found to be significantly maximizing the YTA (Fig. 1C and D). Both factors at lower and higher values led to lower YTA as compared to other factors. For sonication time, 80 min of sonication time resulted in higher YTA than that of other tested levels (Fig. 1E). Based on these results, the centre level for BBD was set as 80% concentration of PEG 200, 40 mL/g liquid-tosolid ratio, 60 °C of sonication temperature and 80 min of sonication time, which was further tested in multi-factorial experimental design.
YTA = + 82.95 + 3.95X1 + 0.50X2 + 2.56X3 - 0.71X 4 + 0.43X1X2 + 6.53X1 X3 - 0.92X1X 4 + 2.42X2X3 + 2.27X2X 4 + 1.29X3X 4 - 15.15X12 - 4.38X22 (3)
YTP = + 984.00 + 48.53X1 + 6.71X2 + 24.39X3 - 0.22X 4 - 23.01X1 X2 + 70.77X1X3 - 10.31X1X 4 - 16.11X2X3 + 10.17X2X 4 + 11.25X3 (4)
YAA = + 43.17 + 2.46X1 + 0.68X2 + 1.41X3 - 0.34X 4 + 0.22X1X2 + 5.07X1 X3 + 1.88X1X 4 + 2.13X2X3 + 0.33X2X 4 - 0.20X3X 4 - 9.70X12 - 1.62X22 - 5.31X32 - 0.87X24
Regression coefficient (β) 3.95** 0.50 2.56* −0.71
48.53** 6.71 24.39 −0.22
2.46 0.68 1.41 −0.34
Quadratic X12 X22 X32 X42
−15.15*** −4.38 −6.64** −4.04
−209.68*** −66.28 −69.26** −51.96
−9.70*** −1.62 −5.31** −0.87
Interaction X1X2 X1X3 X1X4 X2X3 X2X4 X3X4
0.43 6.53** −0.92 2.42 2.27 1.29
−23.01 70.77* −10.31 −16.11 10.17 11.25
0.22 5.07* 1.88 2.13 0.33 −0.20
p-value Model Lack of Fit C.V. R2 Adj. R2
< 0.0001 0.1146 4.95 0.9248 0.8496
< 0.0001 0.0925 6.17 0.9085 0.8169
0.0100 0.0811 7.92 0.8871 0.7941
**
p < 0.01,
***
p < 0.0001.
3.3.2. Effect of UAE factors on the responses As shown in Table 1, YTA under ultrasonic extraction was significantly affected by the linear, quadratic and interaction effect of X1 (concentration of PEG) and X3 (sonication temperature). Similar to YTA, the linear effect of X1, the quadratic and interaction effect of X1 and X3 significantly affected YTP and YAA. The 3D response surface plots were developed for the graphical presentation of the significant interactive effect of independent variables and the optimal levels of variables for the satisfactory YTA (Fig. 2A), YTP (Fig. 2B) and YAA (Fig. 2C). The 3D graphs depicted that YTA, YTP and YAA increased as the concentration of PEG 200 increased from 65 to near 83%, however a continued increase in the concentration of PEG 200 resulted in decrease in the response values. The polarity, viscosity and other characteristics of the PEG 200 solution varied with changing concentration, leading to differences in the stability and mass transfer of bioactive compounds. Based on ‘like dissolve like’ principle, extraction solvent extracted phenolics and anthocyanins having similar polarity to that of the solvent (Feng et al., 2015). With the increase in concentration of PEG 200, the polarity of PEG solvent became closer to that of anthocyanins and phenolic compounds which might result in higher value of YTA and YTP. However, higher viscosity at higher PEG 200 concentration results in decrease in polarity which significantly affects the extraction efficiency (Zhang & Wang, 2016), as seen in the present study in term of negative quadratic regression coefficient value (Table 1). Similarly, as sonication temperature increased from 45 to 75 °C, the YTA significantly increased and reached to the highest YTA at 64 °C (positive regression coefficient) and later significantly decreased with increase in temperature (negative regression coefficient). Moreover, YTP and YAA were significantly positively affected by the linear effect of sonication temperature, however a significant decrease at higher temperature were also seen (Fig. 2B and C). Higher temperature results in increase in plant cell permeability, thus increase the solvent uptake and consequently dissolution and solubility of compounds (Yuan, Lu, Eskridge, Isom, & Hanna, 2018). However, too high temperature may also lead to the degradation of thermo-sensitive anthocyanins and phenolic content (Wang, Wang, & Li, 2013), as also seen during the present study. The interactive effect of PEG 200 concentration and sonication temperature (X1X3) was found to
3.3.1. Fitting the model The polynomial equation for YTA, YTP and YAA were estimated and the same has been shown as Eqs. (3)–(5), respectively:
X 4 - 209.68X12 - 66.28X22 - 69.26X32 - 51.96X24
YAA
Source Linear X1-Concentration of PEG X2-Liquid-to-solid ratio X3-Sonication temperature X4-Sonication time
Level of significance, *p < 0.05,
3.3. Response surface optimization
- 6.64X32 - 4.04X24
YTP
(5)
where, X1, X2, X3 and X4 represented the coded variables for concentration of PEG, liquid-to-solid ratio, sonication temperature and sonication time, respectively. Table 1 summarized the regression coefficient values with significant terms for response surface quadratic model. For YTA, YTP and YAA, the p-values (< 0.0001) of the models demonstrated that the models were significantly fitted to the model design, while lack of fit were found non-significant (p > 0.05), which indicated the developed models were reliable and adequate. The determination coefficient (R2) and adjusted determination coefficients (Adj. R2) were 0.9248 and 0.8496 for YTA, 0.9085 and 0.8169 for YTP, 0.8871 and 0.7941 for YAA, respectively, implying that the observed YTA, YTP and YAA fitted to the predicted ones. The variation coefficient of YTA (4.95%), YTP (6.17%) and YAA (7.92%) accounted for good reproducibility of the models. 62
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be significant for all the responses (Table 1). With increasing PEG 200 concentration (up-to 80%) along with sonication temperature (up-to 65 °C), an increase in YTP, YTA and YAA were recorded, which was significantly higher than other combinations. It was already mentioned that increasing temperature results in the breakdown of cells and also increase the diffusibility and solubility power of the solvent and along with increasing concentration up-to a certain level (80% in this case), the mass transfer and extraction efficiency was improved. The relationship among YAA, YTA and YTP was explored using the Pearson correlation coefficient. A significant positive correlation was detected between YAA and YTA (r = 0.9520, p < 0.001), and YAA and YTP (r = 0.9253, p < 0.001), which showed the effective role of these compounds in determining the antioxidant potential of purple sweet potato extract. Studies also confirmed that anthocyanins (Pedro, Granato, & Rosso, 2016) and phenolic compounds (Bilgin, Elhussein, Özyürek, Güçlü, & Şahin, 2018) from other plants contributed to the antioxidant activity of the extract.
