Optimization of Ultrasonic-Assisted Extraction by Response Surface Methodology with Maximal Phenolic Yield and Antioxidant Activity from Acer truncatum Leaves

This study was designed for the first time to improve phenolic yield and antioxidant activity of ultrasonic-assisted extraction from Acer truncatum leaves (ATL) using response surface methodology, and phenolic composition in ATL extracted under the optimized condition were characterized by UPLC-QTOF-MS/MS. Solvent and extraction time were selected based on preliminary experiments, and a four-factors-three-levels central composite design was conducted to optimize solvent concentration (X1), material-to-liquid ratio (X2), ultrasonic temperature (X3) and power (X4) for an optimal total phenol yield (Y1) and DPPH• antioxidant activity (Y2). The results showed that the optimal combination was ethanol: water (v:v) 66.21%, material-to-liquid ratio 1:15.31 g/mL, ultrasonic temperature 60 °C, power 267.30 W, and time 30 min with three extractions, giving a maximal total phenol yield of 7593.62 mg gallic acid equivalent /100 g d.w. and a maximal DPPH• antioxidant activity of 74241.61 μmol Trolox equivalent/100 g d.w.. Furthermore, 22 phenolics were first identified in ATL extract obtained under the optimized conditions, indicating that gallates, gallotannins, quercetin, myricetin and chlorogenic acid derivatives were the main phenolic composition in ATL. What’s more, a gallotannins pathway existing in ATL from gallic acid to penta-O-galloyl-glucoside was interpreted. All these results provided practical information aiming at full utilization of phenolics in ATL, together with fundamental knowledge for further research.


Introduction
Acer truncatum is a prominent maple (Aceraceae) species widely cultivated in China, Korea and Japan, and is also found in Europe and Northern America [1,2].In northern China, maple leaves, mainly A. truncatum leaves (ATL), are often used as healthy drinks and folk medicines treating coronary artery cirrhosis, cerebrovascular diseases and angina pectoris [3].Previous investigations of ATL indicated that it possessed various biological functions, such as antioxidant [4], antibacterial [5,6], and antitumor [7,8] due to its high contents of tannins, flavonoids, and chlorogenic acid.However, little effort has been made to the optimization of extraction method of phenolics in ATL.Therefore, in order to ensure the full utilization of ATL, an ultrasound-assisted extraction (UAE) method was established through response surface methodology (RSM) in the current study.
UAE is well recognized to exhibit positive effects on the rate of various extraction processes in food, pharmaceutical and cosmetic industries.Comparing to other conventional methods, UAE offers a net advantage in terms of productivity and selectivity with shorter processing time, enhanced quality, reduced chemical and physical hazards, and it is environmentally friendly and causes less damage to the structural and molecular properties of compounds in plant materials [9,10].For these reasons, UAE from plant materials has been widely used lately to facilitate extractions of phenols from Chrysanthemum morifolium flower head [10], Olea europaea fruit [11], Morus nigra must [12], and Curcuma longa rhizome [13], lipids from rice [14], polysaccharides from pumpkin [15] and mycelial fermentation of Phellinus igniarius [16], proteins from Jatropha curcas seed [17], lignans from Schisandra chinensis fruit [18] and other value-added compounds from various natural resources.However, to the best of our knowledge, no effort is available regarding improvements of phenolic yield by itself, let alone the phenolic yield together with antioxidant activity from ATL by ultrasonic stimulation.Thus the present study could provide economic information for the industrial manufacture of ATL as phenolic and antioxidant resource.Nevertheless, the extraction yield of chemical constituents from crude plant materials was affected by many factors.When utilizing an UAE method, factors such as extraction solvent, extraction time, material-to-liquid ratio, ultrasound power and extraction temperature were generally considered to have significant effects on the extraction efficiency.Therefore, when a new UAE method for ATL is developed, optimization of these extraction conditions is indispensable for the best extraction effect.
Being a valuable tool to investigate the interaction among factors and quantitatively depict the effects of given parameters on their measured responses [19], RSM is a collection of statistical and mathematical methods for developing, improving, and optimizing a process [20].In comparison with single variable optimization methods, RSM is a time-and cost-effective means of simultaneously evaluating interactions as well as the key experimental parameters [21], thus have been wildly applied to optimize many bioprocesses [17,[22][23][24].Central composite design (CCD) used in RSM, is more superior comparing with the classical approach in terms of the comprehensiveness of information gained and the accuracy of the experiment conducted [22,25], thus is applied in the present study to optimize the UAE process.
LC-MS/MS technique is gaining increasing interest in the characterization of phenolic compositions due to its high selectivity and sensitivity [26][27][28].For example, Melguizo-Melguizo et al. [29] analyzed 22 compounds from Artemisia vulgaris leaves, 15 of them were phenolics; and Kolniak-Ostek [30] tentatively identified 65 phenolic components in ten pear cultivars.Therefore, the variety of phenolics in ATL extract, determined with UPLC-QTOF-MS/MS analysis under the optimized extraction condition, was analyzed to investigate the phenolic composition as well as the quality of the extraction.
Overall, the present study was designed to optimize the UAE process with RSM and CCD for the maximum phenolic yield and antioxidant activity from ATL.And phenolic composition of the ATL extract obtained under the optimized UAE condition was analyzed with the UPLC-QTOF-MS/MS technique.These results would contribute to the base-line data for industrial manufacture as well as further exploitation of ATL as a phenolic and antioxidant resource.

