Abstract
This study was conducted to obtain annatto extracts with both high antioxidant capacity and colour potential using solvents of different polarities (water, ethanol/water, ethanol, ethanol/ethyl acetate and ethyl acetate). The highest levels of total phenolic compounds were found in the water, ethanol/water and ethanol extracts (0.5 mg GAE mL−1), and the highest level of bixin was found in the ethanol/ethyl acetate extract (5.2 mg mL−1), which was characterised as the reddest and the most vivid one (a* = 40.5, h° = 46.1, C* = 58.4). The ethanol/ethyl acetate extract also showed the highest antioxidant activity (4.7 μM TEAC mL−1) and the highest percentage of tryptophan protection against singlet oxygen (63.6%). On the other hand, ethyl acetate and ethanol/water were the least effective solvents for the extraction of phenolic compounds and bixin, respectively. According to the multivariate statistical analysis, ethanol/ethyl acetate and ethyl acetate were the most promising solvents to obtain annatto extracts with both antioxidant and colour properties.
Introduction
Annatto is a natural colouring agent obtained from the outer coats of the seeds of the tropical shrub Bixa orellana. Annatto and its extracts are designated collectively as E160b and permitted as a food additive all around the world (Scotter, 2009). Brazil is one of the largest producers and exporters of seeds and annatto preparations (Balaswamy et al., 2006), which are used in the food, pharmaceutical and cosmetic industries.
Bixin (methyl (9-cis)-hydrogen-6,6′-diapo-Ψ,Ψ-carotenedioate) is the main carotenoid responsible for the orange-red colour in the seeds and extracts of annatto (Fig. 1), representing approximately 80% of the total carotenoids (Preston & Rickard, 1980). Besides the colourant property, bixin is known to be a very efficient quencher of singlet oxygen (1O2) and triplet state of sensitisers (Montenegro et al., 2004; Rios et al., 2007). In photosensitised reactions, photons are absorbed by a sensitiser (S), resulting in a long-lived, energy-rich state(s) (typically triplet state) of the sensitiser (3S*) (eqn 1). The 3S* can react directly with molecular oxygen (3O2) giving singlet molecular oxygen (1O2) (eqn 2). Both reactive species (3S* and 1O2) can be efficiently quenched by carotenoids (Bix, bixin in this case) because of an efficient energy-transfer process, resulting in the carotenoid triplet state (3Bix*) (eqns 3 and 4). Once formed, 3Bix* returns harmlessly to its ground state (Bix) with the liberation of heat (eqn 5). In general, the reactive quenching reaction between 1O2 and carotenoids, yielding oxidation products, is several orders of magnitude smaller than the physical quenching process (eqn 4). The bleaching of all-trans carotenoids by 1O2-mediated oxidation is only commonly observed after prolonged photosensitisation periods.
Chemical structure of bixin (C25H30O4) and hypolaetin (C15H10O7).
Besides the presence of carotenoids, annatto seed extracts also contain phenolic compounds, namely hypolaetin (Fig. 1), as the major compound, and a caffeoyl acid derivative, as the minor one (Chistéet al., 2011a). The levels of phenolic compounds in annatto seed extracts were 20–30 times lower than the bixin contents (Chistéet al., 2011b). Therefore, annatto extracts can be used as an easy accessible source of natural antioxidants, because they showed an efficient scavenger capacity against the reactive oxygen and nitrogen species (Chistéet al., 2011b). Carotenoids and phenolic compounds have been associated with the reduction in the risks of various chronic degenerative disorders, such as cancer, inflammations, cardiovascular diseases, cataracts and macular degeneration (Krinsky, 1994; Serdula et al., 1996; Huang et al., 2005). In general, these risk-preventing effects are claimed to be related to the inhibition of oxidation reactions. In parallel, the food can benefit from the addition of these natural compounds, because of their potential antioxidant, preventing the oxidation of foods and raw foods, such as oils, meat, milk and dairy products, extending its shelf life (Decker et al., 2005).
In addition, natural extracts may show, depending on the type of extraction and solvent employed, other compounds with beneficial actions to human health, in opposite to the colouring agents obtained by chemical synthesis with a degree of high purity. Our research group produced annatto extracts with different solvents (methanol, ethanol, ethyl acetate and hexane) and performed the classification based on their antioxidant (TEAC assay) and colourant properties (Cardarelli et al., 2008). Ethanol and ethyl acetate are regarded as presenting more applicability for food use considering the good efficiency in the extraction of bixin and phenolic compounds (Cardarelli et al., 2008) and lower toxicity and risk to human health (FDA, 1997). Because the main food industry application of annatto preparations is directed to impart colour to several products, such as butter, cheese, bakery products, oils, ice creams, sausages, cereals and extruded products (Rios & Mercadante, 2004), which may possess natural photosensitisers in its composition, the protection against singlet oxygen is an important issue in such products. Therefore, the aim of this study was to obtain the extracts from annatto seeds with both high antioxidant properties, including protection against singlet oxygen, and colour parameters by the extraction using solvents with different polarities, all allowed to be applied in the food industries.