3.3.3. Effect of viscosity on the responses The effect of PEG 200 concentrations along with other UAE conditions such as sonication temperature, time, sample to solvent ratio were significantly affect the overall viscosity of the solvent (Table S2). Solvent viscosity during extraction played a significant role in determining the extraction efficiency by affecting mass transfer (Duan, Dou, Guo, Li, & Liu, 2016). Similar effect has also been seen during the present study. As the viscosity increased there was a significant decrease in YTA and YTP, with highly significant (p < 0.0001) negative correlation coefficient value (Fig. S1A and B). These results revealed that extraction capacity of PEG 200 at different concentration might be significantly restricted by high viscosity due to the decrease in mass transfer rate. Ultrasound affected the plant matrix by a chain detexturation mechanism in a special order: local erosion, shear forces, sonoporation, fragmentation, capillary effect and detexturation (Khadhraoui et al., 2018). As reported by Sicaire et al. (2016), compared with conventional extraction, the action of ultrasound cavitation on the matrix resulted in the formation of debris. Ultrasound formed the cavitation bubbles in solvent which on collapse generated high pressure and temperature and thus resulting in loosen the sample matrix and dissolution of the compounds (Fig. 3A). Higher viscosity of the solvent at higher PEG 200 concentration resists the movement of molecules and prevents agitation around the collapsing bubbles (Fig. 3B). It has been reported that the influence of viscosity is more prominent in cavitation threshold than the influence of other factors during ultrasonication (Antti, Pentti, & Hanna, 2008; Antti, 2010). Moreover, the lower viscosity of PEG 200 solvent had lower density and higher diffusivity, which led to completely leach out of the bioactive content from plant matrix (Naczk & Shahidi, 2006). Similar to the effect of viscosity on YTP and YTA values, the antioxidant activity was also showed a significant negative correlation with viscosity (Fig. S1C). Also, the higher viscosity of the solvent prevents the cavitation bubble formation and thus decreased the ultrasonic effect and extraction yield (Zhang & Wang, 2016). It has been noted that the sonication temperature up to a certain level increases the extraction yield which is an effect of degradation of PEG, which results in lower viscosity and increase the frictional forces between the solvent molecules and sample and support the formation and distribution of cavitation bubbles. However, higher temperature resulted in decrease of extraction yield, which may be due the direct effect of heat on the bioactive compounds, which are sensitive to higher temperature. Moreover, the sonication time showed negative non-significant effects on extraction yield, which generally reduced the viscosity of solvent at prolong ultrasonic exposure (Mason and Peters, 2002). The non-significant effect of sonication time may be due to very long ultrasonic exposure of 65 min (lower level) in the experimental model which may exert similar effect on solvent viscosity as prolong sonication time.
Fig. 2. Response surface plots showing the interaction effects of PEG 200 concentration (X1) and sonication temperature (X3) on YTA (A), YTP (B), and YAA (C).
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Fig. 3. Effect of low viscosity (A) and high viscosity (B) solvent on extraction efficiency under ultrasonic-assisted extraction.
study as compared to other solvents and extraction methods showed promising results in term of good number of anthocyanin compounds and extraction yield. Moreover, some of the non-anthocyanin bioactive compounds such as, subulatin (caffeic acid derivative), 3,5-Dicaffeoyl quinic acid (quinic acid derivative), gluco-6-O-caffeoyl sucrose (cinnamic acid derivative), eucommicin A (quinic acid diester) and 3,4,5Tri-O-caffeoyl quinic acid (phenylpropanoids) were also detected and characterized from purple sweet potato under PEG-based-UAE optimized condition. The results revealed that the PEG type, concentration and UAE conditions affected the extraction efficiency for anthocyanins in term of total number of anthocyanin compounds and its concentration from purple sweet potato extract. The extracted anthocyanins and non-anthocyanins in the present study using PEG as green solvent was also reported to possess antioxidant, antidiabetic, anti-inflammatory and anticancer activity (Putta et al., 2017). Nevertheless, some of the previous studies reported that ultrasound might increase degradations of natural products. For instance, Meullemiestre, Breil, Abert-Vian, and Chemat (2016) found ultrasound treatment induced a degradation of diacylglycerol into free fatty acids. Also, Pingret, Fabiano-Tixier, and Chemat (2013) reported that ultrasounds could lead to degradation of anthocyanins, especially when used at high frequencies (358 and 850 kHz) or high power (750 W). However, ultrasound of low frequency (20 kHz) presented a minimal effect on degradation of blueberry anthocyanins (3.2%), even with high power of 1500 W (Tiwari, O’Donnell, Patras, & Cullen, 2008). In the present study the ultrasound frequency was used as 40 KHz with 100 W of actual power, which might limited the degradation effect of ultrasound. Moreover, the selection of these extraction conditions is of greater importance for the better recovery and less degradation of valuable compounds of interest under ultrasonic extraction condition.
3.4. Validation of the model Under the optimized PEG-based-UAE conditions, 1.0 g of sample extracted in 42 mL of 83% of PEG 200 at a sonication temperature of 64 °C for 80 min of sonication time, resulted in maximum YTA (83.78 ± 2.36 mg CE/100 g DW), YTP (994.88 ± 6.01 mg GAE/100 g DW) and YAA (42.07 ± 1.97 mg AAE/100 g DW). The experimental values of these responses obtained under optimum condition were very close to the model predicted values (81.66 mg CE/100 g DW for YTA, 989.93 mg GAE/100 g DW for YTP, 43.61 mg AAE/100 g for YAA) with relative percent deviation values as 2.59% for YTA, 0.50% for YTP and 3.66% for YAA, which validated the optimum condition. Also the viscosity at optimum extraction condition was found to be lower (0.038 Pa.s), which further justify the role of viscosity in better extraction of anthocyanins and phenolic compounds from purple sweet potatoes. 3.5. UPLC-Triple-TOF/MS analysis of anthocyanins The compounds profile detected under UPLC-Triple-TOF/MS condition under the optimum UAE condition was presented in Fig. S2. The MS detected and characterized a total of ten anthocyanins (Fig. 4) along with six non-anthocyanin compounds (Table 2). The spectral data of the identified compounds were compared with the published literature, standards and MS databases (Zhu et al., 2017; Cai et al., 2016). The peak area percentage of anthocyanins was also calculated and presented in Table 2. The major components identified by UPLC-TripleTOF/MS were cyanidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5glucoside (25.9%), peonidin-3-caffeoyl-p-hydroxybenzoylsophoroside5-glucoside (14.9%) and peonidin-3-caffeoylsophoroside-5-glucoside (12.6%). Various studies have been conducted on extraction of anthocyanins from purple sweet potato using green solvent (Table S3). However, the use of PEG has not been examined so far and our present 64
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(caption on next page)
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Fig. 4. MS and MS2 spectra of different anthocyanins from purple sweet potato optimized extract.
