1. Introduction
The properties of polyphenols as antioxidants have been widely recognized. They are associated with reduced risk of cancer, cardiovascular diseases, diabetes and Alzheimer’s disease [
1]. Most polyphenols in the human diet are supplied by plants and fruits [
2]. Furthermore, antioxidants from natural sources could be used to increase the stability of food, such as the ability to prevent lipid peroxidation [
3]. This damage could be catalyzed by different metals present in food (especially in meat), because the metals can participate directly or indirectly in the reaction of oxidation of lipids [
4]. In addition, these metals promote the creation of reactive oxygen species (ROS) prejudicial to health [
5]. Polyphenols are also used as antimicrobial agents in food preservation [
6].
The worldwide demand for food has been increasing. Nowadays, fresh fruit and vegetable production is, approximately, 800,000 t/year, without taking into account losses and waste [
7]. In some studies polyphenols were found in pulp and other waste remaining from the production of fruit juices and wines [
8,
9]. Polyphenols can be excellent antioxidants and in some cases are better than synthetics ones [
1]. New technology to treat food waste was required in order to obtain raw materials or ingredients for other processes and products [
10].
Many health effects have been attributed to the borage (
Borago officinalis L.) plant, such as: antispasmodic, antihypertensive, antipyretic, aphrodisiac, demulcent, and diuretic properties. It is also considered useful to treat asthma, bronchitis, cramps, diarrhea, palpitations, and kidney ailments [
11]. In the food industry borage seed extracts have been used as effective antioxidants in the preparation of gelatin films from fish [
12]. It was also shown to be effective in preventing oxidation in fermented dry sausages enriched with ω-3 polyunsaturated fatty acids (PUFA). As well as maintaining organoleptic properties, the borage extract was an economical and safe antioxidant source [
1]. The antioxidant activity of borage meal extract was also demonstrated by Wettasinghe
et al. (1999) [
13] in a model meat system, where the inhibition of oxidation assessed by 2-thiobarbituric acid-reactive substances (TBARS), hexanal and total volatile formation was reported. Borage seed extracts exhibited strong metal chelating activity in an aqueous assay medium, that suggested it is a good chelating agent for food and non-food applications [
4]. Bandoniene
et al. (2002) [
14] reported a study that showed that borage leaf extract was an effective antioxidant in rapeseed oil. The polyphenols found in borage include rosmarinic acid, which is responsible for some of the antioxidant properties of rosemary extracts, which is also widely used by the food industry. Rosmarinic acid has a high antioxidant capacity and it is present in the majority of Lamiaceae species [
4,
14,
15,
16].
Borage leaves are a cheap raw material for the production of polyphenols, because it is a by-product of an industrial process, and in addition, the disposal of this material incurs a cost, which can be minimized by its use [
1].
Response surface methodology (RSM) is a useful tool for process optimization [
17], that allows the influence of independent variables on a response variable to be represented by a mathematical model that is able to reproduce the behavior of these parameters, with only a few experiments [
18,
19]. An experimental design commonly used in the food industry is the central composite design (CCD), which involves evaluation of the factors at various levels [
20].
Several foods such as: milk, sauces and soup have an emulsion structure. This could be oil in water (O/W) or water in oil (W/O) or a combination of both. Oxidation is a principal problem of this model [
21]. The oxidation of emulsions differs from oil oxidation, due to the presence of oil or water droplets and an interface between oil and water, where components partition between the phases and interact with effects on chemical reactions [
22]. Furthermore, in foods, there may be synergy between antioxidants and the protein present; which may increase the antioxidant capacity and enhance the stabilization of the emulsion [
23,
24].
In this work, we modeled and optimized the extraction of polyphenols from borage leaves based on the total polyphenols, antiradical activity (ORAC), and the amount of rosmarinic acid. The response surface method has not been used before, but it allowed the extraction parameters to be studied for optimization of antioxidant effects in a model emulsion system.
