Quantification of Flavonoids in Brazilian Orange Peels and Industrial Orange Juice Processing Wastes

The flavonoid content in orange peels of different Brazilian citrus varieties such as bahia, lima, lima-of-persian, morcote, pera, ponkan, seleta, cravo, kinkan and pomelo was assessed. Industry processing juice wastes such as bagasse, bagasse residues, animal feeding bagasse, pulp WEUE and CORE-wash were also analyzed. The HPLC analysis indicates that the most abundant flavonoids found in these Brazilian citrus peels are hesperidin and naringin. The solvents used are selective for flavonoid extraction, and depending on their polarity, glycoside or aglycone flavonoids are extracted. The use of multivariate analysis shows that DMSO is the best solvent to extract glycosides flavanones while hexane displays high selectivity in the extraction of polymethoxylated flavones. The flavonoids present in the orange wastes, obtained at different stages of the industrial processing, are qualitative and quantitatively different. The identification and quantification of the flavonoid composition in each Brazilian citrus variety were evaluated and allowed the selection of the best solvent for the extraction of each specific class of flavonoids. These compounds were found to be more abundant in the fruit peels than in their juices, revealing their great industrial potential. The residual portion of the processing juices is also rich in flavonoids, depending on the processing step.


Introduction
Flavonoids in citrus are a major class of secondary metabolites that have significant impact in human life [1]- [6]. These compounds are present in many sources, including citrus fruits [7] [8] [9] [10] [11]. The therapeutic or even toxic activity, e.g., antioxidant, anti-inflammatory, antibacterial, antimutagenic, antiviral and antimetastatic effects, has been reported in a great number of these flavonoids [12] [13] [14] [15]. Hesperidin, the most abundant flavonoid in Citrus aurantium, influences vascular permeability [16] [17]; naringin and hesperetin inhibit the in vitro proliferation of human breast cancer cells [6] [8] [18]; tangeretin and nobiletin are the most active antimutagen flavonoids tested so far, and many display chemopreventive potential [19] [20] [21]. The pharmacological potential of these polyphenolic compounds is the reason why the interest of the pharmaceutical industry on these compounds has increased over the last years [14] [22].
Therefore, the analysis of citrus flavonoids has become essential. There are several studies published on the HPLC analysis of citrus flavonoids [8] [23] [24] [25]. However, there are few reports on the HPLC analysis of citrus Brazilian fruit peels [10] [26].
In Brazil, there are many different species and varieties of citrus. The most common are: lima orange, lima-of-persian, seleta, morcote, mexerica poncan, bahia and pera.
The purpose of this paper is to assess different methods for the extraction of the flavanones: hesperidin, hesperetin, naringin, naringenin and the polymathoxylated flavone tangeretin, in ten different Brazilian citrus. The compounds were identified by comparison with standards and quantified by HPLC analysis.
Furthermore, these results were analyzed using multivariate analysis, allowing the identification of the best extraction method. We also analyzed and quantified flavonoids in industrial juice processing waste named "bagacilio" (BCGD), bagasse (BCD), animal feeding bagasse (BRD), pulp WEUE (PUD) and COREwash (CRD).

Chemicals and Reagents
The standards used in the identification and quantification of the peaks in orange peels by HPLC were purchased from Sigma (USA) (quercetin, hesperidin, hesperetin, naringin and naringenin) and tangeretin from Chromadex (USA). Solvents such as DMSO (dimethylsulfoxide) were purchased from Ecibra (Brazil), hexane from Synth (Brazil) and methanol from Merck (Germany).

Sample Preparation
The orange peels were further grounded to a fine powder using a blender (Arno, Brazil) and dried at 70˚C using drying oven (Fanem™, Brazil). This fine powder (60 mesh, 0.25 mm) was extracted for 24 hours in 500 mg portions with 10 mL of DMSO, hexane, methanol or DMSO after hexane extraction. This last portion, after extracting with hexane, filtering and drying, was re-extracted with DMSO (500 mg with 10 mL of DMSO). The extracts were filtered using a 0.45 µm filter (minisart, Sartorius, Germany) and analyzed by HPLC in triplicate. Samples of orange peel residues were dried at 70˚C and processed using the same procedure as the orange peels.