Table 2 Identification of anthocyanins in optimized PEG-based-UAE condition by UPLC-Triple-TOF/MS. Peak
Rt (min)
M+ (m/z)
MS2 ions (m/z)
Compound
Peak area of anthocyanins (%)
6 8 9 10 11 12 13 14 15 16
5.989 6.612 7.105 7.324 7.605 11.064 11.536 11.762 12.913 14.744
773 787 893 907 949 935 1055 1069 949 1125
611, 625, 731, 745, 787, 773, 893, 907, 787, 963,
449, 463, 449, 463, 449, 449, 449, 463, 463, 463,
Cyanidin-3-sophoroside-5-glucoside (A) Peonidin-3-sophoroside-5-glucoside (B) Cyanidin-3-p-hydroxybenzoyl sophoroside-5-glucoside (C) Peonidin-3-p-hydroxybenzoyl sophoroside-5-glucoside (D) Cyanidin-3-feruloyl sophoroside-5-glucoside (E) Cyanidin-3-caffeoyl sophoroside-5-glcoside (F) Cyanidin-3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside (G) Peonidin-3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside (H) Peonidin-3-caffeoyl sophoroside-5-glucoside (I) Peonidin-3-caffeoyl-feruloyl sophoroside-5-glucoside (J)
3.5 3.8 2.9 4.3 4.8 3.9 25.9 14.9 12.6 4.7
705 515 503 707 677 625
513, 353, 341, 353, 515, 301,
338, 191 191 179 191 353, 191 255
Subulatin 3,5-Dicaffeoyl quinic acid Gluco-6-O-caffeoyl sucrose Eucommicin A 3,4,5-Tri-O-caffeoyl quinic acid Unknown
3.4 4.2 3.0 4.3 2.2 1.6
Non-anthocyanins 1 1.012 2 1.545 3 2.324 4 3.605 5 4.744 7 6.288
287 301 287 301 287 287 287 301 301 301
4. Conclusion
carboxymethylcellulose: Effect of viscosity, molecular mass, and concentration. Ultrasonics Sonochemistry, 15, 644–648. Antti, G. (Ed.). (2010). Ultrasonically Enhanced Disintegration: Polymers, Sludge, and Contaminated Soil. VTT. Azmir, J., Zaidul, I. S. M., Rahman, M. M., Sharif, K. M., Mohamed, A., Sahena, F., ... Omar, A. K. M. (2013). Techniques for extraction of bioactive compounds from plant materials: A review. Journal of Food Engineering, 117, 426–436. Belwal, T., Ezzat, S. M., Rastrelli, L., Bhatt, I. D., Daglia, M., Baldi, A., ... Anandharamakrishnan, C. (2018). A critical analysis of extraction techniques used for botanicals: Trends, priorities, industrial uses and optimization strategies. TrAC Trends in Analytical Chemistry, 100, 82–102. Bilgin, M., Elhussein, E. A. A., Özyürek, M., Güçlü, K., & Şahin, S. (2018). Optimizing the extraction of polyphenols from Sideritis montana L. using response surface methodology. Journal of Pharmaceutical and Biomedical Analysis, 158, 137–143. Both, S., Chemat, F., & Strube, J. (2014). Extraction of polyphenols from black tea–conventional and ultrasound assisted extraction. Ultrasonics Sonochemistry, 21, 1030–1034. Cai, Z., Qu, Z., Lan, Y., Zhao, S., Ma, X., Wan, Q., ... Li, P. (2016). Conventional, ultrasound-assisted, and accelerated-solvent extractions of anthocyanins from purple sweet potatoes. Food Chemistry, 197, 266–272. Challis, R. E., & Pinfield, V. J. (2014). Ultrasonic wave propagation in concentrated slurries–the modelling problem. Ultrasonics, 54, 1737–1744. Chemat, F., Rombaut, N., Meullemiestre, A., Turk, M., Perino, S., Fabiano-Tixier, A. S., & Abert-Vian, M. (2017). Review of green food processing techniques. Preservation, transformation, and extraction. Innovative Food Science & Emerging Technologies, 41, 357–377. Chemat, F., Rombaut, N., Sicaire, A. G., Meullemiestre, A., Fabiano-Tixier, A. S., & AbertVian, M. (2017). Ultrasound assisted extraction of food and natural products. Mechanisms, techniques, combinations, protocols and applications. A review. Ultrasonics Sonochemistry, 34, 540–560. Duan, L., Dou, L. L., Guo, L., Li, P., & Liu, E. H. (2016). Comprehensive evaluation of deep eutectic solvents in extraction of bioactive natural products. ACS Sustainable Chemistry & Engineering, 4, 2405–2411. Esatbeyoglu, T., Rodríguez-Werner, M., Schlösser, A., Winterhalter, P., & Rimbach, G. (2017). Fractionation, enzyme inhibitory and cellular antioxidant activity of bioactives from purple sweet potato (Ipomoea batatas). Food Chemistry, 221, 447–456. Feng, S., Luo, Z., Tao, B., & Chen, C. (2015). Ultrasonic-assisted extraction and purification of phenolic compounds from sugarcane (Saccharum officinarum L.) rinds. LWT-Food Science and Technology, 60, 970–976. Fishburn, C. S. (2008). The pharmacology of PEGylation: Balancing PD with PK to generate novel therapeutics. Journal of Pharmaceutical Sciences, 97, 4167–4183. Harris, J. M. (2013). Poly (ethylene glycol) chemistry: Biotechnical and biomedical applications. Springer Science & Business Media. Khadhraoui, B., Turk, M., Fabiano-Tixier, A. S., Petitcolas, E., Robinet, P., Imbert, R., ... Chemat, F. (2018). Histo-cytochemistry and scanning electron microscopy for studying spatial and temporal extraction of metabolites induced by ultrasound. Towards chain detexturation mechanism. Ultrasonics Sonochemistry, 42, 482–492. Liu, L., Liu, R. L., Zhang, J., & Zhang, Z. Q. (2012). Study on the PEG-based microwaveassisted extraction of flavonoid compounds from persimmon leaves. Journal of Separation Science, 35, 3412–3420. Marták, J., & Schlosser, S. (2017). Density, viscosity, and structure of equilibrium solvent phases in butyric acid extraction by phosphonium ionic liquid. Journal of Chemical & Engineering Data, 62, 3025–3035. Mason, T. J., & Peters, D. (2002). Practical sonochemistry: Power ultrasound uses and applications. Woodhead Publishing. Meullemiestre, A., Breil, C., Abert-Vian, M., & Chemat, F. (2016). Microwave, ultrasound,
The present study successfully developed a greener extraction method for anthocyanin extraction from purple sweet potatoes using PEG as a green solvent under UAE conditions. The optimized UAE conditions (64 °C, 80 min and 42 mL/g) along with optimized PEG 200 concentration (83%) were found to be the best for the recovery of total anthocyanins and phenolics and also possess higher antioxidant activity. Moreover, the extract obtained under optimized condition was found to have lower viscosity, which supports the higher recovery of anthocyanins. A total of ten anthocyanins along with six non-anthocyanin bioactive compounds were identified and characterized using UPLC-Triple-TOF/MS, which significantly added value to the existing knowledge of its nutraceutical composition. Our results also highlighted the basis of selection of suitable PEG and its concentration under UAE for the recovery of anthocyanins and other compounds. Moreover, as a green solvent, PEG is less toxic and also cheaper, compared to organic solvents. It is also a base material found in food and pharmaceuticals, which is safe and thus highly recommended for eco-friendly extraction and solvent waste reduction. The present work is a fundamental research on the effect of ultrasonication on PEG viscosity and establishes correlation with its extraction efficiency for the greener recovery of anthocyanins from purple sweet potato. Acknowledgements The research was financially supported by the Key Research and Development Program of Zhejiang province (2018C02049) and Hangzhou Science and Technology Development Program (20180432B31). Conflict of interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.01.017. References Antti, G., Pentti, P., & Hanna, K. (2008). Ultrasonic degradation of aqueous
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batatas (L.) Lam.). Scientific Reports, 8, 5018. Tiwari, B. K., O’Donnell, C. P., Patras, A., & Cullen, P. J. (2008). Anthocyanin and ascorbic acid degradation in sonicated strawberry juice. Journal of Agricultural and Food Chemistry, 56, 10071–10077. Wang, L., Wang, Z., & Li, X. (2013). Optimization of ultrasonic-assisted extraction of phenolic antioxidants from Malusbaccata (Linn.) Borkh. using response surface methodology. Journal of Separation Science, 36, 1652–1658. Yousuf, B., Gul, K., Wani, A. A., & Singh, P. (2016). Health benefits of anthocyanins and their encapsulation for potential use in food systems: A review. Critical Reviews in Food Science and Nutrition, 56, 2223–2230. Yuan, B., Lu, M., Eskridge, K. M., Isom, L. D., & Hanna, M. A. (2018). Extraction, identification, and quantification of antioxidant phenolics from hazelnut (Corylus avellana L.) shells. Food Chemistry, 244, 7–15. Zhang, J. L., Luo, C. L., Zhou, Q., & Zhang, Z. C. (2018). Isolation and identification of two major acylated anthocyanins from purple sweet potato (Ipomoea batatas L. cultivar Eshu No. 8) by UPLC-QTOF-MS/MS and NMR. International Journal of Food Science & Technology, 53, 1932–1941. Zhang, L., & Wang, M. (2016). Polyethylene glycol-based ultrasound-assisted extraction and ultrafiltration separation of polysaccharides from Tremella fuciformis (snow fungus). Food and Bioproducts Processing, 100, 464–468. Zhou, T., Xiao, X., Li, G., & Cai, Z. W. (2011). Study of polyethylene glycol as a green solvent in the microwave-assisted extraction of flavone and coumarin compounds from medicinal plants. Journal of Chromatography A, 1218, 3608–3615. Zhou, X. Y., Liu, R. L., Ma, X., & Zhang, Z. Q. (2014). Polyethylene glycol as a novel solvent for extraction of crude polysaccharides from Pericarpium granati. Carbohydrate Polymers, 101, 886–889. Zhu, Z., Guan, Q., Koubaa, M., Barba, F. J., Roohinejad, S., Cravotto, G., ... He, J. (2017). HPLC-DAD-ESI-MS2 analytical profile of extracts obtained from purple sweet potato after green ultrasound-assisted extraction. Food Chemistry, 215, 391–400.
