Ultrasound-assisted extraction of polyphenols from native plants in the Mexican desert

Ultrasonics Sonochemistry 22 (2015) 474–481

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Ultrasound-assisted extraction of polyphenols from native plants in the Mexican desert Jorge E. Wong Paz a, Diana B. Muñiz Márquez a, Guillermo C.G. Martínez Ávila b, Ruth E. Belmares Cerda a, Cristóbal N. Aguilar a,⇑ a b

Department of Food Science and Technology, School of Chemistry, Autonomous University of Coahuila, 25280 Saltillo, Coahuila, Mexico Laboratory of Biotechnology, Autonomous University of Nuevo Leon, 66050 General Escobedo, Nuevo Leon, Mexico

a r t i c l e

i n f o

Article history: Received 27 December 2012 Received in revised form 19 March 2014 Accepted 2 June 2014 Available online 24 June 2014 Keywords: Ultrasound-assisted extraction Extracts Antioxidants Environmentally friendly Alternatives techniques

a b s t r a c t Several plants that are rich in polyphenolic compounds and exhibit biological properties are grown in the desert region of Mexico under extreme climate conditions. These compounds have been recovered by classic methodologies in these plants using organic solvents. However, little information is available regarding the use of alternative extraction technologies, such as ultrasound. In this paper, ultrasoundassisted extraction (UAE) parameters, such as the liquid:solid ratio, solvent concentration and extraction time, were studied using response surface methodology (RSM) for the extraction of polyphenols from desert plants including Jatropha dioica, Flourensia cernua, Turnera diffusa and Eucalyptus camaldulensis. Key process variables (i.e., liquid:solid ratio and ethanol concentration) exert the greatest influence on the extraction of all of the phenolic compounds (TPC) in the studied plants. The best conditions for the extraction of TPC involved an extraction time of 40 min, an ethanol concentration of 35% and a liquid:solid ratio ranging from 8 to 12 ml g1 depending on the plant. The highest antioxidant activity was obtained in the E. camaldulensis extracts. The results indicated the ability of UAE to obtain polyphenolic antioxidant preparations from desert plants. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Mexico is one of the five richest countries in a large variety of plants in the world. Mexico contains 25,000 registered species and approximately 30,000 species that have not yet been described [1,2]. In addition, more than 3500 native medicinal plant species have been identified and classified [3]. In this context, the desert region in northern Mexico is important due to the great variety of plants that have developed the ability to grow under extreme climate conditions with a variety of chemical compounds that are used as defence mechanisms [4]. Desert scrub covers three semiarid areas with the greatest biodiversity in the world [5]. Several studies have been performed using plant species from the Mexican desert to study the potential biological activities of these plants as inhibitors with pathogenic bacterial [6], postharvest fungal [7–12] and antioxidants properties [13,14]. These compounds have been extracted using conventional techniques, such as maceration, infusion or reflux, that exhibit disadvantages, such as high solvent requirements, long extraction ⇑ Corresponding author. Tel.: +52 844 4161238; fax: +52 844 4159534. E-mail address: [email protected] (C.N. Aguilar). http://dx.doi.org/10.1016/j.ultsonch.2014.06.001 1350-4177/Ó 2014 Elsevier B.V. All rights reserved.

times and risks of degradation of the thermo-labile constituents [15,16]. In addition, the solvents commonly employed include methanol, hexane, and acetone, and in recent years, unconventional solvents, such as lanolin and cocoa butter, as well as organic solvents, such as water and ethanol [4,17], have been used, showing the same effects against bacterial and fungal pathogens. Therefore, within the context of being environmentally friendly and sustainable, alternative technologies and solvents are being studied to perform extractions at same or lower cost while increasing the extraction quality [18,19]. Various novel extraction techniques have been investigated that resolve some of the shortcomings of the conventional techniques. Ultrasound-assisted extraction (UAE) is an example of an alternative extraction technique [20]. UAE offers many advantages, such as selectivity, high efficiency and productivity, enhanced quality, low energy, reduced extraction time and solvent consumption, reduced chemical and physical hazards, environmentally friendly, inexpensive and high level of automation, compared to conventional extraction techniques [21–24]. Research on antioxidants has received increasing interest because natural antioxidants are gaining importance due to their benefits for human health compared to synthetic antioxidants,