2. Experimental Section
2.1. Materials
2,2′-Azo-bis(2-amidinopropane) dihydrochloride (AAPH), was used as peroxyl radical source. Pyrogallol red (PGR), Trolox (6-hydroxy-2,5,8-tetramethylchroman-2-carboxylic acid), rosmarinic acid, ethanol, fluorescein, AAPH, BSA, p-anisidine (4-amino-anisole; 4-methoxy-aniline), isooctane, potassium persulfate, acetic acid (glacial) and polyoxyethylene sorbitan monolaurate (Tween-20) were purchased from Sigma-Aldrich Company Ltd. (Gillingham, UK). Folin–Ciocalteu reagent and sodium carbonate were supplied by Merck (Darmstadt, Germany). Refined sunflower oil, of a brand known to lack added antioxidants, was purchased from a local retail outlet. All compounds were of reagent grade.
2.2. Borage Preparation
The borage plant (Borago officinalis L.) was obtained in the local market, washed and the leaves were separated from other edible parts. This waste was homogenized and frozen at −80 °C for lyophilization. Then the leaves were ground into a powder by using a Moulinex mill (A5052HF, Moulinex, Lyon, France), then the particle size was standardized with a number 40 mesh sieve. Finally, the powder was stored in a dark bottle in a desiccator until use.
2.4. Total Phenolic Content (TPC)
TPC was determined spectrophotometrically following the Folin–Ciocalteu colorimetric method [
25]. Sample diluted 1:4 with milli-Q water was stirred in triplicate. The final concentration in the well (96 wells plate was used) was: 7.7% v/v sample, 4% v/v Folin-Ciocalteu’s reagent, 4% saturated sodium carbonate solution and 84, 3% of milli-Q water were mixed. The solution was allowed to react for 1 h in the dark and the absorbance was measured at 765 nm using a Fluorimetrics Fluostar Omega (BMG Labtech, Ortenberg, Germany). The total phenolic content was expressed as mg Gallic Acid Equivalents (GAE)/g dry weight.
2.5. ORAC Assay
Antioxidant activities of Borage extracts were determined by the ORAC assay, as reported by Ninfali
et al. [
26]. The assay was carried out using a Fluorimetrics Fluostar Omega (Perkin–Elmer, Paris, France) equipped with a temperature-controlled incubation chamber. The incubator temperature was set at 37 °C. The extract samples were diluted 1:20 with milli-Q water. The assay was performed as follows: 20% of sample was mixed with Fluorescein 0.01 mM, and an initial reading was taken with excitation wavelength, 485 nm and emission wavelength, 520 nm. Then, AAPH (0.3 M) was added measurements were continued for 2 h. This method includes the time and decrease of fluorescence. The area under the curve (AUC) was calculated. A calibration curve was made each time with the standard Trolox (500, 400, 250, 200, 100, 50 mM). The blank was 0.01 M phosphate buffered saline (pH 7.4). ORAC values were expressed as mg Trolox Equivalents (TE)/mg of dry borage.
2.6. HPLC
Identification and quantification of rosmarinic acid was performed using a Waters 2695 separations module (Meadows Instrumentation Inc., Bristol, WI, USA) system with a photodiode array detector Waters 996 (Meadows Instrumentation Inc., Bristol, WI, USA). The column was a Kinetex C18 100A, 100 × 4.6 mm (Phenomenex, Torrence, CA, USA). Solvents used for separation were 0.1% acetic acid in water (v/v) (eluent A) and 0.1% acetic acid in methanol (v/v) (eluent B). The gradient used was: 0–12 min, linear gradient from 40% to 50% B; 12–15 min, linear gradient from 50% to 40 B. The flow rate was 0.6 mL/min, and the detection wavelength was 330 nm. The sample injection volume was 10 μL. The chromatographic peak of rosmarinic acid was confirmed by comparing its retention time and diode array spectrum against that of a reference standard. Working standard solutions were injected into the HPLC and peak area responses obtained.
Standard graphs were prepared by plotting concentration (mg/L) versus area. Quantification was carried out from integrated peak areas of the samples using the corresponding standard graph.
2.7. Statistical Analysis
RSM was used to determine the optimal conditions of polyphenol extraction. A central composite design (CCD) was used to investigate the effects of three independent variables with two levels (solvent concentration, extraction temperature, and extraction time) with the dependent variables (TPC, ORAC activity, rosmarinic acid concentration). CCD uses the method of least-squares regression to fit the data to a quadratic model. The quadratic model for each response was as follows:
where,
Y is the predicted response; 0 is a constant;
i is the linear coefficient;
ii is the quadratic coefficient,
ij is the interaction coefficient of variables
i and
j; and
Xi and
Xj are independent variables.