thermal treatments, and bead milling as intensification techniques for extraction of lipids from oleaginous Yarrowia lipolytica yeast for a biojetfuel application. Bioresource Technology, 211, 190–199. Montilla, E. C., Hillebrand, S., Butschbach, D., Baldermann, S., Watanabe, N., & Winterhalter, P. (2010). Preparative isolation of anthocyanins from Japanese purple sweet potato (Ipomoea batatas L.) varieties by high-speed countercurrent chromatography. Journal of Agricultural and Food Chemistry, 58, 9899–9904. Naczk, M., & Shahidi, F. (2006). Phenolics in cereals, fruits and vegetables: Occurrence, extraction and analysis. Journal of Pharmaceutical and Biomedical Analysis, 41, 1523–1542. Pandey, A., Belwal, T., Sekar, K. C., Bhatt, I. D., & Rawal, R. S. (2018). Optimization of ultrasonic-assisted extraction (UAE) of phenolics and antioxidant compounds from rhizomes of Rheum moorcroftianum using response surface methodology (RSM). Industrial Crops and Products, 119, 218–225. Pedro, A. C., Granato, D., & Rosso, N. D. (2016). Extraction of anthocyanins and polyphenols from black rice (Oryza sativa L.) by modeling and assessing their reversibility and stability. Food Chemistry, 191, 12–20. Pingret, D., Fabiano-Tixier, A. S., & Chemat, F. (2013). Degradation during application of ultrasound in food processing: A review. Food Control, 31, 593–606. Putta, S., Yarla, N. S., Peluso, I., Tiwari, D. K., Reddy, G. V., Giri, P. V., & Reddy, D. (2017). Anthocyanins: Multi-target agents for prevention and therapy of chronic diseases. Current Pharmaceutical Design, 23, 6321–6346. Rommel, A., Heatherbell, D. A., & Wrolstad, R. E. (1990). Red raspberry juice and wine: Effect of processing and storage on anthocyanin pigment composition, color and appearance. Journal of Food Science, 55, 1011–1017. Sicaire, A. G., Vian, M. A., Fine, F., Carré, P., Tostain, S., & Chemat, F. (2016). Ultrasound induced green solvent extraction of oil from oleaginous seeds. Ultrasonics Sonochemistry, 31, 319–329. Sun, H., Zhang, P., Zhu, Y., Lou, Q., & He, S. (2018). Antioxidant and prebiotic activity of five peonidin-based anthocyanins extracted from purple sweet potato (Ipomoea
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Ultrasonic impact on viscosity and extraction efficiency of polyethylene glycol: A greener approach for anthocyanins recovery from purple sweet potato
T
⁎
Hao Huanga, Qin Xua, Tarun Belwala, Li Lia, Halah Aalima, Qiong Wua, Zhenhua Duanb, , ⁎ Xuebing Zhangc, Zisheng Luoa, a
Zhejiang University, College of Biosystems Engineering and Food Science, Key Laboratory of Agro-Products Postharvest Handling of Ministry of Agriculture and Rural Affairs, Zhejiang Key Laboratory for Agri-Food Processing, Hangzhou 310058, People’s Republic of China Institute of Food Science and Engineering, Hezhou University, Hezhou, People’s Republic of China c Hangzhou Wanxiang Polytechnic, Huawu Road 3, Hangzhou 310023, People’s Republic of China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Polyethylene glycol Ultrasound-assisted extraction Anthocyanins Purple sweet potato Viscosity
Purple sweet potatoes are known for its vibrant purple color due to high level of anthocyanins. A polyethylene glycol based ultrasonic-assisted green extraction (PEG-UAE) of anthocyanins from purple sweet potato was proposed. Different types of PEG were tested for anthocyanin extraction along with PEG concentration, liquid-tosolid ratio, ultrasonic temperature and time were investigated for its impact on viscosity and extraction efficiency. The optimum extraction condition, 42 mL/g of ratio, 83% of PEG 200 concentration, 64 °C of ultrasonic temperature and 80 min of sonication time, resulted in better extraction of anthocyanins (83.78 mg CE/100 g DW) and phenolics (994.88 mg GAE/100 g DW). Using UPLC-Triple-TOF/MS, ten anthocyanin and six nonanthocyanin compounds were identified and characterized, with the highest peak area for cyanidin-3-caffeoyl-phydroxybenzoyl sophoroside-5-glucoside (25.9%). Moreover, the anthocyanins and phenolics extraction yield along with antioxidant activity were negatively correlated with PEG viscosity, on which ultrasonication has profound effects.
1. Introduction Purple sweet potato (Ipomoea batatas) is an important staple food mainly cultivated in Asia, especially in China (Zhang, Luo, Zhou, & Zhang, 2018). Although worldwide it is among the seven largest crops (in term of production) after sugar cane, rice, wheat, potatoes, maize, and cassava, it is still underutilized in term of nutraceutical exploration and applications (Esatbeyoglu, Rodríguez-Werner, Schlösser, Winterhalter, & Rimbach, 2017). Previous studies have reported that purple sweet potato contains abundant anthocyanins, including cyanidin and peonidin with acylated p-hydroxybenzoic acid, ferulic acid and caffeic acid (Montilla et al., 2010). Anthocyanins are secondary metabolites, responsible for the developement of purple, blue, red colors in plants (Rommel, Heatherbell, & Wrolstad, 1990). In recent years, there is an increasing interest in anthocyanins research due to their health benefits against cardiovascular diseases, neurodegenerative diseases, cancer and many others (Yousuf, Gul, Wani, & Singh, 2016). Concurrently, Sun, Zhang, Zhu, Lou, and He (2018) also reported that
⁎
anthocyanins of purple sweet potato are attributed to their antioxidant, anticancer, antidiabetic, anti-inflammatory, anti-bacteria, and hepatoprotective activities. Recently, the utilization of agricultural products for extracting value-added bioactive compounds have generated interests among researchers and nutraceutical industry (Azmir et al., 2013), and purple sweet potato could be considered as one of the potential source of bioactive compounds, especially anthocyanins. Anthocyanins are sensitive to high temperature, pH and other extraction conditions, along with the kind of instrument used, which together played an important role in anthocyanin yield and its quality attributes (Belwal et al., 2018). For valorization of purple sweet potato, it is of paramount importance to find novel methodologies to improve the extraction efficiency of anthocyanins. Recently, the special interest on ultrasonic-assisted extraction (UAE) has been drawn, due to its green impacts on extraction process of bioactive compounds, in term of higher yield, shorter processing time and lower maintenance cost (Chemat, Rombaut, & Meullemiestre et al., 2017). The majority of research studies employ UAE focused on the extraction conditions such as type of
Corresponding authors. E-mail addresses: [email protected] (Z. Duan), [email protected] (Z. Luo).