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which can produce serious side effects [25]. Polyphenols are the major plant compounds associated with healthy properties due to their antioxidant activity and free radical scavenging ability [26,27], and several studies have demonstrated their antimicrobial activity, which make them a good alternative to chemical preservatives [28]. For example, desert Mexican plants are rich in polyphenols (i.e., tannin) [4,17]. To the best of our best knowledge, there are no reports on the use of UAE to extract phenolic compounds from semiarid Mexican plants, and only poor information about the antioxidants is known. Therefore, the aims of the current study were to study the extraction efficiency of UAE of polyphenolic compounds from the Jatropha dioica, Flourensia cernua, Turnera diffusa and Eucalyptus camaldulensis plants and to evaluate the antioxidant potential of the obtained extracts. In UAE, several extraction variables, such as ultrasonic power, temperature, sonication time, and solvent concentration and ratio, could be explored. In the current research, we chose to study the extraction time, liquid–solid ratio and ethanol concentration to evaluate the feasibility of the method. 2. Materials and methods 2.1. Reagents and vegetable materials The J. dioica stem and root, F. cernua stem and leaves, E. camaldulensis leaves and T. diffusa stem and leaves were collected from January to June 2011 in places near Saltillo City, Coahuila, Mexico. The plants were dried (48 h at 60 °C, Oven LABNET International, Inc.), pulverized into powder and sieved (0.6–0.8 mm particle size). The fine and dried powder was stored at room temperature in hermetically sealed bags under darkness prior to use. The acetonitrile (ACN), acid acetic, methanol and ethanol solvents were of analytical grade. The standards used (i.e., pyrogallol (PG), gallic acid (GA), resorcinol (RS), chlorogenic acid (CHA), methyl gallate (MG), coumaric acid (CUA), catechin (CAT), 2-hydroxycinnamic acid (HA), ellagic acid (EA), quercetin (QE), cinnamic acid (CA) and 1,1-diphenyl-2-picrylhydrazil (DPPH) free radical, linoleic acid, Folin–Ciocalteu (FC) reagent) were purchased from Sigma–Aldrich.

liquid–solid ratio and ethanol concentration (Table 1). All of the treatments were performed in triplicate. Response surface methodology (RSM) is a collection of statistically based experimental designs that have been established as a convenient method for optimizing several processes [29]. Therefore, RSM was used to analyse the results along with the Statistica software (Statsoft version 7.0). The calculations were performed at the 95% confidence level. The optimal extraction conditions were estimated with the regression analysis performed on the data for the dependent variable obtained and were fitted to an empiric polynomial model, as shown in the following general equation:

Y ¼ b0 þ b1 x1 þ b2 x2 þ b3 x3 þ b11 x21 þ b22 x21 þ b22 x22 þ b33 x23 þ b12 x1 x2 þ b13 x1 x3 þ b23 x2 x3 where y is the predicted response, b0 is the interception, b1, b2 and b3 are the linear coefficients for the ethanol concentration (x1), ultrasonic time (x2) and liquid–solid ratio (x3), respectively, b11, b22 and b33 are the squared coefficients for the ethanol concentration, ultrasonic time and liquid–solid ratio, respectively, and b12, b13 and b23 are the interaction coefficients for the ethanol concentration, ultrasonic time and liquid–solid ratio, respectively. 2.4. Yield of total phenolic content (TPC) The total phenolic compounds were analysed using the traditional Folin–Ciocalteu method with some modifications [8,30]. First, the traditional method was adapted for small volumes with microplate assays to provide exhaustive standardization for the modified technique (data not shown). Then, 20 lL of each extract (1 mg ml1) was mixed in a well with 20 lL of the Folin and Ciocalteu’s reagent. After 5 min, 20 lL of Na2CO3 (0.01 M) was added to each sample and allowed to stand for 5 min. Next, the solution was diluted with 125 lL of distilled water, and the absorbance was read at 790 nm using a spectrophotometer microplate reader