The adequacy of the model was determined by evaluating the lack of fit, coefficient of determination (
R2) obtained from the analysis of variance (ANOVA) that was generated by the software. Statistical significance of the model and model variables were determined at the 5% probability level (α = 0.05). The software uses the quadratic model equation shown above to build response surfaces. Three-dimensional response surface plots and contour plots were generated by keeping one response variable at its optimal level and plotting that against two factors (independent variables). Response surface plots were determined for each response variable. The coded values of the experimental factors and factor levels used in the response surface analysis are shown in
Table 1. The graphics and the RSM analysis were made by software Matlab version R2013b (The MathWorks Inc., Natick, MA, USA).
Table 1.
Design variable and code.
Table 1.
Design variable and code.
Extraction | Code | Temperature (°C) | Time (min) | Ethanol Concentration (%) |
---|
Ethanolic | | | | |
| −1 | 60 | 10 | 30 |
| 0 | 70 | 15 | 45 |
| 1 | 80 | 20 | 60 |
Aqueous | | | | |
| −1 | 50 | 10 | |
| 0 | 70 | 15 | |
| 1 | 90 | 20 | |
All responses were determined in triplicate and are expressed as average ± standard deviation. The answers have a percentage deviation less than 10%.
2.8. Oil-Water Emulsions
Oil-in-water emulsions (20.2 g) were prepared by dissolving Tween-20 (1%) in acetate buffer (0.1 M, pH 5.4), either with or without protein, namely BSA (0.2%), and borage extracts (3% v/v, 1% v/v, 0.3% v/v, 0.06% v/v). The emulsion was prepared by the dropwise addition of oil (sunflower oil) to the water phase, cooling in an ice bath with continuous sonication with a Vibracell sonicator (Sonics & Materials Inc., Newtown, CT, USA) for 5 min. All emulsions were stored in triplicate in 60 mL glass beakers in the dark (inside an oven) at 30 °C in an incubator. Two aliquots of each emulsion (0.005–0.1 g, depending on the extent of oxidation) were removed periodically for determination of peroxide value (PV) and p-anisidine value.
2.9. Peroxide Value (PV)
PV was determined by the ferric thiocyanate method (Frankel, 1998) [
27] (after calibrating the procedure with a series of oxidized oil samples analyzed by the AOCS Official Method Cd 8-53). Data from the PV measurements were plotted against time.
2.10. p-Anisidine Value (p-AV)
The test was performed according to the methods reported by Singh
et al. (2007) [
28], with some modifications. In a 10 mL volumetric flask, 0.05 g of emulsion was taken and dissolved in 25% (v/v) of isooctane at 1% of acetic acid (glacial). From this solution, 2 mL was treated with 5% (v/v) of
p-anisidine reagent and kept in the dark for 10 min and absorbance was measured at 350 nm using a UV-Vis spectrophotometer (Zuzi, AUXILAB, S.L., Beriain, Navarra, Spain).
4. Conclusions
A model has been developed to describe the effect of several variables on extraction of polyphenols from borage leaves. The antioxidant activity (ORAC value) demonstrated optimal values for ethanolic and for aqueous extraction. The increase in antioxidant capacity can be related to the increase of the amount of rosmarinic acid; however other polyphenols may also contribute, as seen by the decrease of ORAC values with increase of extraction temperature. The decomposition of these polyphenols may explain these results. The ethanolic or aqueous extraction conditions can be chosen according to the type of phenolic acids we want to enrich in the extract, or the effect we want to obtain. The use of water for extraction of polyphenols made the extraction process more environmentally friendly, but increased the energy used. The time variable was of little significance for the model.
Figure 5.
The p-anisidine values for the samples with BSA and without BSA.
Figure 5.
The p-anisidine values for the samples with BSA and without BSA.
The use of the borage extract and the extract in combination with BSA decreased the rate of increase of PV and p-AV in an emulsion, with a synergistic effect demonstrated. This effect could be associated with the presence of rosmarinic acid in the extract and the function of BSA as a metal chelating agent at the interface.
The application of innovative technologies such as ultrasound, electromagnetic pulses, subcritical water extraction, among other, could be applied for further study in order to enhance the extraction of polyphenols [
38,
51,
52] of the borage leaves, as well as its influence on preservation of the antioxidant properties on the extract obtained.