Solvent types
Phenolic yield obtained with different extraction solvent [water, methanol, ethanol, methanol: water (1:1, v:v), or ethanol : water (1:1, v:v)] was firstly determined to select the best solvent (combination).Results show that different solvents significantly affected phenolic yield (Figure 1A).The phenolic yield with methanol (5112.17GAE/100 g d.w.) was slightly higher than that with ethanol (5049.23),and both of them were significantly higher than that with water (602.63).However, when ethanol was combined with water at the ratio of 1:1 (v:v), phenols extracted was significantly increased to 7262.61.This result was consistent with several previous studies that aqueous organic solvents exhibit a higher extraction efficiency than absolute organic solvents [33][34][35].Cujic et al. [36] explained this phenomenon as water takes response for swelling of plant material while ethanol is responsible for disrupting the bonding between the solutes and plant matrix, and just this synergistic effect leads to a higher phenolic yield.However, phenolic yield with methanol: water (1:1, v:v) exhibited a worse result (2369.13)comparing with that extracted merely with methanol.It might resulted from its unappropriated polarity for extraction targeting phenols in ATL.Therefore, ethanol but not methanol, and aqueous ethanol, instead of pure ethanol, was selected as the best solvent for phenolic extraction.

Solvent concentration
Aqueous ethanol of different concentrations was used to conduct the single factor analysis under the following uniform conditions for material-to-liquid ratio, extraction temperature, ultrasonic power, and extraction time at 1:20 g/mL, 50 °C, 240 W, and 30 min, respectively.As shown in Figure 1B, phenolic yield increased from 6850.04 mg GAE/100 g d.w. to 7329.02 at ethanol concentration between 50% and 60%, and to the maximum (7388.07)at 70%.The efficiency then decreased with further increase of ethanol concentrations, and reach the minimum (5026.35)at 100%.Therefore, it can be concluded that the aqueous ethanol exhibited the closest polarity to the phenols in ATL at the range between 60 and 80% as the efficiency reached the maximum value with ethanol concentration at 70%.Thus, the variable range of ethanol concentration used in the RSM experiments was 60-80%.

Material-to-liquid ratio
Phenolic yields with different material-to-liquid ratios were evaluated in the single factor analysis under the following uniform conditions for aqueous ethanol concentration, extraction temperature, ultrasonic power, and extraction time at 70%, 50 °C, 240 W, and 30 min, respectively.As shown in Figure 1C, phenolic yield increased slightly from 6772.95 mg GAE/100 g d.w. to 6858.25 at ratios between 1:5 and 1:10, and much rapidly to the maximum (7320.00)at 1:20.The efficiency declined at ratios between 1:20 and 1:30, then increased to a second peak at 1:40 and reached the minimum (5086.68)at 1:50.Based on the mass transfer principles occurred in UAE, the driving force for mass transfer is considered to be the concentration gradient between the solid and solvent [37].These results revealed that the optimal ratio leading to the strongest driving force was 1:20 g/mL for phenolic extraction in ATL.Thus, the variable range of material-to-liquid ratio used in the RSM experiments was determined as 1:15 -1:25 g/mL.