https://doi.org/10.1016/j.foodchem.2019.01.017 Received 23 October 2018; Received in revised form 10 December 2018; Accepted 4 January 2019 Available online 14 January 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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for 80 min in an ultrasonic bath (SB-4200D, Scientz Biotechnology Co., Ningbo, China). Calorimetric measurements were applied to assess actual ultrasound power (Both, Chemat, & Strube, 2014; Sicaire et al., 2016), calculated by Eq. (1).
solvents, its concentration, liquid-to-solid ratio, sonication temperature and time (Belwal et al., 2018; Chemat, Rombaut, & Sicaire et al., 2017). However, for green extraction of bioactive compounds, the selection of non-toxic, high efficiency, low cost and environmental friendly solvent is of paramount importance (Chemat, Rombaut, & Meullemiestre et al., 2017). Polyethylene glycol (PEG) is one of such kind of green solvent which is non-toxic, low cost and safe to the environment (Zhang & Wang, 2016). Moreover, a number of bioactive compounds have been successfully extracted using PEG and the yield of many of these are higher as compared to other solvents. For instance, Zhou, Liu, Ma, and Zhang (2014) reported PEG offered higher extraction yield of polysaccharides than that of the water-based extraction. It has also been reported that PEG had higher efficiency for the extraction of flavonoid compounds, compared with ethanol (Liu, Liu, Zhang, & Zhang, 2012) and methanol (Zhou, Xiao, Li, & Cai, 2011). PEG has also been applied in food (Harris, 2013) and pharmaceutical products (Fishburn, 2008), due to its non-toxic nature. However, no study has been reported for its use in the extraction of anthocyanins from purple sweet potato. Moreover, the relationship study between extraction efficiency and various PEG associated factors including viscosity is still lacking. In the present study, for efficient extraction of anthocyanins from purple sweet potatoes, we investigated the effect of different PEG at various concentrations and other extraction conditions to develop a polyethylene glycol-based ultrasonic-assisted green extraction method (PEG-based-UAE). The relationship between anthocyanin yield and PEG viscosity was also be established during the investigation. For the identification and characterization of anthocyanin compounds, a highend UPLC-Triple-TOF/MS analytical method was also employed. Although, many researches have been conducted on anthocyanins extraction from purple sweet potatoes, however the current proposed research work is in continuation of developing greener extraction method for anthocyanins, which can further be utilized for its multiindustrial applications.
P(W) = m × Cp ×
dT dt
(1)
where Cp is the heat capacity of the solvent at constant pressure (J/g/ °C), m is the mass of solvent (g) and dT/dt is temperature rise per second (°C/s). The actual ultrasound power in present study was around 100 W. The total anthocyanin yield (YTA) was determined to select the best type of PEG and viscosity of each was also estimated. 2.3.2. Yield of total anthocyanin (YTA) YTA was determined according to pH differential method (Zhu et al., 2017) with some modifications. An aliquot (1 mL each) of the extract was mixed with 0.025 M potassium chloride buffer (pH 1.0, 3 mL) and 0.4 M sodium acetate buffer (pH 4.5, 3 mL), separately. The absorbance of the mixture was measured at 510 and 700 nm by UV–Vis spectrophotometer (UV-1750, Shimadzu Co., Ltd., Japan). YTA was calculated as cyanidin-3-glucoside equivalents according to Eq. (2).
YTA (mg CE/100g DW) = [(A510 - A700)pH1.0 - (A510 - A700)pH4.5] × V× n×M × 100 ε × L×m
(2)
where, A510 and A700 represent the absorbance at 510 nm and 700 nm, respectively. V as volume of extract (mL), n is the dilution factor, M is the molecular weight of cyanidin-3-glucoside (449.2 g/mol), ε is the extinction coefficient of cyanidin-3-glucoside (26,900 L/mol/cm), L is the width of the cuvette (1 cm), and m is the weight of sample (1 g). 2.3.3. Yield of total polyphenol (YTP) YTP was determined according to the Folin-Ciocalteu procedure (Feng, Luo, Tao, & Chen, 2015) with some modifications. Briefly, 0.125 mL of extract was mixed with 0.5 mL of Folin-Ciocalteu reagent and 2.125 mL of distilled water. Thereafter, 1.250 mL of 70 mg/mL sodium carbonate was added. After 90 min of incubation in dark, the absorbance was measured at 760 nm by a spectrophotometer (UV-1750, Shimadzu Co., Japan). YTP was expressed as milligrams of gallic acid equivalents per 100 g dry weight (mg GAE/100 g DW) of sample.
2. Materials and methods 2.1. Chemicals and reagents Folin-Ciocalteu reagent was purchased from Dingguo Changsheng Biotechnology Co. (Beijing, China). PEG (200, 300, 400, 600), potassium chloride, sodium acetate, sodium carbonate, ascorbic acid, 2,2diphenyl-1-picrylhydrazyl (DPPH) and standard of gallic acid were purchased from Aladdin Industrial Co. (Shanghai, China). Hydrochloric acid and ethanol were provided by Sinopharm Chemical Reagent Co. (Shanghai, China). All solvents and standards used for chromatographic analyses were of HPLC grade.
2.3.4. Antioxidant activity (YAA) The antioxidant activity was determined by DPPH assay (Pandey, Belwal, Sekar, Bhatt, & Rawal, 2018). Briefly, 2 mL of extract was mixed with 4 mL of 0.1 mM DPPH solution and kept in dark for 30 min at room temperature. The absorbance was measured at 517 nm using UV–Vis spectrophotometer (UV-1750, Shimadzu Co., Japan). The antioxidant activity was expressed as ascorbic acid equivalent per gram dry weight (mg AAE/100 g DW).
2.2. Materials and preparation Purple sweet potatoes cv. Yamakawamurasaki were collected from a commercial vegetable market of Hangzhou City, Zhejiang province, China. The purple sweet potatoes were peeled and cut into slices of 0.3–0.6 cm. The slices were then dried in an oven at 45 °C for 24 h. The dried samples were grounded into powder with a pulveriser (Huangchen HC-280T, Zhejiang, China) and passed through a 60-mesh sieve and stored at 4 °C until use.
2.3.5. Viscosity The viscosity of different PEG solvents and model experimental runs were measured using a rheometer (Haake Co., Coesfeld, Germany) according to the protocol of Marták and Schlosser (2017). A cone and plate system was applied at 25 °C with shear rates of 0.1 s−1. The correlation of viscosity with the YTA, YTP and YAA was calculated using SPSS 20.0 software.