Materials and methods
Chemicals
Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), ABTS (2,2′azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)), potassium persulphate and methylene blue (MB) were purchased from Sigma-Aldrich (Steinheim, Germany). l-Tryptophan was purchased from Fisher Scientific (Pittsburgh, PA, USA), and Folin-Ciocalteau reagent, from Dinamica (São Paulo, Brazil). Dichloromethane, methanol, ethanol, ethyl acetate, sodium carbonate (Na2CO3), sodium chloride (NaCl), potassium chloride (KCl), sodium phosphate (NaH2PO4) and potassium phosphate (KH2PO4) were all of analytical grade and purchased from Synth (São Paulo, Brazil). The gallic acid standard was purchased from Extrasynthèse (Lyon Nord, France).
Sample
In August 2008, annatto seeds were obtained from the local market in Campinas, São Paulo, Brazil (22°50′S; 47°00′W). The seeds were portioned (500 g), vacuum-packed and stored under light-free conditions at room temperature until analysis.
Preparation of annatto extracts
Triplicate extractions were performed in a completely randomised design, using the following solvents: water, ethanol/water (1:1, v/v), ethanol, ethanol/ethyl acetate (1:1, v/v) and ethyl acetate. These solvents were chosen considering the permissibility of residues in the extracts after evaporation, according to the Commission Directive 95/45/EC. (1995) from European Communities. The intact annatto seeds were weighed (20 g), and the solvents were added in the mass/solvent ratio of 1:2 (m/v) and stirred on an orbital shaker MA 140/CFT (Marconi, São Paulo, Brazil) at 168 r.p.m. for 15 h at room temperature (25 °C). After the extraction time, only the liquid extract was transferred to a volumetric flask and filled with the respective solvent to 50 mL. Each liquid annatto extract was transferred to ambar flasks, sealed under N2 flow and stored at −36 ºC until analysis.
Bixin quantification
In a previous study, bixin separated by HPLC comprised from 80% to 96% of the total peak area of the carotenoids in annatto extracts (Chistéet al., 2011b). Thus, bixin concentration in seeds and annatto extracts was determined using the methodology described by FAO/WHO (1982). Because bixin is located on the outer coats of the seeds, the exhaustive extraction was performed by washing the intact seeds (1 g) with dichloromethane until the seeds were colourless, the extracts were pooled, and the volume was made up to 100 mL with the same solvent. For the extracts obtained with solvents of different polarities, aliquots of 0.1 mL were evaporated under N2 flow and resuspended in 10 mL of dichloromethane. Absorbance was measured with an UV–Visible spectrophotometer (Agilent, Santa Clara, CA, USA) at 487 nm, and bixin concentration was calculated according to the Lambert–Beer’s law, using 
(FAO/WHO, 1982). All measurements were taken in triplicate.
Total phenolic compounds quantification
For the annatto seeds, 5 g of intact seeds was weighed and extracted five times with methanol/water (8:2, v/v) in ultrasound equipment (Unique model, São Paulo, Brazil) for 10 min at 25 °C. After extraction, the extract was evaporated under vacuum (T < 40 °C), dissolved with 5 mL of methanol, transferred to 25-mL volumetric flask and filled with distilled water. For the extracts obtained according to item 2.3, an aliquot (2.5 mL) was evaporated under N2 flow, resuspended in 2.5 mL of methanol, transferred to 10-mL volumetric flask and filled with distilled water. These extracts were then kept in the freezer for 20 min before centrifugation at 290 g for 20 min. The total phenolic compound content from seeds and extracts was determined using the Folin–Ciocalteau colorimetric method (Singleton et al., 1999), with an UV–visible spectrophotometer at 750 nm, and the results were expressed as milligrams of gallic acid equivalent (GAE) per mL of extract or g of seeds. All measurements were taken in triplicate.
Colour measurements
Colour measurements in annatto extracts were taken using a spectrocolorimeter Color Quest XE (HunterLab, Reston, VA, USA) using reflectance with specular excluded, equipped with the light source D65 and observation angle of 10°. Using the values obtained in the CIELAB system, L* (lightness), the chromatic coordinates a* (red-green component) and b* (yellow-blue component), and the values of C* (chroma) and h° (hue angle) were calculated according to eqns 6 and 7. All measurements were taken in triplicate with the crude extract.