Extraction temperature
To study the effect of extraction temperature on phenolic yield, UAE was implemented at different temperatures under the following uniform conditions for ethanol concentration, materialto-liquid ratio, ultrasonic power, and extraction time at 70%, 1:20 g/mL, 240 W, and 30 min, respectively.As shown in Figure 1D, phenolic yield increased from the minimum (6840.00mg GAE/100 g d.w.) at 30 °C to the maximum (7376.17)at 50 °C, decreased to 6890.21 at 60 °C, then slightly increased up to 80 °C thereafter with no significant differences between 60 and 80 °C.These results suggest that a relative high temperature increased phenolic extraction efficiency as it increased the number of cavitation nucleus formed [38], leading an enhanced mass transfer and therefore a better access of solvent to cell components [39].However, this increase declined, namely, brought a negative effect on the efficiency when the temperature exceeded a certain value.This was mainly due to the decreased cavitation intensity under increased temperatures [40,41].In addition, the accelerated evaporation would also be induced at high temperatures, which led to the decrease of driving force [42].The yield affected by temperature with the same pattern in a UAE process were previously reported, such as phenols from olive fruit [11], oil from pomegranate seed [42], and carotene from citrus peels [43].Thus, an extraction temperature range from 40 to 60 °C was chosen in RSM process of ATL.

Ultrasonic power
The effect of ultrasonic power on phenolic yield was determined under the following uniform conditions for ethanol concentration, material-to-liquid ratio, extraction temperature, and extraction time at 70%, 1:20 g/mL, 50 °C, and 30 min, respectively.As shown in Figure 1E, phenolic yield increased from 6693.94 mg GAE/100 g d.w. at 150 W to the maximum (7327.12)at 240 W. The yield decreased to the minimum (6456.36)at 270 W, then increased to 6959.13 at 300 W.There exhibited no significant differences among 150, 180, and 210 W or between 270 and 300 W. According to a report by Hemwimol et al. [44], with the larger amplitude ultrasonic wave traveling through extraction solvent, more bubbles would be created, and more collapse and violent shock wave and high-speed jet might be generated to disrupt the cell walls.Therefore, the penetration of extraction solvent into cells became stronger, resulting in more release of target components from cells into the mass medium.Consequently, the mass transfer rate was thus enhanced and led to an appropriate increase in yield.However, extraction efficiency tended to decrease when ultrasonic power was higher than 240 W, and this might be caused by degradation of bioactive compounds and overproduced bubbles which could hamper the propagation of ultrasound waves at a too high ultrasonic power [45].Thus, an ultrasonic power range from 210 to 270 W was chosen in RSM process of ATL.

Extraction time
The effect of extraction time on phenolic yield was determined under the following uniform conditions for ethanol concentration, material-to-liquid ratio, extraction temperature, and ultrasonic power at 70%, 1:20 g/mL, 50 °C, and 240 W, respectively.As shown in Figure 1F, a biphasic increase in phenolic yield was observed with increases of the extraction time.Phenolic yield increased from 6760.65 mg GAE/100 g d.w. to 7421.41 with the sonication time prolonged from 10 to 30 min, then declined to the lowest (6411.25)at 60 min, and the yield increased again to the maximum (7458.61)at 120 min.There was no significant difference between phenolic yields at 10 and 30 min.The sonication time was recognized as a significant factor increasing the extraction efficiency on UAE process, however, extraction efficiency decreased in some cases due to a prolonged sonication time as it will lower the permeability of solvent into the cell walls due to oversuspended impurities [14].Moreover, prolonged extraction time may increase the chances of decomposition of phenolics [46] and also potentially increase the loss of solvent by vaporization [47], which can directly affect the loss of mass transfer during extraction.Considering energy-saving, 30 min was chosen to conduct all the experimental design below.