2.3. Polyethylene glycol-based ultrasonic-assisted extraction (PEG-UAE) 2.3.6. Design of experiments (DoE) 2.3.6.1. Single-factor experiments. Single-factor experiments were firstly conducted to determine the optimal level of individual factors i.e., concentration of PEG, liquid-to-solid ratio, sonication temperature and sonication time on YTA (Table S1). Briefly, about 1.0 g of purple sweet potato powder was extracted in 50-mL centrifuge tube with different concentration of PEG and varying liquid-to-solid ratio. The ultrasonication was carried out in an ultrasonic bath at different
2.3.1. Selection of PEG PEG was considered to show various extraction efficiency depends on its composition and molecular weight. Thus, different types of PEG such as PEG 200, PEG 300, PEG 400 and PEG 600 were tested in a preliminary experiment for selecting the most efficient type. About 1.0 g of purple sweet potato powder was mixed with 30 mL of PEG (60%, v/v) and ultrasonicated (maximum up to 400 W, 40 KHz) at 50 °C 60
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temperatures with varying time. Thereafter, the extract was centrifuged at 12,000 rpm for 15 min and the supernatant was collected and stored at 4 °C for further analysis. 2.3.6.2. Multi-factor experiments. According to single-factor experiments, the optimal level of each factor was chosen as center point for the multi-factorial experimental design to determine the linear, quadratic and interactive effects between the variables. The response surface methodology (RSM) was used to optimize the extraction process parameters. A four-factor (X1, X2, X3 and X4) and three levels (−1, 0 and 1) Box–Behnken design (BBD) was applied. X1, X2, X3 and X4 represented concentration of PEG (%, v/v), liquid-to-solid ratio (mL/g), sonication temperature (℃) and sonication time (min), respectively. The responses i.e., YTA, YTP and YAA along with viscosity of each experimental run were presented in Table S2. Design Expert software (V. 8.0.6) was used for the experimental design, regression and graphical analysis. Analysis of variance (ANOVA) was used to determine the significant difference of linear, quadratic and interactive variables, statistical significance of the model and the regression coefficients. 2.3.7. Validation of the model For model validation, the optimized condition which showed highest desirability was selected using Design Expert software (V. 8.0.6) and tested for its validation. The model predicted value was compared with the experimental values. 2.4. UPLC-triple-TOF/MS analysis The ultra-high pressure liquid chromatography (Waters Co., Milford, USA) combined with triple-TOF mass spectrophotometry (UPLC-Triple-TOF/MS) 5600+ System (AB SCIEX Co., Framingham, USA) for anthocyanins were conducted according to Cai’s protocol with minor modifications (Cai et al., 2016). The PEG extract was filtered through 0.5 μm fluoropore membrane filter (Millipore Co., Shanghai, China). The injection volume was 5 μL and the separation was carried out using a ZORBAX-SB C18, 100 mm × 4.6 mm, 1.8 µm column (Agilent Technologies Co., Santa Clara, USA), and the temperature was maintained at 30 °C. The UV detector was set at 530 nm. The mobile phase was selected as solvent A (formic acid: water = 1:1000) and solvent B (formic acid: acetonitrile = 1:1000). The elution gradient was as follows: 0–2 min, 5% B; 2–25 min, 5% to 50% B; 25–35 min, 50% to 95% B; 35–37 min, 95% B, 37–40 min 95% to 5% B and the flow rate was set at 0.8 mL min−1. The MS condition was set at the positive ion mode as follows: scan range m/z 200–2000, capillary voltage 3.0 kV, sampling cone 35 V, collision energy 4 eV, source temperature 100 °C, desolvation temperature 300 °C, desolvation gas 500 L/h. 2.5. Statistical analysis All experiments were performed in triplicates and the data were statistically analyzed by the SAS 9.4 program and presented as means ± SD. Significant differences were calculated using ANOVA and p < 0.05 was considered as significant. 3. Results and discussion 3.1. Selection of PEG Among the tested PEG, PEG 200 was found to be the most effective solvent for the extraction of anthocyanins (YTA) (Fig. 1A). With increasing molecular weight of PEG from 200 to 600 Da, the extraction efficiency was significantly decreased. It was also recorded that the viscosity of PEG increased with increasing molecular weight, which played significant role in lower down extraction efficiency. During ultrasonication the cavitation bubbles were formed which disrupted the (caption on next page) 61
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Fig. 1. Effect of different PEG on YTA and viscosity (A), effects of PEG 200 concentration (B), liquid-to-solid ratio (C), sonication temperature (D) and sonication time (E) on YTA. Values are presented as means ± SD (n = 3). Different letters above the bar showed significant difference at p < 0.05.
Table 1 Regression coefficient (β) and analysis of the variance (ANOVA) of the response surface quadratic models for the investigated parameters. YTA
cells and increase the release of bioactive compounds. However, the viscous medium prevent the distribution of ultrasonic waves and formation of cavitation bubbles (Challis & Pinfield, 2014), thus decreases the mass transfer and extraction yield, as seen during the present study. Some previous studies were also support this phenomenon while extracting different bioactive compounds using PEG (Zhang & Wang, 2016). 3.2. Single-factor experiments The effect of various PEG-based-UAE factors i.e., PEG concentration, liquid-to-solid ratio, sonication temperature and time on YTA were estimated. All these factors showed significant differences in YTA under different tested levels (Fig. 1). For instance, at lower concentration of PEG 200, the YTA was significantly lower than that at higher concentration of 80%, however, when concentration increased to 100%, a significant decrease in YTA has been seen (Fig. 1B). Thus, 80% was considered as the optimum concentration of PEG 200. In case of liquidto-solid ratio and sonication temperature, 40 mL of PEG 200 for 1 g of powder sample and 60 °C sonication temperature was found to be significantly maximizing the YTA (Fig. 1C and D). Both factors at lower and higher values led to lower YTA as compared to other factors. For sonication time, 80 min of sonication time resulted in higher YTA than that of other tested levels (Fig. 1E). Based on these results, the centre level for BBD was set as 80% concentration of PEG 200, 40 mL/g liquid-tosolid ratio, 60 °C of sonication temperature and 80 min of sonication time, which was further tested in multi-factorial experimental design.
YTA = + 82.95 + 3.95X1 + 0.50X2 + 2.56X3 - 0.71X 4 + 0.43X1X2 + 6.53X1 X3 - 0.92X1X 4 + 2.42X2X3 + 2.27X2X 4 + 1.29X3X 4 - 15.15X12 - 4.38X22 (3)
YTP = + 984.00 + 48.53X1 + 6.71X2 + 24.39X3 - 0.22X 4 - 23.01X1 X2 + 70.77X1X3 - 10.31X1X 4 - 16.11X2X3 + 10.17X2X 4 + 11.25X3 (4)
YAA = + 43.17 + 2.46X1 + 0.68X2 + 1.41X3 - 0.34X 4 + 0.22X1X2 + 5.07X1 X3 + 1.88X1X 4 + 2.13X2X3 + 0.33X2X 4 - 0.20X3X 4 - 9.70X12 - 1.62X22 - 5.31X32 - 0.87X24
Regression coefficient (β) 3.95** 0.50 2.56* −0.71
48.53** 6.71 24.39 −0.22
2.46 0.68 1.41 −0.34
Quadratic X12 X22 X32 X42
−15.15*** −4.38 −6.64** −4.04
−209.68*** −66.28 −69.26** −51.96
−9.70*** −1.62 −5.31** −0.87
Interaction X1X2 X1X3 X1X4 X2X3 X2X4 X3X4
0.43 6.53** −0.92 2.42 2.27 1.29
−23.01 70.77* −10.31 −16.11 10.17 11.25
0.22 5.07* 1.88 2.13 0.33 −0.20
p-value Model Lack of Fit C.V. R2 Adj. R2
< 0.0001 0.1146 4.95 0.9248 0.8496
< 0.0001 0.0925 6.17 0.9085 0.8169
0.0100 0.0811 7.92 0.8871 0.7941
**
p < 0.01,
***
p < 0.0001.