Table 1 Complete factorial design employed for the extraction of TPC. Run

2.2. Ultrasound-assisted extraction (UAE) The ultrasound-assisted extraction was performed in an ultrasonic bath device (Model 2510, BRANSON). The bath consisted of a rectangular container (34.29 cm  10.16 cm  30.5 cm) with 40 kHz transducers annealed to the bottom. The samples were processed at room temperature. The ultrasonic wave provided a slight increase in the temperature (i.e., between 40 and 50 °C). The powder of the dried plants (4 mg) was placed in a capped tube (100 mL) and mixed with an appropriate amount of the extraction solution (according to experimental design). Next, the tube with the suspension was immersed in water in the ultrasonic device and irradiated for the pre-set extraction time. Then, the obtained extracts were filtered (Whatman No. 41 paper) and centrifuged at 3500 rpm for 10 min. Next, the extracts were dehydrated at 60 °C for 48 h and stored under refrigeration prior to the TPC analysis. For all of the determinations, the extracts were resuspended in water (1 mg of dried extract ml1). 2.3. Optimization of process parameters The extraction of the total phenolic content of the plants by ultrasound was performed by employing various extraction conditions. In this study, a complete factorial (33) with three levels and three factors was applied to determine the best combination of extraction variables. The factors selected included extraction time,

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

Code variables

Decoded variables

X1

X2

X3

Ethanol concentration (%)

Time (min)

Liquid:solid ratio (ml/g)

1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1

1 1 1 0 0 0 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 0 0 0 1 1 1

1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1

0 0 0 0 0 0 0 0 0 35 35 35 35 35 35 35 35 35 70 70 70 70 70 70 70 70 70

20 20 20 40 40 40 60 60 60 20 20 20 40 40 40 60 60 60 20 20 20 40 40 40 60 60 60

4 8 12 4 8 12 4 8 12 4 8 12 4 8 12 4 8 12 4 8 12 4 8 12 4 8 12

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(Epoch, BioTek, Instruments, Inc.) controlled with a Gen5 Data Analysis software interface. The TPC measurements were performed in triplicate, and mean values were expressed as mg gallic acid equivalents g1 of dry plant material according to the standard curve from 0 to 500 mg L1, R2 0.9963.

linear; 18–25 min, 100% B linear. Then, the column was washed and reconditioned. All of the standard solutions of the different phenolic compounds were injected under the same conditions [34].

2.5. Antioxidant activity

3.1. Effect of UAE process and modelling behaviour

2.5.1. DPPH (1,1-diphenyl-2-picryl-hydrazil) radical scavenging activity DPPH free radical-scavenging activity was measured using a modified colorimetric method [31]. The experiment was performed as follows: the methanolic solution containing the free radical DPPH (193 lL, 60 lM) was added to the sample extract (7 lL) and allowed to stand in the dark for 30 min at room temperature. Then, the absorbance was measured at 517 nm using a spectrophotometer microplate reader (Epoch, BioTek, Instruments, Inc.) controlled with a Gen5 Data Analysis software interface. The free radical-scavenging activity of the extracts was expressed as a percentage of reduced DPPH and was calculated according to the following equation:

The experimental values of TPC for each set of variable combinations and vegetable material used in this study are presented in Table 2. TPC extraction ranged from 0.70 to 3.32, from 0.52 to 3.10, from 3.9 to 15.71 and from 1.12 to 3.52 (mg g1) for J. dioica, F. cernua, E. camaldulensis and T. diffusa, respectively. To maximize the UAE conditions for the TPC from semiarid plants, regression analysis was performed on the results, and a polynomial equation was derived using the significant values of the estimated regression coefficients (Table 3). The adequacy of the model to compare the experimental and predicted values was verified using ANOVA and R2 values, which were statistically acceptable at a 95% confidence level. For F. cernua and E. camaldulensis, the fit of the resulting model exhibited a slight loss of goodness of fit (i.e., R2 more than 0.8). For J. dioica and T. diffusa, the fit of the resulting model was low (R2 less than 0.7). It is well known that studies using vegetable material are laborious and the high variation obtained in most of the cases is due to the nature of the material. Among the variables screened, the liquid:solid ratio linear and ethanol concentration quadratic effects were identified as the most significant variables influencing the TPC yield from the extraction in all of the studied plants. Three-dimensional plots (Figs. 1–4) were constructed to show the maximum levels of the variables for the UAE process of TPC according to the fitted model obtained for each vegetable material.