Statistical analysis and model fitting
To optimize the operating parameters, all 30 random sequential experiments were performed under designed UAE conditions based on ranges of every single factors (independent variable) determined above to study the reciprocal influence of independent variables (i.e., solvent concentration, material-to-liquid ratio, extraction temperature and ultrasonic power) on the two dependent (response) variables (i.e., phenolic yield and its corresponding antioxidant activity).The experimental values, together with predicted values obtained by their response surface central composite design, were presented in Table 1.Where, X1, X2, X3, X4, Y1, Y2 were aqueous ethanol concentration, material-to-liquid ratio, extraction temperature, ultrasonic power, response of phenolic yield, response of antioxidant activity, respectively.
The linear effect of solvent concentration (X1) was found to be significant for both response variables while X4 was only significant for phenol yield (Y1), and X2 and X3 were only significant for antioxidant activity (Y2).It can be concluded that solvent concentration was the vital parameter in both responses.However, the quadratic effect (X1 2 , X2 2 , X3 2 ) was found to produce extremely significant (P < 0.01) positive effect on both Y1 and Y2, but X4 2 was only significant for Y2.ANOVA results for multiple regression analysis and response surface quadratic model of Y1 and Y2 were evaluated using the corresponding F and P values (Table 2).F values of Y1 and Y2 were calculated to be 25.95 and 50.34, both leading to a P value < 0.0001, suggesting that both the models were statistically extremely significant.The models' coefficient of determination (R 2 ) were 0.9604 and 0.9792, indicating that more than 96.04% and 97.92% of the response variability is explained, and supporting a good accuracy and ability of the established model within the range limits used [48].Correlation coefficients of 0.9800 and 0.9898 also indicate a good positive correlation between the actual data and the predicted values obtained using CCD.The F-values of Lack of Fit of Y1 and Y2 were 0.6032 and 0.2015, respectively, implying that the Lack of Fit is not significantly relative to the pure error, thus the model can be used to predict the phenols and corresponding antioxidant activity of ATL.In addition, Adj-R 2 , Pre-R 2 and the coefficient of variation (C.V.) were calculated to check the model's adequacy.Pre-R 2 of Y1 and Y2 were 0.8341 and 0.8968, which were in reasonable agreement with their Adj-R 2 of 0.9233 and 0.9597, respectively (Adj-R 2 − Pre-R 2 < 0.2), indicating a high degree of correlation between the measured and predicted data from the regression model [49].The C.V. expressed the standard deviation as a percentage of the mean, and was found to be 1.5169% (< 5.00%) for the phenolic yield, and 2.3491% (< 5.00%) for antioxidant activity, implying that the models were reproducible [24].Adequate precision measures the signal to noise ratio, which is desirable when the value is larger than 4. The ratios of 22.3775 and 28.1801 both referred an adequate signal and illustrated that the models (Y1 and Y2) were applicative for the present UAE process [50].These correlation analyses between predicted values and actual data can be used to evaluate the suitability of the response surface model [51].Thus, the model can be used to predict the phenolic yield and corresponding antioxidant activity under various extraction conditions during UAE process.To further verify the models obtained from RSM, ATL was extracted under the predicted optimal UAE conditions, and its phenolic yield and antioxidant ability were evaluated and compared to the predicted maximum yield.For operational convenience, the optimal parameters were modified slightly in the verification experiment as follows: solvent concentration 66.20%, material-to-liquid ratio 1:15.30g/mL, ultrasonic power 270.00 W, temperature 60 °C, and extraction time 30min.The predicted phenolic yield and antioxidant activity under the optimal conditions were 7589.19 mg GAE/100 g and 74010 μmol TE/ 100 g, and the experimental values under the optimal conditions were 7579.56 ± 354.44 mg GAE/100 g and 73585.78± 790.74 μmol TE/ 100 g, respectively.No significant differences were observed between predicted and experimental values (P > 0.05), indicating that the experimental results confirmed the adequate fitness of the predicted model.