3.3.2. Effect of UAE factors on the responses As shown in Table 1, YTA under ultrasonic extraction was significantly affected by the linear, quadratic and interaction effect of X1 (concentration of PEG) and X3 (sonication temperature). Similar to YTA, the linear effect of X1, the quadratic and interaction effect of X1 and X3 significantly affected YTP and YAA. The 3D response surface plots were developed for the graphical presentation of the significant interactive effect of independent variables and the optimal levels of variables for the satisfactory YTA (Fig. 2A), YTP (Fig. 2B) and YAA (Fig. 2C). The 3D graphs depicted that YTA, YTP and YAA increased as the concentration of PEG 200 increased from 65 to near 83%, however a continued increase in the concentration of PEG 200 resulted in decrease in the response values. The polarity, viscosity and other characteristics of the PEG 200 solution varied with changing concentration, leading to differences in the stability and mass transfer of bioactive compounds. Based on ‘like dissolve like’ principle, extraction solvent extracted phenolics and anthocyanins having similar polarity to that of the solvent (Feng et al., 2015). With the increase in concentration of PEG 200, the polarity of PEG solvent became closer to that of anthocyanins and phenolic compounds which might result in higher value of YTA and YTP. However, higher viscosity at higher PEG 200 concentration results in decrease in polarity which significantly affects the extraction efficiency (Zhang & Wang, 2016), as seen in the present study in term of negative quadratic regression coefficient value (Table 1). Similarly, as sonication temperature increased from 45 to 75 °C, the YTA significantly increased and reached to the highest YTA at 64 °C (positive regression coefficient) and later significantly decreased with increase in temperature (negative regression coefficient). Moreover, YTP and YAA were significantly positively affected by the linear effect of sonication temperature, however a significant decrease at higher temperature were also seen (Fig. 2B and C). Higher temperature results in increase in plant cell permeability, thus increase the solvent uptake and consequently dissolution and solubility of compounds (Yuan, Lu, Eskridge, Isom, & Hanna, 2018). However, too high temperature may also lead to the degradation of thermo-sensitive anthocyanins and phenolic content (Wang, Wang, & Li, 2013), as also seen during the present study. The interactive effect of PEG 200 concentration and sonication temperature (X1X3) was found to
3.3.1. Fitting the model The polynomial equation for YTA, YTP and YAA were estimated and the same has been shown as Eqs. (3)–(5), respectively:
X 4 - 209.68X12 - 66.28X22 - 69.26X32 - 51.96X24
YAA
Source Linear X1-Concentration of PEG X2-Liquid-to-solid ratio X3-Sonication temperature X4-Sonication time
Level of significance, *p < 0.05,
3.3. Response surface optimization
- 6.64X32 - 4.04X24
YTP
(5)
where, X1, X2, X3 and X4 represented the coded variables for concentration of PEG, liquid-to-solid ratio, sonication temperature and sonication time, respectively. Table 1 summarized the regression coefficient values with significant terms for response surface quadratic model. For YTA, YTP and YAA, the p-values (< 0.0001) of the models demonstrated that the models were significantly fitted to the model design, while lack of fit were found non-significant (p > 0.05), which indicated the developed models were reliable and adequate. The determination coefficient (R2) and adjusted determination coefficients (Adj. R2) were 0.9248 and 0.8496 for YTA, 0.9085 and 0.8169 for YTP, 0.8871 and 0.7941 for YAA, respectively, implying that the observed YTA, YTP and YAA fitted to the predicted ones. The variation coefficient of YTA (4.95%), YTP (6.17%) and YAA (7.92%) accounted for good reproducibility of the models. 62
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be significant for all the responses (Table 1). With increasing PEG 200 concentration (up-to 80%) along with sonication temperature (up-to 65 °C), an increase in YTP, YTA and YAA were recorded, which was significantly higher than other combinations. It was already mentioned that increasing temperature results in the breakdown of cells and also increase the diffusibility and solubility power of the solvent and along with increasing concentration up-to a certain level (80% in this case), the mass transfer and extraction efficiency was improved. The relationship among YAA, YTA and YTP was explored using the Pearson correlation coefficient. A significant positive correlation was detected between YAA and YTA (r = 0.9520, p < 0.001), and YAA and YTP (r = 0.9253, p < 0.001), which showed the effective role of these compounds in determining the antioxidant potential of purple sweet potato extract. Studies also confirmed that anthocyanins (Pedro, Granato, & Rosso, 2016) and phenolic compounds (Bilgin, Elhussein, Özyürek, Güçlü, & Şahin, 2018) from other plants contributed to the antioxidant activity of the extract.
3.3.3. Effect of viscosity on the responses The effect of PEG 200 concentrations along with other UAE conditions such as sonication temperature, time, sample to solvent ratio were significantly affect the overall viscosity of the solvent (Table S2). Solvent viscosity during extraction played a significant role in determining the extraction efficiency by affecting mass transfer (Duan, Dou, Guo, Li, & Liu, 2016). Similar effect has also been seen during the present study. As the viscosity increased there was a significant decrease in YTA and YTP, with highly significant (p < 0.0001) negative correlation coefficient value (Fig. S1A and B). These results revealed that extraction capacity of PEG 200 at different concentration might be significantly restricted by high viscosity due to the decrease in mass transfer rate. Ultrasound affected the plant matrix by a chain detexturation mechanism in a special order: local erosion, shear forces, sonoporation, fragmentation, capillary effect and detexturation (Khadhraoui et al., 2018). As reported by Sicaire et al. (2016), compared with conventional extraction, the action of ultrasound cavitation on the matrix resulted in the formation of debris. Ultrasound formed the cavitation bubbles in solvent which on collapse generated high pressure and temperature and thus resulting in loosen the sample matrix and dissolution of the compounds (Fig. 3A). Higher viscosity of the solvent at higher PEG 200 concentration resists the movement of molecules and prevents agitation around the collapsing bubbles (Fig. 3B). It has been reported that the influence of viscosity is more prominent in cavitation threshold than the influence of other factors during ultrasonication (Antti, Pentti, & Hanna, 2008; Antti, 2010). Moreover, the lower viscosity of PEG 200 solvent had lower density and higher diffusivity, which led to completely leach out of the bioactive content from plant matrix (Naczk & Shahidi, 2006). Similar to the effect of viscosity on YTP and YTA values, the antioxidant activity was also showed a significant negative correlation with viscosity (Fig. S1C). Also, the higher viscosity of the solvent prevents the cavitation bubble formation and thus decreased the ultrasonic effect and extraction yield (Zhang & Wang, 2016). It has been noted that the sonication temperature up to a certain level increases the extraction yield which is an effect of degradation of PEG, which results in lower viscosity and increase the frictional forces between the solvent molecules and sample and support the formation and distribution of cavitation bubbles. However, higher temperature resulted in decrease of extraction yield, which may be due the direct effect of heat on the bioactive compounds, which are sensitive to higher temperature. Moreover, the sonication time showed negative non-significant effects on extraction yield, which generally reduced the viscosity of solvent at prolong ultrasonic exposure (Mason and Peters, 2002). The non-significant effect of sonication time may be due to very long ultrasonic exposure of 65 min (lower level) in the experimental model which may exert similar effect on solvent viscosity as prolong sonication time.