In vitro antioxidant capacity determination
Scavenging capacity
The scavenging capacity against ABTS˙+ radical was determined according to the method described by Re et al. (1999). The radical cation ABTS˙+ (7 mm solution) was formed by chemical reaction with potassium persulphate (2.45 mm). In a cuvette, 1980 μL of radical cation ABTS˙+ solution and an aliquot of 20 μL of the different annatto extracts or of the Trolox standard solution were added. The absorbance was determined at 734 nm after 10 min using an UV–visible spectrophotometer. The results were analysed through the inhibition percentage curves as a function of extracts or Trolox standard solution (0.3–1.5 mm) concentration, and the free radical scavenging capacity was expressed as Trolox equivalent antioxidant activity (TEAC). All measurements were taken in triplicate.
Percentage of protection against singlet oxygen (1O2)
The percentage of protection against 1O2 was evaluated according to methods described by Montenegro et al. (2004) with adaptations. The reactions were performed using 950 μL of l-tryptophan (TRP) (100 μm) as the actinometer and 950 μL of MB (10 μm) as sensitiser, both solutions prepared in phosphate-buffered saline (pH 7.2). In addition, an aliquot (100 μL) of the different annatto extracts was evaporated under N2 flow, resuspended in 100 μL of ethanol/ethyl acetate (1:1, v/v) and added to the solution under moderate agitation, air atmosphere and temperature set at 25 ± 1 °C. The blank was maintained under the same conditions, replacing 100 μL of annatto extract by 100 μL of ethanol/ethyl acetate (1:1, v/v). The excitation source used was a 150-W filament lamp coupled with red and orange cut-off filters to avoid direct excitation of the carotenoids. The excitation light (>620 nm) was focused into the sample cell at right angle, and the intensity decay of fluorescence emission of TRP (excitation at 276 nm and emission at 356 nm) was monitored during 20 min using a fluorescence spectrophotometer (Varian Cary Eclipse, Santa Clara, CA, USA). Kinetics data obtained from the intensity decay of TRP fluorescence emission were fitted to a first-order reaction (eqn 8), and the rate constants were calculated (eqn 9). The protection percentage that annatto extracts (EXT) offered to the actinometer (TRP) was calculated through eqn 10.
where Y is the intensity of TRP fluorescence; Y∞ is the intensity of TRP fluorescence at infinite time; A is pre-exponential factor; k is pseudo-first-order rate constant; x is reaction time; t1/2 is half-life (min); 
is the observed pseudo-first-order rate constant fitted to TRP decay curve (obtained in the blank experiment); and 
is the observed pseudo-first-order rate constant fitted to TRP decay curve in the presence of annatto extract (EXT).
Stability of annatto extracts
After extraction, all annatto extracts were stored at −36 °C, and to verify the stability of bixin and phenolic compounds in solution, the contents of these compounds were evaluated during 8 months. For this purpose, two extracts with opposite polarities were selected: the least polar one – ethyl acetate (EtOAc) – and the most polar one – water (H2O). The quantification of bixin and phenolic compounds was performed, in triplicate, according to the methodologies previously described (FAO/WHO, 1982; Singleton et al., 1999).
Statistical analysis
The results (mean ± SD) of bixin, phenolic compounds, antioxidant capacity and colour parameters were analysed with the Statistica 6.0 software (Statsoft Inc., 2001) using nonlinear regression analysis, analysis of variance (Anova) and Tukey test (P < 0.05). In addition, two multivariate exploratory techniques, principal components analysis (PCA) and hierarchical cluster analysis (HCA), were applied for characterisation of the extracts, using the Statistica 6.0 software package. For PCA, colour parameters (L* and C*) and bixin and phenolic compounds were the active variables used in the derivation of the principal components; the supplementary variables (antioxidant capacity responses from two methodologies and the chemical characteristics of the solvents) were projected onto the factor space. The hierarchical tree was obtained considering the active variables of PCA and the extracts were joined by unweighted pair-group average as the linkage rule, considering the Euclidian distances as the coefficient of similarity.