Inhibition of DPPH ð%Þ ¼

ðAc  As Þ  100 Ac

where Ac is the absorbance of the control (0.7 lL of water instead of sample) and As is the absorbance of the sample. 2.5.2. Inhibition of lipid oxidation in a model solution This test is used to measure the extraction potential for the protection or stimulation of linoleic acid oxidation in a model solution [32]. In addition, this test is accurate and closely related to food or biological system. First, the linoleic acid solution was prepared in ethanol (0.56 g of linoleic acid and 1.5 g of Tween 20 in 8 mL of 96% ethanol). Briefly, the linoleic acid solution (100 lL) was mixed with each extract (50 lL) or the control (50 lL of distilled water) and 1.5 mL of a 0.02 M acetate buffer (pH 4.0). The samples were homogenized and incubated at 37 °C for 1 min. After 1 min, 750 lL of a 50 M FeCl2 solution (0.0994 g FeCl2 and 0.168 g EDTA diluted to 1 L with distilled water) were added to initiate the oxidation of linoleic acid. Then, two aliquots (250 lL) were removed at 1 and 24 h after initiating the incubation. Each aliquot was processed instantaneously as follows: the aliquot was added to 1 mL of 0.1 M NaOH in 10% ethanol to terminate the oxidation process followed by dilution with 2.5 mL of 10% ethanol. The absorbance was measured at 232 nm against a 10% ethanol blank. The percent inhibition of linoleic acid oxidation was calculated with the following equation:

Lipid oxidation inhibition ð%Þ ¼

AB  100 A

where A is the difference between the absorbance of the control sample (distilled water) after 24 h and after 1 h of incubation and B is the difference between the absorbance of each extracted sample after 24 h and after 1 h of incubation [33]. 2.6. HPLC analysis The extracts used in the antioxidant activity were injected into an HPLC system (Varian Pro-Star 330), equipped with a DAD detector. The samples (10 lL) were injected onto a Pursuit XRs C18 column (5 lm, 150  4.6 mm, Varian). The temperature of the oven was 30 °C, and the detection was performed at 280 nm. A gradient of A (0.1% acetic acid in water) and B (acetonitrile) was used as the eluent. The following gradient was applied at a flow rate of 1 mL/ min: initial, 0% B; 0–1 min, % B linear; 1–3 min, 10% B linear; 3–8 min, 20% B linear; 8–13 min, 30% B linear; 13–18 min, 50% B

3. Results and discussion

3.2. Maximization of UAE conditions Fig. 1 shows the surface response plots of the J. dioica extracts. The three extraction process factors screened exhibit significant Table 2 Effect of the experimental conditions on the yields of TPC. Run

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

TPC yield (mg g1) J. dioica

F. cernua

E. camaldulensis

T. diffusa

0.70 ± 0.26 1.25 ± 0.40 1.59 ± 0.98 1.31 ± 0.53 2.88 ± 0.63 2.53 ± 1.26 0.76 ± 0.29 1.31 ± 0.44 1.96 ± 0.32 1.18 ± 0.31 2.33 ± 0.76 2.18 ± 0.48 1.67 ± 0.58 2.68 ± 0.36 3.32 ± 0.43 1.11 ± 0.24 1.78 ± 0.08 2.50 ± 0.59 1.02 ± 0.17 1.75 ± 0.22 1.77 ± 0.06 1.42 ± 0.14 2.31 ± 0.63 2.43 ± 0.20 0.92 ± 0.15 1.90 ± 0.12 1.52 ± 0.27

0.65 ± 0.11 0.96 ± 0.23 0.81 ± 0.03 0.61 ± 0.13 1.18 ± 0.40 0.71 ± 0.02 0.52 ± 0.10 0.69 ± 0.07 0.78 ± 0.03 1.46 ± 0.24 2.57 ± 0.57 2.10 ± 0.19 1.93 ± 0.23 2.98 ± 0.41 3.07 ± 0.14 2.10 ± 0.14 2.70 ± 0.28 2.53 ± 0.41 1.49 ± 0.34 2.32 ± 0.38 1.81 ± 0.36 1.67 ± 0.19 3.10 ± 1.36 2.63 ± 0.33 1.88 ± 0.27 2.15 ± 0.10 2.46 ± 0.14