Effect of interactions among variables on phenolic yield and antioxidant activity in ATL
To visualize interactions of two operational parameters on extraction efficiency, the responses were generated as triaxial response surfaces (Figure 2) and planar contour plots (Figure 3).Two variables unshown in the Figures were kept constant at their respective central experimental values and the other two variables presented on the two horizontal axis varied within their experimental ranges in order to understand their main and interactive effects on the dependent variables.Figs.2af and 3a-f show the results of interactive influence of solvent concentration, material-to-liquid ratio, extraction temperature, and ultrasonic power on phenolic yield, while Figs.2g-h and 3g-h exhibited the impact of these variables on antioxidant activity.Figs.2a-c, g-i and 3a-c, g-i show the phenols and antioxidant activities as responses of aqueous ethanol concentration (X1) and the other factors (X2, X3, X4).The extraction efficiencies first increased then decreased with increase of aqueous ethanol concentration from 50% to 90%, material-to-liquid ratio from 1:10 to 1:30 g/mL, ultrasonic power from 180 to 300 W, and extraction temperature from 30 to 70 °C.
The interaction of solvent concentration and material-to-liquid ratio (X1X2) showed extremely significant positive effect (P < 0.001) on both responses (see Table 2).At lower ethanol concentration with increasing material-to-liquid ratio, total phenols and antioxidant activity kept generally mild.However, the total phenols with antioxidant activity first increased, then decreased at lower material- to-liquid ratio with increasing ethanol concentration (Figures 2a and g and 3a and g).The possible interaction mechanism between solvent concentration and material-to-liquid ratio might be interpreted as that the positive interaction caused by appropriate solvent concentration and materialto-liquid ratio affected the polarity and viscosity of aqueous ethanol and solubility of target phenolic compounds in the extraction solvent, thus influenced the yield of phenols.
As shown in Figures 2f and 2l, and 3f and 3l, the interaction between sonication power and temperature (X3X4) also showed significant positive effect (P < 0.05) on total phenols and antioxidant activity (Figures 2f and 3f).The possible interaction mechanism between temperature and power may be the change of cavitation threshold affected by changing temperature, which is responsible for acoustic cavitation and also results in the formation of a cavitational nucleus.The influence of relatively greater forces ruptures and erupts the formed cavitational nucleus and disrupts the cell tissues during extraction, which in turn enhances the mass transfer rate [39].
With respect to antioxidant activity (Y2), the interaction effect of solvent concentration and sonication power (X1X4) was also found to be significant (P < 0.05).According to the study of Hemwimol et al. [44], only a small fraction of the electric energies from the ultrasound actually entered the extraction solvent in the ultrasonic bath system, and most of them were absorbed by the water in the bath.Under this circumstances, the rise of the solvent concentration might increase the utilization efficiency of the limited electric energies, then play a vital role in the improvement of the extraction yield.

Characterization of phenolic compositions in ATL
Table 3 shows a list of 29 phenolic compounds identified in ATL prepared under the optimal extraction conditions obtained above through UPLC-QTOF-MS/MS experiments, along with their retention times (RT), experimental m/z, calculated m/z, error values (ppm), molecular formula and MS/MS fragments.As signals in negative mode was stronger than those in the positive mode, data collected in negative mode was thus chosen to conduct the identification.Mass spectra in negative ion mode (Figure 4A), together with MS (Figure 4B) and MS/MS (Figure 4C) fragments of the peak with retention time at 26.51 min were presented as follows.