Results and discussion
Characteristics of annatto seeds
The commercial annatto seeds used in this study presented 14.9 ± 0.5 mg bixin g−1 of seeds and 1.7 ± 0.1 mg GAE g−1 of seeds for total phenolic compounds, both in wet basis (moisture of 10.2 ± 0.1%). The average concentration of bixin is reported to vary from 12 to 23 mg g−1 seeds, depending on edaphic–climatic (such as temperature, illumination, rainfall and soil) and genetic (cultivar) factors (Shuhama et al., 2003). Cardarelli et al. (2008) and Costa & Chaves (2005) found lower levels of bixin (7.5 and 11.8 mg g−1) in annatto seeds collected in Campinas and Teresina (Piauí, Brazil), respectively. However, Balaswamy et al. (2006) reported values varying from 10 to 17 mg bixin g−1 in annatto seeds from India, after applying different extraction procedures. The phenolic compound level detected in the annatto seeds was similar to that reported by Chistéet al. (2011a,b).
Stability of annatto extracts under storage
The levels of bixin and total phenolic compounds in the solutions tested (ethyl acetate and water extracts) were not statistically different (P < 0.05) during storage for eight months at −36 °C (Fig. 2), showing that these compounds are highly stable in solution in both polar and nonpolar solvents. Balaswamy et al. (2006) observed 15% loss of bixin in annatto seeds stored during 360 days at 30 ºC. The same authors also reported that the stability was higher when bixin was added to an oil solution and stored in the dark at lower temperature (5–8 °C).
Stability of bixin (■) and total phenolic compounds (○) in annatto extract solutions stored at −36 °C.
Characteristics of annatto extracts obtained by different solvents
According to Table 1, the extracts obtained with water, ethanol/water mixture and ethanol presented the highest values of total phenolic compounds with no statistical difference (P < 0.05) between them. Considering the content of total phenolic compounds found in annatto seeds (1.7 mg GAE g−1), these solvents showed the highest efficiency in the extraction of phenolic compounds (69–77%), whilst the lowest yield was obtained with ethyl acetate (39%).
Total phenolic compounds (TPC), bixin, CIELAB parameters, free radical scavenging as Trolox equivalent antioxidant activity (TEAC) and protection percentage against singlet oxygen of annatto extracts obtained with solvents of different polarities
| Characteristics | Annatto extracts | ||||
|---|---|---|---|---|---|
| H2O | EtOH/H2O | EtOH | EtOH/EtOAc | EtOAc | |
| Bioactive compounds | |||||
| TPC (mg GAE mL−1) | 0.47 ± 0.05ab | 0.46 ± 0.04ab | 0.52 ± 0.05a | 0.36 ± 0.03bc | 0.26 ± 0.06c |
| Bixin (mg mL−1) | 1.84 ± 0.29b | 0.23 ± 0.02c | 4.63 ± 0.35a | 5.20 ± 0.42a | 4.64 ± 0.74a |
| Colour parameters (CIELAB) | |||||
| L* | 28.90 ± 0.05a | 20.72 ± 0.81b | 28.56 ± 1.43a | 27.93 ± 0.44a | 30.08 ± 1.63a |
| a* | 36.83 ± 1.00a | 24.24 ± 1.54b | 36.41 ± 3.09a | 40.49 ± 0.95a | 38.67 ± 3.16a |
| b* | 37.17 ± 1.83ab | 26.81 ± 3.30b | 37.74 ± 2.35ab | 42.12 ± 1.87a | 37.90 ± 8.86ab |
| h° | 43.83 ± 3.57a | 47.70 ± 1.83a | 46.06 ± 0.76a | 46.11 ± 0.73a | 43.09 ± 3.87a |
| C* | 52.33 ± 1.75a | 36.13 ± 3.41b | 52.45 ± 3.38a | 58.43 ± 1.73a | 54.26 ± 7.26a |
| Antioxidant capacity | |||||
| TEAC (μm of Trolox equivalent mL−1 of extract) | 3.63 ± 0.08b | 3.18 ± 0.19b | 4.71 ± 0.16a | 4.74 ± 0.05a | 4.63 ± 0.03a |
| Protection against singlet oxygen (1O2) (%) | 20.12 ± 0.91c | 8.53 ± 0.51d | 45.14 ± 2.45b | 63.62 ± 2.22a | 49.31 ± 2.71b |
| Characteristics | Annatto extracts | ||||
|---|---|---|---|---|---|