Statistical Analysis
In the research for possible differences between the groups, analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison test (parametric data) and Kruskal-wallis (nonparametric data) was used. When the comparison was only between two groups Student unpaired t test was used. P < 0.05 was considered statistically significant (GraphPad, Prism 6.0, San Diego, CA, USA).

Multivariate Statistical Analysis
Principal component analysis (PCA) is an analysis that allows to describe the variation (or dispersion) of one determined data set. Samples (orange peels) were represented by a row vector while the variables (different extracting solvents) were represented by a column vector. This matrix can be decomposed in two different matrices, the scores that represent the position of a sample in this new system of cartesian coordinates and the loadings that represents the weight of each variable in this new axles of coordinates. In the present work, the analysis of the composition of the orange peels and the industry processing wastes was assessed using The Unscrambler 10.2 software (CAMO, USA). PCA and Hierarchical Clustering Analysis (HCA) using Ward`s method on previously normalized data, were performed.

Extraction of Flavonoids in Orange Peels
Orange peels are a good source of flavonoids such as glycosides and polymethoxylated flavones [11]. Orange peel extracts of different citrus varieties were prepared in methanol, hexane and DMSO. The flavonoid content in the orange peels was quantified using external standards such as quercetin, hesperidin, hesperetin, naringin, naringenin and tangeretin ( Figure 1).
Typical chromatograms of the extracts are shown in Figure 2. The most abundant flavonoid extracted by DMSO and methanol, in all orange peels studies, was hesperidin (Table 1).
More polar solvents like methanol or DMSO display better extraction efficiency of glycoside flavanones such as naringin, hesperidin and naringenin. Quercetin (aglycone flavonol) was identified in some orange peels extracted with these same solvents. Quercetin was identified in peels of Navel variety citrus [27]. However, hexane is the solvent of choice to extract polymethoxylated flavones, such as tangeretin. Nobiletin, which is also a polymethoxylated flavone, Figure 1. Chromatogram of standards with respective retention time (Rt, in minutes) and chemical structures.
was identified in the extracts by comparison with the UV-Vis spectra library, but it was not quantified ( Figure 2).

Effect of the Extracting Solvents on the Orange Peels Composition
The multivariate analysis, showed a selective extraction of flavonoids using different solvents. The extractions of the orange peels were named in accordance with the solvent used in the extraction and type of orange (Table 1). Figure 2. Typical HPLC-DAD chromatogram of baia orange peels extracted with DMSO (a), hexane (b) and methanol (c). Peak 1 is naringin, 2 is hesperidin, 3 is narigenin, 4 is tangeretin, 5 is tangeretin and 6 nobiletin (chemical structure showed in (b)), identified by comparison with the UV-Vis spectrum of the spectrum library, not quantified).
PC1 and PC2 can explain 97% of the data variance, and the accumulated variance of PC3 is 99%. The hexane extracts are clustered together while there is no clear separation between the methanol and DMSO extracts. The loadings plot show that tangeretin is responsible for gathering all the hexane extracts together  Table 1. revealing a selective separation with these solvents ( Table 1).