3.90 ± 0.50 7.76 ± 1.31 8.81 ± 0.72 4.36 ± 0.36 8.01 ± 0.30 9.89 ± 0.48 4.30 ± 0.46 7.96 ± 0.82 10.75 ± 0.96 7.56 ± 0.60 11.96 ± 2.10 13.39 ± 1.43 8.24 ± 0.47 11.98 ± 1.20 15.71 ± 0.46 8.24 ± 0.82 12.84 ± 1.10 13.33 ± 0.49 4.76 ± 0.21 7.58 ± 1.34 7.46 ± 0.53 5.76 ± 0.39 8.79 ± 1.16 9.39 ± 0.39 5.43 ± 0.01 7.59 ± 0.90 9.68 ± 3.90

1.56 ± 0.17 2.10 ± 0.28 2.75 ± 0.40 1.12 ± 0.05 2.36 ± 0.45 2.52 ± 0.57 1.94 ± 0.30 2.31 ± 0.33 3.30 ± 0.53 1.74 ± 0.33 2.77 ± 0.52 3.50 ± 0.13 2.44 ± 0.26 2.84 ± 0.60 3.05 ± 0.59 2.21 ± 0.30 3.11 ± 0.76 3.52 ± 0.73 1.36 ± 0.02 1.84 ± 0.31 2.19 ± 0.61 1.65 ± 0.44 2.08 ± 0.01 2.00 ± 0.44 1.53 ± 0.25 2.41 ± 0.25 2.14 ± 0.21

Mean values ± standard deviation (n = 3).

J.E. Wong Paz et al. / Ultrasonics Sonochemistry 22 (2015) 474–481 Table 3 Regression coefficients. Variables

b0 b1 b2 b3 b11 b22 b33 b12 b13 b23

Estimated coefficients J. dioica

F. cernua

E. camaldulensis

T. diffusa

2.8301a 0.0416 0.0013 0.5399a 0.4542a 0.7521a 0.3612a 0.0567 0.0794 0.0459

2.9027a 0.7000a 0.0915 0.2552a 0.9142a 0.3208a 0.4498a 0.1083 0.1109 0.0123

12.4830a 0.0391 0.4846a 2.4487a 3.9295a 0.7078a 1.0968a 0.0301 0.5254a 0.3444

2.8380a 0.1530a 0.1470a 0.5230a 0.7330a 0.1216 0.1735 0.0377 0.1791a 0.0415

(R2) = 66.23%, 82.42%, 83.55% and 69.85% for J. dioica, F. cernua, E. camaldulensis and T. diffusa, respectively. a Significance at p < 0.05.

quadratic effects in response to TPC, which indicated that an increase in the effects near the midpoint resulted in the highest yield in the response when another corresponding factor was fixed. However, the TPC yields in this plant were negatively affected when the ethanol concentration and extraction time level factors were increase beyond the midpoint (Fig. 1a and b). Similarly, the increase in the liquid:solid ratio at a fixed extraction time led to a substantial increase in TPC and reached a maximum at the

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midpoint of the tested liquid:solid ratios. In addition, the increase between the midpoint and the highest level in liquid:solid ratio resulted in a slight decrease in TPC (Fig. 1c). According to the polynomial equation, the maximum TPC yield for J. dioica was predicted to be 3.03 mg TPC g1 vegetable material under extractions conditions involving 35% ethanol, a liquid:solid ratio of 11 ml g1 with an extraction time of 40 min. For the J. dioica plant, the liquid:solid ratio was the most important factor affecting TPC yield. In contrast, in the F. cernua extracts, the ethanol concentration was the most significant factor in the extraction (Table 3). Fig. 2 shows the effects of the variables process on TPC in the F. cernua extracts. Similar to the J. dioica extracts, in the F. cernua extracts, an increase in the ethanol concentration, extraction time and liquid:solid ratio had significant quadratic effects. In addition, only the ethanol concentration and liquid:solid ratio had a significant linear effect. In the plot TPC as a function of extraction time and ethanol concentration, a large increase in response to the increase in the ethanol concentration was observed until the midpoint at a fixed extraction time. An increase in the extraction time at a fixed ethanol concentration also led to an increase in the TPC, which was less substantial (Fig. 2a). According to Fig. 2b and c, TPC yield was affected by the liquid:solid ratio factor, which resulted in a marked increase in the yield with an increase from 1 to 0 (code levels). However, a decrease was observed when the levels were increased from 0 to 1 (code levels). Based on the polynomial equation deduced for the F. cernua extracts, the maximum predicted TPC

Fig. 1. Response surface plots of TPC from the J. dioica extracts as a function of extraction time, ethanol concentration and liquid:solid ratio.