| H2O | EtOH/H2O | EtOH | EtOH/EtOAc | EtOAc | |
| Bioactive compounds | |||||
| TPC (mg GAE mL−1) | 0.47 ± 0.05ab | 0.46 ± 0.04ab | 0.52 ± 0.05a | 0.36 ± 0.03bc | 0.26 ± 0.06c |
| Bixin (mg mL−1) | 1.84 ± 0.29b | 0.23 ± 0.02c | 4.63 ± 0.35a | 5.20 ± 0.42a | 4.64 ± 0.74a |
| Colour parameters (CIELAB) | |||||
| L* | 28.90 ± 0.05a | 20.72 ± 0.81b | 28.56 ± 1.43a | 27.93 ± 0.44a | 30.08 ± 1.63a |
| a* | 36.83 ± 1.00a | 24.24 ± 1.54b | 36.41 ± 3.09a | 40.49 ± 0.95a | 38.67 ± 3.16a |
| b* | 37.17 ± 1.83ab | 26.81 ± 3.30b | 37.74 ± 2.35ab | 42.12 ± 1.87a | 37.90 ± 8.86ab |
| h° | 43.83 ± 3.57a | 47.70 ± 1.83a | 46.06 ± 0.76a | 46.11 ± 0.73a | 43.09 ± 3.87a |
| C* | 52.33 ± 1.75a | 36.13 ± 3.41b | 52.45 ± 3.38a | 58.43 ± 1.73a | 54.26 ± 7.26a |
| Antioxidant capacity | |||||
| TEAC (μm of Trolox equivalent mL−1 of extract) | 3.63 ± 0.08b | 3.18 ± 0.19b | 4.71 ± 0.16a | 4.74 ± 0.05a | 4.63 ± 0.03a |
| Protection against singlet oxygen (1O2) (%) | 20.12 ± 0.91c | 8.53 ± 0.51d | 45.14 ± 2.45b | 63.62 ± 2.22a | 49.31 ± 2.71b |
Mean ± SD (three independent experiments).
Means with the same superscript letters at the same line do not present statistical difference (P < 0.05).
H2O, distilled water; EtOH/H2O, ethanol/water (1:1, v/v); EtOH, ethanol; EtOH/EtOAc, ethanol/ ethyl acetate (1:1, v/v); EtOAc, ethyl acetate; GAE, gallic acid equivalent.
Total phenolic compounds (TPC), bixin, CIELAB parameters, free radical scavenging as Trolox equivalent antioxidant activity (TEAC) and protection percentage against singlet oxygen of annatto extracts obtained with solvents of different polarities
| Characteristics | Annatto extracts | ||||
|---|---|---|---|---|---|
| H2O | EtOH/H2O | EtOH | EtOH/EtOAc | EtOAc | |
| Bioactive compounds | |||||
| TPC (mg GAE mL−1) | 0.47 ± 0.05ab | 0.46 ± 0.04ab | 0.52 ± 0.05a | 0.36 ± 0.03bc | 0.26 ± 0.06c |
| Bixin (mg mL−1) | 1.84 ± 0.29b | 0.23 ± 0.02c | 4.63 ± 0.35a | 5.20 ± 0.42a | 4.64 ± 0.74a |
| Colour parameters (CIELAB) | |||||
| L* | 28.90 ± 0.05a | 20.72 ± 0.81b | 28.56 ± 1.43a | 27.93 ± 0.44a | 30.08 ± 1.63a |
| a* | 36.83 ± 1.00a | 24.24 ± 1.54b | 36.41 ± 3.09a | 40.49 ± 0.95a | 38.67 ± 3.16a |
| b* | 37.17 ± 1.83ab | 26.81 ± 3.30b | 37.74 ± 2.35ab | 42.12 ± 1.87a | 37.90 ± 8.86ab |
| h° | 43.83 ± 3.57a | 47.70 ± 1.83a | 46.06 ± 0.76a | 46.11 ± 0.73a | 43.09 ± 3.87a |
| C* | 52.33 ± 1.75a | 36.13 ± 3.41b | 52.45 ± 3.38a | 58.43 ± 1.73a | 54.26 ± 7.26a |
| Antioxidant capacity | |||||
| TEAC (μm of Trolox equivalent mL−1 of extract) | 3.63 ± 0.08b | 3.18 ± 0.19b | 4.71 ± 0.16a | 4.74 ± 0.05a | 4.63 ± 0.03a |
| Protection against singlet oxygen (1O2) (%) | 20.12 ± 0.91c | 8.53 ± 0.51d | 45.14 ± 2.45b | 63.62 ± 2.22a | 49.31 ± 2.71b |
| Characteristics | Annatto extracts | ||||
|---|---|---|---|---|---|
| H2O | EtOH/H2O | EtOH | EtOH/EtOAc | EtOAc | |
| Bioactive compounds | |||||
| TPC (mg GAE mL−1) | 0.47 ± 0.05ab | 0.46 ± 0.04ab | 0.52 ± 0.05a | 0.36 ± 0.03bc | 0.26 ± 0.06c |
| Bixin (mg mL−1) | 1.84 ± 0.29b | 0.23 ± 0.02c | 4.63 ± 0.35a | 5.20 ± 0.42a | 4.64 ± 0.74a |
| Colour parameters (CIELAB) | |||||
| L* | 28.90 ± 0.05a | 20.72 ± 0.81b | 28.56 ± 1.43a | 27.93 ± 0.44a | 30.08 ± 1.63a |
| a* | 36.83 ± 1.00a | 24.24 ± 1.54b | 36.41 ± 3.09a | 40.49 ± 0.95a | 38.67 ± 3.16a |
| b* | 37.17 ± 1.83ab | 26.81 ± 3.30b | 37.74 ± 2.35ab | 42.12 ± 1.87a | 37.90 ± 8.86ab |
| h° | 43.83 ± 3.57a | 47.70 ± 1.83a | 46.06 ± 0.76a | 46.11 ± 0.73a | 43.09 ± 3.87a |
| C* | 52.33 ± 1.75a | 36.13 ± 3.41b | 52.45 ± 3.38a | 58.43 ± 1.73a | 54.26 ± 7.26a |
| Antioxidant capacity | |||||