The extraction of hesperetin in bahia (LBM) and lima-of-persian (LIPM)
oranges, was only observed in methanol, which supports the statement that methanol is the best solvent to extract hesperidin aglycones, when compared to DMSO and hexane.
The amount of naringenin found in all the orange peels studied was very low, only being observed in the extraction with DMSO. This solvent was also more efficient than methanol in the extraction of tangeretin, however, hexane was the most efficient and selective solvent in the extraction of tangeretin and other polymetoxylated flavanones. None of the flavanones were detected in the hexane extract ( Figure 2). This study also shows that hexane is a very selective solvent to extract polymethoxy flavones.
An attempt to increase the flavonones extraction was made using DMSO, after Figure 4. HCA separating the extraction methods for the orange peels. For abbreviations see Table 1. . This study shows that this consecutive extraction was more efficient in the extraction of naringin, hesperidin and naringenin, than when using DMSO alone (Table 1).
The amount of flavanones and polymethoxyflavones found in orange peels was always higher than those found in their corresponding juices according to the literature. That is the case of the pera variety (Citrus sinensis Osbeck) whose concentration of hesperidin found in the orange juice was 0.269 mg•g −1 [28] which is lower than the amount found in the respective peel that was 8.61 mg•g −1 ( Table 1). The bahia variety orange juice presented 0.427 mg•g −1 of this substance [28] while the concentration in the orange peels was 41.17 mg•g −1 (LBD, Table 1). The same situation was observed in the lima variety juice which presented 0.223 mg•g −1 of hesperidin and its peel contained 28.30 mg•g −1 (LLD, Table 1). The hesperidin content of the orange juice of the Citrus sinensis Osbeck variety, was similar to that reported in the literature, however the amount of naringin and naringenin was much lower [29]. Similar behavior was observed in other citrus fruit juices from industries all over the world [30].
Molina-Calle et al. [21] reported that the most abundant glycoside flavanones in citrus peels from Spain were naringin, hesperidin and neohesperidin. M'hiri et al. [31] used different operating conditions, such as ultrasound, microwave, supercritical CO 2 and high pressure for the flavonoid extraction from oranges (Citrus sinensis) of the Maltese variety. They found that the best condition to extract hesperidin was ultrassonic extration at 125W (8.362 ± 0.296 mg•g −1 ) and microwave, 200 W (9.289 ± 0.007 mg•g −1 ). The most abundant flavonoids were hesperidin and neohesperidin.
It was reported that capillary electrophoresis (CE) coupled to mass spectro- Chen et al. [34] reported that the most abundant flavonoid in oranges peels from different places such as China, Canada, and the United States, was hesperidin, as it was observed in the current study. Orange peels showed both antioxidant and anti-inflammatory activities. The presence of hesperidin as a major component in Citrus peels was confirmed by Guccione et al. [35] using HPLC-DAD and HPLC-MS techniques.

Flavonoids in Industrial Orange Juice Processing Wastes
In the orange juice production industry, the process to obtain final products from available raw materials, involves a large amount of wastes. Several steps are performed and beyond juice, everything is used with a commercial purpose.
Industrial orange juice processing wastes were extracted in DMSO. In this study, naringin, hesperidin and tangeretin, and flavonoids found in different amounts at each stage of the process were quantified. Naringenin and quercetin were not present at any of the steps (considering a detection limit of 0.003 mg/g) and hesperetin was only found in the CORE. The portion used in animal feed (BRD) was rich in hesperidin (greater amount), naringin and tangeretin ( Table   2).  The finding that the concentration of these important compounds is higher in these fruit peels than in their juice clearly indicates that orange peels are an important industry source [37] [38]. The demonstrated possibilities of simple and selective extraction of these compounds entitles them as an alternative for industrial production.

Conclusion
The extraction of flavanones and polymethoxylated flavones from orange peels of different citrus species, using different solvent systems, allowed the identification and quantification of the flavonoid composition in each Brazilian citrus variety and the selection of the best solvent for the extraction of each specific class of flavonoids. It was determined that hexane is a selective solvent to extract polymethoxy flavones like tangeretin. The consecutive extraction using hexane and then DMSO was more efficient in the extraction of naringin, hesperidin and naringenin, than when using DMSO alone. The extracting procedures used in this work showed that these compounds are more abundant in the fruit peels than in their juices, revealing their great industrial potential. Industrial orange juice processing wastes, in all the processing steps, are also rich in hesperdin, with similar (or proportional) amounts as the ones found in the peels, and thus they are a promising source of this flavonoid. The combination of techniques such as HPLC-DAD and PCA is a powerful tool to evaluate clusters and confirm, in this case, what solvent is most effective extracting flavonoids in orange peels.