Gallates and gallotannins derivatives
Compound 2 (RT = 4.03 min) was identified as theogallin (a galloylquinic acid) with a [M-H] − ion at m/z 343.0654.It has a major fragment at m/z 191 (quinic acid) due to the loss of a galloyl moiety (152 amu) and another fragment at m/z 169 (gallic acid) due to the loss of a part of quinic acid moiety (174 amu).Compound 3 (4.29 min) gave a [M-H] − ion at 169.0143 and a major fragment ion at m/z 125 caused by the loss of a -CO2 group, and was identified as gallic acid.Compound 4 was assigned as methyl gallate with a [M-H] − ion at m/z 183.0323 and a major fragment ion at m/z 124 due to the loss of -CO2CH3 (59 amu).The parent ions and MS/MS profiles of these three gallates accorded with the data reported in the literature [52].
Moreover, the gallic acid ion at m/z 169 occurred in not only compounds 2 and 3, but compounds 16, 21, 22, and 26, indicating that all these compounds share the gallic acid moiety.Compound 16 (21.47 min) with deprotonated ion at m/z 787.1001 was identified as tetra-O-galloyl-glucoside. It fragmented at m/z 615 with a loss of one gallic acid moiety (170), and fragmented at m/z 465 with another loss of a galloyl (152).Meanwhile, compounds 21 gave [M−H] − ion at m/z 939 with molecular formula C41H32O26.It fragmented at m/z 769 with a loss of a gallic acid moiety (170), and fragmented at m/z 617 with another loss of galloyl (152) (Figure 4B and 4C).Thus compound 21 (26.51 min) was identified as a penta-O-galloyl-glucoside isomer.Possessing the same parent ion and fragments, compound 22 (28.23 min) was identified as another penta-O-galloyl-glucoside isomer.One of them should be the 1,2,3,4,6-pentakis-O-galloyl-β-D-glucose identified in ATL before [53].Compound 26 (56.11 min) with deprotonated ion at m/z 1091.12 was identified as hexa-O-galloyl-glucoside.It fragmented at m/z 939 and 769 following the mode described above.The parent ions and fragmentation mode of these gallotannins were reported in the literature [54].
Accordingly, we hypothesized that there is a gallotannins pathway existing in the ATL, leading to the synthesis from gallic acid to tetra-O-galloyl-glucoside, penta-O-galloyl-glucoside and hexa-Ogalloyl-glucoside. Grundhöfer et al. [55] elucidated the pathway forming complex gallotannins in Rhus typhina through enzyme studies.They reported the detailed converting process from gallic acid to β-glucogallin, digalloyglucose, trigalloyglucose, tetragalloyglucose, pentagalloylglucose, and hexagalloyglucose.As most of galloylglucoses mentioned above were also found in ATL, we conjectured the gallotannins pathway existing in ATL might be quite similar with the one in Rhus typhina.In addition, 1,2,3,4,6-pentakis-O-galloyl-β-D-glucose was the common precursor of two subclasses of hydrolyzable tannins, gallotannins and the related ellagitannins [52].Thus the pathway from gallic acid to the penta-O-galloyl-glucoside might be the tip of the iceberg.Unfortunately, gallotannins with higher degree of polymerization than hexagalloyglucose was not found in the present study.
Compound 11 (16.39 min) was identified as catechin, as its molecular ion was at m/z 289.0717.It fragmented at m/z 245 and 211, corresponding with its MS/MS data in the literature [26].Compound 27 (60.30min) possessed a deprotonated ion at m/z 431.0974, and was identified as kaempferol-3-Orhamnoside [58].Its fragment ion at 285 was assigned as the kaempherol moiety.

Chlorogenic acid derivatives
Compound 1 (2.35 min) with deprotonated ion at m/z 191.0564 was identified as quinic acid by comparing its MS/MS data with the literature [29].Quinic acid is a cyclic polyol, instead of phenol itself, however, it is an important part consisting caffeoylquinic acid derivatives.Compounds 4 (6.91 min) and 5 (7.85 min) were found to share a deprotonated ion at m/z 353, indicating the presence of monocaffeoylquinic acid isomers.According to Ncube et al. [62] and Melguizo-Melguizo et al. [29], the common base peak in their MS/MS spectra at m/z 191 illustrated the presence of quinic acid moiety, and the fragment ion at m/z 179 occurred in compound 4 indicated that caffeoyl group is linked to the 3-OH position of quinic acid.Therefore, compound 4 was identified as 3-Ocaffeoylquinic acid while compound 5 was identified as 5-O-caffeoylquinic acid due to their different fragmentation pattern.
Compounds 7 (12.64 min) and 8 (12.93 min) were identified as p-coumaroylquinic acid isomers as they shared a deprotonated ion at m/z 337.According to Ncube et al. [62], trans-and cis-5-pcoumaroylquinic acids possess a base peak at m/z 191, while trans-and cis-4-p-coumaroylquinic acid own one at m/z 173.As they shared a base peak at m/z 191 in the present study, and considering their reported eluting order, compounds 7 and 8 were identified as trans-and cis-5-p-coumaroylquinic acids, respectively.