| TEAC (μm of Trolox equivalent mL−1 of extract) | 3.63 ± 0.08b | 3.18 ± 0.19b | 4.71 ± 0.16a | 4.74 ± 0.05a | 4.63 ± 0.03a |
| Protection against singlet oxygen (1O2) (%) | 20.12 ± 0.91c | 8.53 ± 0.51d | 45.14 ± 2.45b | 63.62 ± 2.22a | 49.31 ± 2.71b |
Mean ± SD (three independent experiments).
Means with the same superscript letters at the same line do not present statistical difference (P < 0.05).
H2O, distilled water; EtOH/H2O, ethanol/water (1:1, v/v); EtOH, ethanol; EtOH/EtOAc, ethanol/ ethyl acetate (1:1, v/v); EtOAc, ethyl acetate; GAE, gallic acid equivalent.
In relation to the bixin contents, the extracts obtained with ethanol, ethanol/ethyl acetate mixture and ethyl acetate showed the highest values, with no statistical difference (P < 0.05) between them. Based on the bixin level found in annatto seeds (14.9 mg bixin g−1), these solvents showed the highest yield of bixin (77–87%), whilst ethanol/water mixture presented the lowest efficiency (4%). The structure of bixin with 25 carbons and an acid and methyl ester end-groups is more polar than the carotenoids usually found in foods (chain with 40 carbons) and showed more affinity for medium polar solvents (Cardarelli et al., 2008).
The characteristics of the solvents used in this study, such as solvent polarity index (SPI) and the solvent selectivity triangle, described by Snyder et al. (1993) (Table 2), were considered to explain the extraction efficiency of phenolic compounds and bixin from annatto seeds. Considering the results of Tables 1 and 2, in general, the extraction of bioactive compounds in annatto seeds seems to be dependent on the polarity of the solvent applied, i.e. the higher the polarity of solvent, the higher the extraction of phenolic compounds (more polar compounds) and, on the other hand, the lower the polarity, the higher the extraction of bixin (less polar compound). The selectivity triangle, in which organic solvents are classified according to their ability to interact with the solute either as a dipole (dipolarity, π*), as a proton acceptor (acidity, α), or as a proton donor (basicity, β), (Table 2), also explained the yield obtained for the different compounds with the different solvents. The solvents that presented predominant property of acidity (water, ethanol/water and ethanol) showed to be more efficient for the extraction of phenolic compounds. However, none of the solvent characteristics alone explained the bixin yield found in all different extracts. The fact that the amount of bixin in the water extract was higher than that found in the ethanol/water mixture indicated that predominant dipolar property might play an important role in the extraction of bixin with water-based solvents. Because the dipolarity value of water is higher than that of the ethanol/water mixture, water was able to extract higher amounts of bixin than the ethanol/water mixture.
Characteristics of solvents used for the preparation of annatto extracts
| Solvents | Characteristics | |||
|---|---|---|---|---|
| SPI* | Normalised selectivity factors† | |||
| π* | α | β | ||
| H2O | 9.0 | 0.45 | 0.43 | 0.48 |
| EtOH/H2O | 7.1 | 0.35 | 0.41 | 0.42 |
| EtOH | 5.2 | 0.25 | 0.39 | 0.36 |
| EtOH/EtOAc | 4.7 | 0.40 | 0.19 | 0.41 |
| EtOAc | 4.3 | 0.55 | 0.00 | 0.45 |
| Solvents | Characteristics | |||
|---|---|---|---|---|
| SPI* | Normalised selectivity factors† | |||
| π* | α | β | ||
| H2O | 9.0 | 0.45 | 0.43 | 0.48 |
| EtOH/H2O | 7.1 | 0.35 | 0.41 | 0.42 |
| EtOH | 5.2 | 0.25 | 0.39 | 0.36 |
| EtOH/EtOAc | 4.7 | 0.40 | 0.19 | 0.41 |
| EtOAc | 4.3 | 0.55 | 0.00 | 0.45 |
*SPI, solvent polarity index (Snyder, 1974).