Fig. 2. Response surface plots showing the interaction effects of PEG 200 concentration (X1) and sonication temperature (X3) on YTA (A), YTP (B), and YAA (C).
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Fig. 3. Effect of low viscosity (A) and high viscosity (B) solvent on extraction efficiency under ultrasonic-assisted extraction.
study as compared to other solvents and extraction methods showed promising results in term of good number of anthocyanin compounds and extraction yield. Moreover, some of the non-anthocyanin bioactive compounds such as, subulatin (caffeic acid derivative), 3,5-Dicaffeoyl quinic acid (quinic acid derivative), gluco-6-O-caffeoyl sucrose (cinnamic acid derivative), eucommicin A (quinic acid diester) and 3,4,5Tri-O-caffeoyl quinic acid (phenylpropanoids) were also detected and characterized from purple sweet potato under PEG-based-UAE optimized condition. The results revealed that the PEG type, concentration and UAE conditions affected the extraction efficiency for anthocyanins in term of total number of anthocyanin compounds and its concentration from purple sweet potato extract. The extracted anthocyanins and non-anthocyanins in the present study using PEG as green solvent was also reported to possess antioxidant, antidiabetic, anti-inflammatory and anticancer activity (Putta et al., 2017). Nevertheless, some of the previous studies reported that ultrasound might increase degradations of natural products. For instance, Meullemiestre, Breil, Abert-Vian, and Chemat (2016) found ultrasound treatment induced a degradation of diacylglycerol into free fatty acids. Also, Pingret, Fabiano-Tixier, and Chemat (2013) reported that ultrasounds could lead to degradation of anthocyanins, especially when used at high frequencies (358 and 850 kHz) or high power (750 W). However, ultrasound of low frequency (20 kHz) presented a minimal effect on degradation of blueberry anthocyanins (3.2%), even with high power of 1500 W (Tiwari, O’Donnell, Patras, & Cullen, 2008). In the present study the ultrasound frequency was used as 40 KHz with 100 W of actual power, which might limited the degradation effect of ultrasound. Moreover, the selection of these extraction conditions is of greater importance for the better recovery and less degradation of valuable compounds of interest under ultrasonic extraction condition.
3.4. Validation of the model Under the optimized PEG-based-UAE conditions, 1.0 g of sample extracted in 42 mL of 83% of PEG 200 at a sonication temperature of 64 °C for 80 min of sonication time, resulted in maximum YTA (83.78 ± 2.36 mg CE/100 g DW), YTP (994.88 ± 6.01 mg GAE/100 g DW) and YAA (42.07 ± 1.97 mg AAE/100 g DW). The experimental values of these responses obtained under optimum condition were very close to the model predicted values (81.66 mg CE/100 g DW for YTA, 989.93 mg GAE/100 g DW for YTP, 43.61 mg AAE/100 g for YAA) with relative percent deviation values as 2.59% for YTA, 0.50% for YTP and 3.66% for YAA, which validated the optimum condition. Also the viscosity at optimum extraction condition was found to be lower (0.038 Pa.s), which further justify the role of viscosity in better extraction of anthocyanins and phenolic compounds from purple sweet potatoes. 3.5. UPLC-Triple-TOF/MS analysis of anthocyanins The compounds profile detected under UPLC-Triple-TOF/MS condition under the optimum UAE condition was presented in Fig. S2. The MS detected and characterized a total of ten anthocyanins (Fig. 4) along with six non-anthocyanin compounds (Table 2). The spectral data of the identified compounds were compared with the published literature, standards and MS databases (Zhu et al., 2017; Cai et al., 2016). The peak area percentage of anthocyanins was also calculated and presented in Table 2. The major components identified by UPLC-TripleTOF/MS were cyanidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5glucoside (25.9%), peonidin-3-caffeoyl-p-hydroxybenzoylsophoroside5-glucoside (14.9%) and peonidin-3-caffeoylsophoroside-5-glucoside (12.6%). Various studies have been conducted on extraction of anthocyanins from purple sweet potato using green solvent (Table S3). However, the use of PEG has not been examined so far and our present 64
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(caption on next page)
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Fig. 4. MS and MS2 spectra of different anthocyanins from purple sweet potato optimized extract.
Table 2 Identification of anthocyanins in optimized PEG-based-UAE condition by UPLC-Triple-TOF/MS. Peak
Rt (min)
M+ (m/z)
MS2 ions (m/z)
Compound
Peak area of anthocyanins (%)
6 8 9 10 11 12 13 14 15 16
5.989 6.612 7.105 7.324 7.605 11.064 11.536 11.762 12.913 14.744
773 787 893 907 949 935 1055 1069 949 1125
611, 625, 731, 745, 787, 773, 893, 907, 787, 963,
449, 463, 449, 463, 449, 449, 449, 463, 463, 463,
Cyanidin-3-sophoroside-5-glucoside (A) Peonidin-3-sophoroside-5-glucoside (B) Cyanidin-3-p-hydroxybenzoyl sophoroside-5-glucoside (C) Peonidin-3-p-hydroxybenzoyl sophoroside-5-glucoside (D) Cyanidin-3-feruloyl sophoroside-5-glucoside (E) Cyanidin-3-caffeoyl sophoroside-5-glcoside (F) Cyanidin-3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside (G) Peonidin-3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside (H) Peonidin-3-caffeoyl sophoroside-5-glucoside (I) Peonidin-3-caffeoyl-feruloyl sophoroside-5-glucoside (J)
3.5 3.8 2.9 4.3 4.8 3.9 25.9 14.9 12.6 4.7
705 515 503 707 677 625
513, 353, 341, 353, 515, 301,
338, 191 191 179 191 353, 191 255
Subulatin 3,5-Dicaffeoyl quinic acid Gluco-6-O-caffeoyl sucrose Eucommicin A 3,4,5-Tri-O-caffeoyl quinic acid Unknown
3.4 4.2 3.0 4.3 2.2 1.6
Non-anthocyanins 1 1.012 2 1.545 3 2.324 4 3.605 5 4.744 7 6.288
287 301 287 301 287 287 287 301 301 301
4. Conclusion
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The present study successfully developed a greener extraction method for anthocyanin extraction from purple sweet potatoes using PEG as a green solvent under UAE conditions. The optimized UAE conditions (64 °C, 80 min and 42 mL/g) along with optimized PEG 200 concentration (83%) were found to be the best for the recovery of total anthocyanins and phenolics and also possess higher antioxidant activity. Moreover, the extract obtained under optimized condition was found to have lower viscosity, which supports the higher recovery of anthocyanins. A total of ten anthocyanins along with six non-anthocyanin bioactive compounds were identified and characterized using UPLC-Triple-TOF/MS, which significantly added value to the existing knowledge of its nutraceutical composition. Our results also highlighted the basis of selection of suitable PEG and its concentration under UAE for the recovery of anthocyanins and other compounds. Moreover, as a green solvent, PEG is less toxic and also cheaper, compared to organic solvents. It is also a base material found in food and pharmaceuticals, which is safe and thus highly recommended for eco-friendly extraction and solvent waste reduction. The present work is a fundamental research on the effect of ultrasonication on PEG viscosity and establishes correlation with its extraction efficiency for the greener recovery of anthocyanins from purple sweet potato. Acknowledgements The research was financially supported by the Key Research and Development Program of Zhejiang province (2018C02049) and Hangzhou Science and Technology Development Program (20180432B31). Conflict of interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.01.017. References Antti, G., Pentti, P., & Hanna, K. (2008). Ultrasonic degradation of aqueous
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