1. Introduction
Free radical biology is a burgeoning discipline that has been investigated more widely in life science in recent years. This area mainly concerns the formation and scavenging of free radicals, as well as the damage caused by free radicals in biological systems. It is now well established that a series of oxygen-centred free radicals and other reactive oxygen species contribute to the pathology of numerous disorders, including atherogenesis, neurodegeneration, chronic inflammation, cancer and physiological senescence [
1], therefore, antioxidants are considered to be important nutraceuticals on account of their many health benefits and they are widely used in the food industry as potential inhibitors of lipid peroxidation [
2]. However, it has been demonstrated that synthetic antioxidants can accumulate in the body and this can result in liver damage and carcinogenesis. These problems are not seen when natural antioxidants, which are extracted from herbs and spices and have high antioxidant activity, are used in food applications. These extracts are considered safe, potentially nutritional and to have therapeutic effects.
Flavonoids are a group of polyphenolic compounds that are present in many natural products and have been studied extensively due to their high antioxidant, anti-microbial and antiproliferative activity [
3,
4]. Apart from their health benefits and nutraceutical value, anthocyanins are also used as food colorants since their color is pH dependent. Fundamental studies have indicated that at different pH levels, anthocyanins undergo structural transformations that are responsible for their color changes in solution.
Potato peel is a waste product generated from potato processing and disposal of this material is a problem. Many varieties of potato contain large amounts of antioxidant pigments in the tuber and, for this reason, potato peel is a good source of functional ingredients. The variety ‘Pinta-Boca’ is cultivated in different part of South America and it has a very high anthocyanin content in the peel.
In recent years, numerous methodologies for the extraction of compounds of relatively high polarity have been developed that employ different extraction methods. In the present work we analyze and compare the viability of two anthocyanin extraction methods: supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE).
SFE was introduced as an environmentally benign and efficient extraction technique for solid materials [
5]. In most of these studies carbon dioxide is used as the solvent because of its relatively low critical temperature, non-toxicity, non-flammability, good solvent power, ease of removal from the product and low cost. However, quantitative extraction of polar analytes, such as anthocyanins, typically requires the addition of an organic modifier [
6].
PLE is based on the use of conventional solvents at controlled temperatures and pressures and this approach for the extraction of valuable compounds from natural sources is well established. PLE requires less solvent and the extraction is complete in a shorter period of time in comparison to traditional organic solvent extraction. Subcritical water at temperatures between 100 and 180 °C has been identified as optimal to recover the maximum amount of anthocyanins from berry substrates, grape skins [
7] and blackcurrants [
8]. Several authors [
9] found that subcritical water extraction at 160 to 180 °C, 6 MPa and 60 min is a good substitute for organic solvents such as methanol and ethanol to obtain phenolic compounds from potato peel. However, at these temperatures there is a greater possibility of thermal degradation of the flavonoid compounds during the extraction from grape pomace.
Studies performed on black carrots using water acidified with citric and lactic acids at temperatures between 50 and 100 °C and 50 bar pressure yielded the maximum amount of anthocyanins, particularly when lactic acid was used as the acidulant [
10]. These studies showed that greater thermal degradation of the anthocyanin extracts occurred at 100 °C, along with higher concomitant browning when sulfuric acid was used as an acidulant. In this study, the extractions at high temperatures were performed for only 10 min, while the sulfuric acid extractions were performed at room temperature over a period of 12 h. It should be noted that in the aforementioned studies with mineral and/or organic acids, the pH of the extraction medium was not reported.
Recently, studies performed by Monrad
et al. [
11] indicated that 50%–70% v/v ethanol in water and temperatures between 80 and 120 °C was optimal for the recovery of anthocyanins and procyanidins from grape pomace.
Several other methods have been applied for extraction of antioxidants from plant matrices, one such novel process being microwave assisted extraction (MAE). MAE presents different advantages regarding to conventional methods: it is faster, selective and lower solvent consumption [
12,
13]. MAE has been used to extract phenolic antioxidant from potato peels; Singh
et al. [
14] used a response surface method for to optimize the MAE parameters such as extraction time, solvent (methanol) concentration and microwave power level for extraction of antioxidants from potato peels. They were able to extract higher levels of phenolic from dried potato peel than the values reported by number of previous studies.
A MAE method coupled with the orthogonal array design was investigated for efficient extraction of the phenolic compounds in potato downstream wastes [
15]. Four parameters were examined for the MAE of the total phenolic content and optimized at 60 % ethanol, 80 °C, 2 min, solid-to-solvent ratio 1:40 (g/mL). The MAE was proven more efficient than the conventional solvent extraction by refluxing.
The advantage of PLE and SFE over MAE is the applicability at different scales. PLE and SFE can be applied to systems on various scales, from the laboratory scale (a few grams) to the pilot plant scale (several hundred grams of sample), through to the industrial scale (tons of raw materials).
It is worth to mention that SFE and PLE have been widely used for extraction process development, that is, to extract bioactive compounds from different matrices. Even though these processes usually offer clear advantages over traditional ones, the main drawback for industrial scale use is the lack of realistic economic studies. In this sense, some articles have been lately published dealing with the assessment of the industrial economic feasibility of some developed process, such as essential oil extraction from rosemary, fennel and anise [
16] and brewery spent grain management [
17]. Moreover, industrial applications of SFE have experienced a strong development since the early 1990s in terms of patents [
18]. Therefore, SFE can be regarded as a possible tool not only from a laboratory point of view but also for the natural products and food industries.