Other phenolic compounds
Compound 6 (11.05 min) with a molecular ion at m/z 285.0612 was identified as uralenneoside as it fragmented at m/z 153 due to the loss of hydroxybenzoic acid unit and at m/z 109 because of another loss of a CO2 group from the carboxylic acid moiety, and its MS/MS data were accordance with the fragmentation pattern reported by Yu et al. [63].Compound 12 (16.91 min), with deprotonated ion at m/z 863.1819, was identified as cinnamtannin B1 as it consisted of three epicatechin units, which was reflected by the daughter ion at m/z 289 [64].
Twenty Acer truncatum Bunge trees, whose tree-age (around 15 years old) and growth environment were approximatively identical, were randomly selected in Bajiajiaoye Park, Haidian District (N 40°00'57.21'';E 116°19'43.36''with altitude 39-41 m) and authenticated by associate professor Zhonghua Liu, Beijing Forestry University, Beijing, China. A. truncatum leaves (ATL) were uniformly collected from the selected trees in April 20, 2015 and taken back to laboratory immediately.The leaves were cleaned with distilled water and air-dried, the dried leaves were grinded and passed a 250 × 250-μm sieve, and the powder was stored at -20 °C in refrigerator for extraction.

Preliminary experiments
To test the impact of solvent type on phenolic yield, 1.000 g leaf powder was mixed thoroughly with different solvents, including water, methanol, ethanol, methanol: water (1:1, v:v) or ethanol: water (1:1, v:v) at a material-to-liquid ratio (g/mL) of 1:20 in a plastic centrifuge tube.The tube was then immersed into a tunable ultrasonic cleaning bath (KQ-300DE type, Kunshan Ultrasonic Instrument Co., Ltd., China) with the liquid level in the tube keeping lower than that of the cleaner tank, and extracted under ultrasonic conditions at 50 °C, 240 W and 30 min.The sample mixture was then centrifuged for 10 min at 6000 rpm and the supernatant was collected.The resulting residue were repeated for extraction twice more, and all the supernatants were combined, filtered, and diluted to a final volume of 60 mL.The resulting solutions were analyzed for total phenols.The optimal solvent with the highest total phenol content was selected for the following experiments.

RSM experiment
After the single factor tests, RSM with CCD was applied to estimate the effect of independent variables (i.e., X1, extraction temperature; X2, ultrasonic power; X3, solvent concentration; X4, liquidto-material ratio) and their interactions on UAE of phenolic yield (Y1) and antioxidant activity (Y2).Based on preliminary single factor analysis and literature data, levels of independent parameters were selected and coded at five levels according to Eq. (1): Where xi and Xi are the coded and actual values of an independent variable, respectively.X0 is the actual value on the center point of Xi, and ΔXi is the value of the step change.The design values of independent variables and their coded values are represented in Table 4.In the present study, CDD conducted 30 experimental points including six replicates at the central point, sixteen factorial points and eight axial points for a full factorial design to study the effect of independent variables on the response.The experiments were randomized and the response values in each trial were analyzed using Design-Expert (Version 8.0.6,Stat-Ease Inc., Minneapolis, USA) and fitted to a second-order polynomial regression model expressing mathematical relationship between independent variables (X1, X2, X3 and X4) and responses (Y1 and Y2): where, Y (Y1 or Y2) is the predicted response, Xi and Xj are diverse input variables that influence the response variable Y, β0 is the constant coefficient, βi is the linear coefficient, βij is the interaction coefficient of two factors (Xi and Xj), and βii is the quadratic coefficient of one factor (Xi 2 ).The obtained model was verified by comparing the phenolic yield and antioxidant ability of the ATL extract obtained under the optimal UAE conditions to those predicted by the models.Furthermore, the resulting solution was analysis by UPLC-QTOF-MS/MS for its phenolic compositions.