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Fig. 2. Response surface plots of TPC from the F. cernua extracts as a function of extraction time, ethanol concentration and liquid:solid ratio.

yield was 3.07 mg g1 at the critical conditions, which involved 48.3% ethanol and a liquid:solid ratio of 9.12 ml g1 for a 40 min extraction time. As shown in the three-dimensional plots for TPC, in the E. camaldulensis extracts, a quadratic effect in the ethanol concentration was observed resulting in a higher extraction yield at both ends of the ethanol concentration range studied (Fig. 3a). Interestingly, an increased in the liquid:solid ratio resulted in positive strong linear and quadratic effects in the TPC yield (Fig. 3b). In addition, the interaction between both factors (i.e., ethanol concentration and liquid:solid ratio) was significant, indicating that the effect of the ethanol concentration is not the same at all liquid:solid ratios (Fig. 3b). In contrast, the extraction time exhibited a weak effect on the TPC yield for E. camaldulensis (Fig. 3c). The E. camaldulensis extracts exhibited the highest TPC value among all of the studied plants. Therefore, the maximal predictive value of TPC yield in E. camaldulensis was 13.91 mg g1 under the follow conditions predicted by the polynomial equation: 35% ethanol, liquid:solid ratio of 12 ml g1and 46.8 min of extraction time. Finally, the magnitude of the effects in the extraction of TPC from T. diffusa is shown in the Table 3 and Fig. 4. Similarly, it was observed that the experimental model had a stationary point in the mid-level when the ethanol concentration was explored, and the maximal value of the predictive TPC yield was located at this stationary point (Fig. 4a–b). In addition, an increase in the

linear positive in the TPC was observed as the liquid:solid ratio increased at constant ethanol concentration (Fig. 4b). In addition, an increase in the extraction time up to the highest level resulted in a slight increase in the TPC yield (Fig. 4c). An interaction effect similar to the E. camaldulensis extracts was observed between the ethanol concentration and the liquid:solid ratio, which had a significant effect on the extraction (Fig. 4b). Then, using the polynomial equation obtained for T. diffusa, the maximum TPC was predicted to be 3.53 mg g1 under the following extraction conditions: a 31.5% ethanol concentration with a liquid:solid ratio of 12 ml g1 and a 60 min extraction time. In the liquid:solid extraction, a mass transport phenomenon (i.e., diffusion) was observed [35] where the solids contained in the matrix migrate into the solvent to achieve equilibrium. In ultrasound, acoustic cavitation occurs, which can accelerate the mass transport phenomenon. Acoustic cavitation is produced in the solvent by the passage of ultrasound waves, which are strong enough to generate voids in the solvent. These voids are cavitation bubbles that are able to increase and decrease in size during pressure fluctuations reaching a critical point where they collapse and release large amounts of energy with an increase in the temperature and pressure of the medium [21,36,37]. Therefore, this phenomenon produces an enlargement in the pore walls or the disruption of the cell walls, which releases active ingredients reducing the particle size to allow greater penetration of the solvent into the sample

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Fig. 3. Response surface plots of TPC from the E. camaldulensis extracts as a function of extraction time, ethanol concentration and liquid:solid ratio.