†π*, dipolarity, α, acidity and β, basicity (Snyder et al., 1993).
The normalised selectivity factors for the mixtures EtOH/H2O and EtOH/EtOAc were calculated considering the solvent proportion and the respective values for pure solvents.
H2O, distilled water; EtOH/H2O, ethanol/water (1:1, v/v); EtOH, ethanol; EtOH/EtOAc, ethanol/ethyl acetate (1:1, v/v); EtOAc, ethyl acetate.
Characteristics of solvents used for the preparation of annatto extracts
| Solvents | Characteristics | |||
|---|---|---|---|---|
| SPI* | Normalised selectivity factors† | |||
| π* | α | β | ||
| H2O | 9.0 | 0.45 | 0.43 | 0.48 |
| EtOH/H2O | 7.1 | 0.35 | 0.41 | 0.42 |
| EtOH | 5.2 | 0.25 | 0.39 | 0.36 |
| EtOH/EtOAc | 4.7 | 0.40 | 0.19 | 0.41 |
| EtOAc | 4.3 | 0.55 | 0.00 | 0.45 |
| Solvents | Characteristics | |||
|---|---|---|---|---|
| SPI* | Normalised selectivity factors† | |||
| π* | α | β | ||
| H2O | 9.0 | 0.45 | 0.43 | 0.48 |
| EtOH/H2O | 7.1 | 0.35 | 0.41 | 0.42 |
| EtOH | 5.2 | 0.25 | 0.39 | 0.36 |
| EtOH/EtOAc | 4.7 | 0.40 | 0.19 | 0.41 |
| EtOAc | 4.3 | 0.55 | 0.00 | 0.45 |
*SPI, solvent polarity index (Snyder, 1974).
†π*, dipolarity, α, acidity and β, basicity (Snyder et al., 1993).
The normalised selectivity factors for the mixtures EtOH/H2O and EtOH/EtOAc were calculated considering the solvent proportion and the respective values for pure solvents.
H2O, distilled water; EtOH/H2O, ethanol/water (1:1, v/v); EtOH, ethanol; EtOH/EtOAc, ethanol/ethyl acetate (1:1, v/v); EtOAc, ethyl acetate.
According to the colour parameters (Table 1), the similar values of h° (43.1–47.7) located all annatto extracts in the CIELAB space corresponding to the orange colour. In addition, no statistical difference (P < 0.05) was verified for L* and a* values in all extracts, excepting for the ethanol/water mixture. The ethanol/water extract, which presented the lowest bixin content, was the darkest (L* = 20.7), the least red (a* = 24.2) and vivid (C* = 36.1). On the other hand, the extract obtained with ethanol/ethyl acetate mixture, which showed the highest bixin level, was characterised by a high intensity of red (a* = 40.5; h° = 46.1) and vivid colour (C* = 58.4).
In relation to the TEAC assay, all extracts showed the same kinetic profile, with fast absorbance decay of ABTS˙+ after 1 min, followed by no significant changes in absorbance after 10 min. The TEAC values of ethanol, ethanol/ethyl acetate and ethyl acetate annatto extracts were higher, with no statistical difference (P < 0.05) between them, as compared to those found in water and ethanol/water ones (Table 1). Because the bixin contents in ethanol, ethanol/ethyl acetate and ethyl acetate extracts were from 9 to 15 times greater than the contents of total phenolic compounds, bixin most likely contributes with greater influence than phenolic compounds to the antioxidant capacity of annatto extracts. Because ABTS assay is based on a single electron transfer reaction between the tested antioxidant and the formed radical (Huang et al., 2005), the carotenoid bixin is able to scavenge the radical cation ABTS˙+ most probably due to the presence of resonant electrons in its many conjugated double bonds (eleven in the bixin structure). The TEAC values of annatto extracts found in the present study were higher than those previously reported for annatto seed extracts (Cardarelli et al., 2008) for EtOH/H2O (0.80), EtOH (0.53) and EtOAc (0.41), expressed in μm of Trolox equivalent mL−1 of extract.