Measurement of total phenols
Total phenols were determined according to the Folin-Ciocalteu method [31] with slight modifications.In brief, 40 μL of 25% Folin-Ciocalteu solution was added to designated wells of a 96well microplate, followed by addition of 20 μL standard (10-400 mg/L gallic acid, R 2 = 0.999), ATL extract solution or blank (MilliQ water).After blending, 140 μL of 700 mM Na2CO3 solution was added to each well, and the plate was shaken for 5 min at 250 rpm and incubated in dark at 40 °C for 30 min, followed by absorbance measurement at 765 nm with a microplate reader (Bio-Rad xMark™ Microplate Absorbance Spectrophotometer, USA).Results were expressed as mg gallic acid equivalent (GAE)/100 g d.w. of ATL powder.

Determination of DPPH• scavenging activity
Assays of DPPH• scavenging activity were performed using the method described by Alañón et al. [32] with slight modifications.In brief, 10 μL standard (0-400 mg/L, R 2 = 0.998), ATL extract solution or blank was added to a designated well of a 96-well microplate.Subsequently, 40 μL of 1 mM freshly prepared DPPH solution was added to each well followed by addition of 190 μL methanol, and the plate was then stood in an orbital shaker setting at 200 rpm for 1 min.After 30 min incubation at room temperature in dark, absorbance was recorded at 517 nm using the microplate reader.Results were expressed as μmol Trolox equivalent (TE)/100 g d.w. of ATL powder.

Statistical analysis
Experiment design was performed using Design-Expert (Version 8.0.6,Stat-Ease Inc., Minneapolis, USA).All experimental results obtained were expressed as means ± SD, and data were analyzed by analysis of variance (P < 0.05) using SPSS software (Version 22.0, SPSS Inc., Chicago, IL, USA).All analyses were performed in triplicate.

Conclusions
The current study is the first report on the ultrasonic stimulation for phenolic yield and antioxidant activity in ATL.The quadratic model well fitted both responses, i.e., total phenol yield and antioxidant activity.Operating parameters were optimized using single factor experiments and response surface design.Optimal conditions were found to be percentage of aqueous ethanol 66.21%, material-to-liquid ratio 1:15.25 g/mL, extraction temperature 60 °C; ultrasonic power 270 W, and ultrasound time 30 min, which gave a maximum total phenol yield of 7593.62 mg GAE equivalent /100 g d.w. and antioxidant activity of 74241.61μmol TE/100 g d.w..And the verified results confirmed the adequate fitness of the predicted model.Furthermore, 22 phenolic compounds were first discovered in the present study with UPLC-QTOF-MS/MS analysis, which indicated the superiority of the extraction condition and provided new insights to the phenolic compositions of ATL.

Figure 2 .
Figure 2. Response surfaces showing the effects of four variables (solvent concentration, material to liquid ratio, ultrasonic power, and extraction temperature) and their interactions on extraction yield of total phenols (TP, a-f) and their DPPH• scavenging capacity (g-l).The other two variables in each of the Figures were kept constant at their respective central experimental values.

Figure 3 .
Figure 3. Contour plots showing the effects of four variables (solvent concentration, material to liquid ratio, ultrasonic power, and extraction temperature) and their interactions on extraction efficiency of total phenols (TP, a-f) and their DPPH• scavenging capacity (g-l).The other two variables in each of the Figures were kept constant at their respective central experimental values.

Figure 4 .
Figure 4. UPLC-QTOF-MS/MS data of the ATL extract obtained under the optimized conditions.Besides the UPLC-MS profile (A), a peak with the retention time at 26.51 min was identified as pentagalloyl glucose based on its MS (B) and MS/MS (C) fragments.

Table 1 .
Response surface central composite design and experimental data and predicted values for extraction yield of phenols in ATL.Upon applying multiple regression analysis to the experimental data, the final quadratic equation obtained in terms of actual factors was given below.

Table 2 .
Analysis of variance for the extraction of phenols yield (Y1) and antioxidant capacity (Y2)., the levels of phenolic yield ranges from 5783.04 to 7591.30mg GAE/100 g d.w., and the levels of antioxidant activity ranges from 42030.84 to 73849.78 μmol TE/100 g d.w..The * 0.01 ≤ P < 0.05; ** P < 0.01 As shown in Table1

Table 4 .
Independent variables and their levels and corresponding coded values used in CCD.