matrix and enhancement of the mass transfer phenomenon on the cell content [20,24,38,39]. In our experiments, the liquid:solid ratio was a crucial factor in the increase of TPC extraction yield. It should be noted that higher liquid:solid ratios generate an increase in the consumption of solvent, the production of waste and an increase in the cost of extraction [40]. For an environmental friendly process and to select extraction conditions that are close to the optimal values predicted, a liquid:solid ratio of 8 ml g1 of vegetable material was selected for the J. dioica and F. cernua extracts. In addition, a liquid:solid ratio of 12 ml g1 of vegetable material was selected for the E. camaldulensis and T. diffusa extracts because a large increase in TPC was observed from 8 to 12 ml g1 in these plants. These differences are due to the mechanism produced by the ultrasound process on the vegetable material. When the ultrasound effect generate changes in the vegetable material (smaller particle size and higher contact surface) increasing the mass transfer phenomenon, there is the possibility to obtain more soluble compounds and consequently reaching saturation of the liquid employed at low levels in the E. camaldulensis and T. diffusa extracts. Therefore, an increase in the liquid:solid ratio results in the prevention of saturation of the liquid and an increase in the yield. The extraction time is an important parameter to optimize to minimize the energy cost of the process [39]. On average, a

medium extraction time of 40 min was determined to be the best time for achieving a maximum TPC yield in all of the studied plants. This result indicates the efficacy of the UAE process for the extraction of the target compound from vegetable material due to the advantages produced by ultrasounds that were discussed above. In addition, in all of the cases, an ethanol concentration close to 35% (v/v) resulted in maximal levels for the TPC yield. In addition, this ethanol concentration is relatively very low. Therefore, this concentration is environmentally friendly and can be used in the food and pharmaceutical industries. In general, similar to our results, Vázquez et al. [41] reported the use of a medium concentration for an ethanol:water mixture (50%, v/v), which resulted in an enhancement of the extraction yield compared to a high concentration for the ethanol:water mixture (80%, v/v) in the infusion method. In addition, these authors demonstrated that the same ethanol concentration effect was observed for the antioxidant activity of the Eucalyptus extracts and an increase in the antioxidant activity in the midpoint tested (50%, v/v) was observed compared to the end levels of the ethanol:water mixture (0% and 80%, v/v). Therefore, 35% ethanol concentration in the extracts was considered the best one in our study. In addition, the Eucalyptus grandis leaf extracts produced antioxidant compounds using emergent technologies, such as

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Fig. 4. Response surface plots of TPC from the T. diffusa extracts as a function of extraction time, ethanol concentration and liquid:solid ratio.

subcritical liquid water extraction (SLWE). Despite the extraction yield being lower than with a conventional extraction process, the extracts obtained by SLWE exhibited higher antioxidant properties than the extracts obtained by the conventional method [42]. To the best of our knowledge, for J. dioica, F. cernua and T. diffusa, this is the first report of the extraction of phenol antioxidant compounds by emergent technologies (i.e., ultrasound). According to the previously discussed extracts, run number 14 for J. dioica and F. cernua and run number 15 for E. camaldulensis and T. diffusa were selected for the following experiments involving antioxidant assays and phytochemical screening by HPLC (Table 2). 3.3. Antioxidant activity of UAE extracts In the current study, the stable DPPH free radical and linoleic acid solution was used to investigate the potential antioxidant properties of the extracts obtained by ultrasound. For the plant species, the following order of antioxidant activity was observed: E. camaldulensis > T. diffusa > F. cernua > J. dioica extracts. The results obtained indicated that a higher antioxidant capacity was observed for the E. camaldulensis extracts for both tests (i.e., DPPH radical scavenging (79.53 ± 0.69%) and lipid oxidation inhibition (70.12 ± 23.09%)) under the studied conditions. The F. cernua

and T. diffusa extracts exhibited similar antioxidant capacity of approximately 62% for DPPH radical scavenging and 23% for lipid oxidation inhibition. However, the J. dioica extracts exhibited a lower antioxidant activity with percentages of 29.25 ± 3.79 for the DPPH radical scavenging and 13.6 ± 6.85 for lipid oxidation inhibition. The amount of phenolic compounds in the extracts correlates with their antioxidant activity, which confirms that these compounds are likely to contribute to the radical scavenging activity of these plant extracts. 3.4. Profile obtained using HPLC in the UAE extracts Only pyrogallol was observed in the F. cernua extracts obtained by UAE. However, for the rest of the plants, any compound could be identified. Previous studies have reported the presence of some phenolic compounds in this type of plant based on HPLC analysis [34]. However, the extraction conditions were different. This absence in our results could be due to the relative concentrations of phenolic compounds in the crude extracts. 4. Conclusions The excellent enrichment performance, simplicity, ease of operation, low cost and smaller consumption of organic solvents and

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