Regarding the percentage of protection against singlet oxygen, illustrated in Fig. 3a, all annatto extracts presented an excellent fit to the first-order reaction (R² = 0.99) for the fluorescence intensity decay of tryptophan, in the presence of the sensitiser and annatto extracts, as shown in Fig. 3b. The extract obtained with ethanol/ethyl acetate showed the highest percentage of protection (63.6%), i.e. this extract delayed more efficiently the degradation of tryptophan by the singlet oxygen generated by the photosensitisation of MB. As expected, the highest value of protection was associated with the highest level of bixin in the annatto extract. Consequently, the half-life of tryptophan was superior in the ethanol/ethyl acetate mixture extract (22.6 min), followed by the ethyl acetate (17.7 min), ethanol (12.4 min), water (10.8 min) and ethanol/water (8.2 min) extracts.
(a) Illustration of the experimental system for the measurement of protection against singlet oxygen, accompanied by a spectrophotometer and (b) fluorescence intensity decay of tryptophan (TRP) in the presence of methylene blue (MB) and annatto extracts.
It is well known that bixin is an efficient quencher of both excited triplet state of sensitiser and singlet oxygen (Montenegro et al., 2004; Rios et al., 2007). Rios et al. (2007) showed that bixin is a very efficient 1O2 quencher in fluid solutions because of an efficient energy-transfer process and confirmed that the energy level of bixin triplet state is lower than that of 1O2 (18 and 22.5 kcal mol−1, respectively).
Classification of annatto extracts by multivariate statistical analysis
In the PCA analysis, the first two components accounted for 93% of the explained variance. A positive correlation between bixin contents and the colour parameters L* and C* (r = 0.75 and r = 0.87, respectively) was observed. Bixin contents in annatto extracts were also positively correlated with TEAC values (r = 0.99) and percentage of protection (r = 0.98), whilst the contents of phenolic compounds did not show any positive correlation with both antioxidant capacity methods (r = −0.56 and r = −0.62, respectively) (Fig. 4a). Considering the correlations obtained by the PCA analysis, the bixin content presented the largest contribution to the antioxidant capacity in annatto extracts.
PCA plot of composition, colour and antioxidant properties of the different annatto extracts. (a) Variable projection and (b) scatterplot for the cases with suggested drawn grouping ellipses.
The phenolic compounds presented positive correlation with the acidity property (r = 0.99) and with a less extend to the SPI (r = 0.69), i.e. the higher the acidity and the SPI values, the higher the yield of total phenolic compounds in the extract. The polarity (r = −0.80), acidity (r = −0.63) and basicity (r = −0.37) properties of the solvents showed negative correlation with bixin yield, whilst dipolar value showed a weak positive correlation (r = 0.08). Thus, the extraction of bixin seems to be more efficient with solvents with both low values of SPI and of acidity property.
Therefore, the annatto extracts were divided into three groups (Fig. 4b): one group was formed by the extracts containing ethyl acetate (EtOAc); another, the extracts containing only ethanol (EtOH) or water (H2O); and the last one, by the extracts containing ethanol/water (EtOH:H2O). Based on the PCA analysis, Figure 4b clearly shows the localisation of the extracts according to the solvent efficiency in the yield of colour (bixin and colour parameters) as PC1 and antioxidant capacity of the extracts (TEAC and percentage of protection against 1O2) as PC2. The annatto extracts obtained with ethyl acetate were characterised by the highest percentage of protection against the singlet oxygen, highest TEAC values and the best colour intensity, probably, again, due to the presence of high levels of bixin.
Figure 5 shows the dendrogram obtained when HCA was applied taking into consideration the the active variables of PCA. The tree diagram provides evidence for three groups, which can be observed in PCA plot (Fig. 4b).
Dendrogram obtained by hierarchical cluster analysis analysis, considering the active variables of PCA for the different annatto extracts.
Conclusion
The extracts obtained with ethyl acetate were characterised by the greatest values of protection against singlet oxygen, free radical scavenger capacity and colour intensity. In annatto extracts, these properties were all closely related to the bixin contents, but not to the phenolic compounds levels. Colour is considered to be the main attribute influencing the acceptance and preference of a food product (Clydesdale, 1993), and antioxidant capacity is related to the prevention of some chronic diseases (Krinsky, 1994; Serdula et al., 1996; Huang et al., 2005); thus, the solvents containing ethyl acetate can accomplish both requirements. This information is important to the industry because annatto is a known accessible source of natural colourants.
Acknowledgment
The authors thank the Brazilian foundation FAPESP for financial support.