In the work described here, a comparison of two high-pressure extraction methods for the recovery of antioxidant compounds (anthocyanins) from Solanum stenotomun has been analyzed. The methods analyzed were Supercritical Fluid Extraction (SFE) studying the effect of ethanol as cosolvent, and Pressurized Liquid Extraction (PLE) using ethanol as solvent. The effects of temperature and pressure on the total extraction yield, anthocyanin and phenolic content were studied. The antioxidant capacity of the extract obtained at the best conditions was measured and compared with data obtained from the literature.
3. Experimental
3.1. Samples and Chemicals
The raw material used was obtained from Bolivian potatoes (Solamun stenotomun) of the variety ‘Pinta-Boca’. Samples were liophilized to constant weight. All results are expressed on dry basis. Carbon dioxide (99.995%) was supplied by Abello-Linde S.A. (Barcelona, Spain). 2,2-Diphenyl-1-picrylhydrazyl, free radical (DPPH), pelargonin chloride, malvin chloride, cyanin chloride and gallic acid standards were supplied by Sigma-Aldrich (Steinheim, Germany). The organic solvents ethanol, methanol and acetic acid, all HPLC gradient grade, were supplied by Panreac (Barcelona, Spain). The water used in all experiments was double-distilled MilliQ grade.
3.3. Anthocyanins Analysis
HPLC analysis of the anthocyanins present in the extracts was performed using the method described by Cho
et al. [
27]. An Agilent Technologies (Palo Alto, CA, USA) 1100 Series chromatograph was used. 5% Formic acid in water (v/v) (A) and methanol (B) were used as solvents in this HPLC method. The HPLC gradient program was executed as follows: 98% A to 40% A in 60 min, 40% A to 98% A in 5 min. The entire HPLC run time was 70 min using a flow rate of 1 mL/min.
The resultant extracts were filtered before HPLC assay using a 0.45 μm PTFE filter (Varian Inc., Palo Alto, CA, USA) and 100 μL of the filtered extract was injected into the column (250 × 4.6 mm) C18 Hypersil ODS (5 μm particle size) (Supelco). The compounds were detected using a UV-Vis detector at a wavelength of 510 nm. The peaks were identified by comparison of retention times with those of the commercial standards (Sigma) and were quantified by means of calibration curves. The anthocyanins in the extract analyzed by HPLC were reported based on malvin chloride. The experiments for each extraction were carried out in triplicate in order to evaluate the variability of the measurements. The results are shown as the average of all the independent analyses with a reproducibility of approximately 4.5% CV (coefficient of variation).
3.4. Phenolic Compounds Analysis
Shimadzu (Tokyo, Japan) UVmini-1240 spectrophotometer was used for analytical determination of phenolic compounds. A mixture of 0.2 mL of each extract and 3.8 mL of hydrochloric acid (1 M) was introduced in the flask [
28]. After 1 h the solutions were measured at 280 nm. Gallic acid was used as standard. The experiments for each extraction were carried out in triplicate in order to evaluate the variability of the measurements. The results are shown as the average of all the independent analyses with a reproducibility of approximately 3.2% CV (coefficient of variation).
3.5. Antioxidant Assay with DPPH
The antioxidant activities were determined using DPPH as a free radical. Different concentrations were tested (expressed as the mg of extract/mg DPPH) for each set of extraction conditions. Extract solution in methanol (0.1 mL) was added to a 6 × 10
−5 mol/L methanol DPPH solution (3.9 mL). The decrease in absorbance was determined at 515 nm at different times until the reaction had ‘reached a plateau’. The exact initial DPPH concentration (CDPPH) in the reaction medium was calculated from a calibration curve with the following equation:
as determined by linear regression (r = 0.9999).
For each set of extraction conditions a plot of % remaining DPPH vs. time (min) was generated. These graphs were used to determine the percentage of DPPH remaining at the steady state and the values were transferred onto another graph showing the percentage of residual DPPH at the steady state as a function of the weight ratio of antioxidant to DPPH. Antiradical activity was defined as the amount of antioxidant required to decrease the initial DPPH concentration by 50% [Efficient Concentration = EC50 (mg extract/mg DPPH)]. The antioxidant activity was expressed as the antioxidant activity index (AAI), which was calculated in terms of 1/EC50.
The experiments were carried out in triplicate in order to evaluate the variability of the measurements. The results are shown as the average of all the independent analyses and the reproducibility was approximately 1.9% CV (coefficient of variation).
3.6. Experimental Design
Given the importance of obtaining extracts with high anthocyanin contents, experimental designs were carried out for the extracts obtained by SFE and PLE. Due to the fact that the objective of the paper was to analyze the influence of operating conditions in the anthocyanins yield, a screening experimental design was selected. The variables studied were pressure and temperature. In the case of SFE, a 2
2 factorial design was carried out, whereas for PLE a multifactorial design was carried out. The factor and physical values are shown in
Table 4. The experiments were performed in randomized order.
Table 4.
Physical values in the experimental design.
Table 4.
Physical values in the experimental design.
Extraction method | Experimental variable | Factor | Levels |
---|
SFE | Pressure | P | 100 bar | 400 bar |
Temperatue | T | 35 °C | 65 °C |
PLE | Pressure | P | 100 bar | 200 bar |
Temperatue | T | 60 °C | 80°C | 100 °C |
Statistical calculations and analyses were performed using STATGRAPHICS Plus 5.1® (1994–2001, Statistical Graphics Corp.). An empirical correlation was developed in order to predict the influence of extraction conditions on the extraction yields of anthocyanins.