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.
Flavonoids in citrus are a major class of secondary metabolites that have significant impact in human life [
Therefore, the analysis of citrus flavonoids has become essential. There are several studies published on the HPLC analysis of citrus flavonoids [
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 polymath- oxylated 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 CORE- wash (CRD).
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).
Ten different types of citrus were analyzed, bahia (Citrus sinensis L.Osbeck var. baía), lima (Citrus aurantifolia or Citrus limetta―Rutaceae), lima-of-persian (Citrus limettioides), morcote (Citrus aurantium × reticulata var. murcote), pera (Citrus sinensis L. Osbeck), ponkan (Citrus reticulata Blanco var. poncan), seleta (Citrus sinensis L. Osbeck var. seleta), cravo (Citrus reticulata Blanco), kinkan (Fortunella margarita) and pomelo (Citrus paradisi Macfayden), and purchased at local markets in São Paulo, Brazil.
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.
Orange samples were analyzed by HPLC in a D-7000 Merck-Hitachi equipment (Merck-Hitachi, Darmstadt, Germany) with a L-7100 pump and a L-7200 autosampler. The chromatographic conditions were: reverse phase column (Lichrochart 100 RP-18; 12.5 ´ 0.4 cm, diameter of particle of 5mm, Merck, Darmstadt, Germany) and oven temperature of 35˚C. The mobile phase was water-formic acid (5% (v/v) of formic acid, solvent A) and methanol (solvent B), with a flow of 1 mL∙min−1, using a linear gradient (starting with 30% of solvent B, increasing to 80% in 20 minutes and going back to 30% of solvent B in 22 minutes remaining in this conditions until 25 minutes). The time of the analysis was 25 minutes and the detection was performed using a diode array detector (HPLC-DAD) (Merck-Hitachi, L-7450) and the wavelengths used were 280 nm and 340 nm. The software used for data analysis was the Chromatography Dates Station - DAD Manager).
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).
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.
Orange peels are a good source of flavonoids such as glycosides and polymethoxylated flavones [
Typical chromatograms of the extracts are shown in
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 [
was identified in the extracts by comparison with the UV-Vis spectra library, but it was not quantified (
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 (
Orange | Abbrev.* | Flavonoid** | ||||
---|---|---|---|---|---|---|
Naringin | Hesperidin | Hesperetin | Naringenin | Tangeretin | ||
DMSO extraction | ||||||
Baia | LBD | 2.06 ± 1.78 | 41.17 ± 0.08 | 0.00 ± 0.00 | 1.78 ± 0.01 | 1.02 ± 0.00S |
Lima | LLD | 0.80 ± 0.02A | 28.30 ± 0.02 | 0.00 ± 0.00 | 0.85 ± 0.01L | 0.84 ± 0.01 |
Lima-of-persian | LIPD | 1.73 ± 0.05 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.19 ± 0.03YZ |
Morcote | MOD | 0.88 ± 0.14A | 2.10 ± 0.29 | 0.00 ± 0.00 | 0.47 ± 0.81LM | 1.59 ± 0.03b |
Morcote (dried) | MSD | 0.96 ± 0.01A | 2.27 ± 0.02H | 0.00 ± 0.00 | 1.80 ± 0.01 | 4.44 ± 0.01 |
Pera | LPD | 0.00 ± 0.00 | 8.61 ± 0.03 | 0.00 ± 0.00 | 0.00 ± 0.00 | 1.22 ± 0.02W |
Ponkan | PKD | 1.37 ± 0.03A | 33.49 ± 0.16 | 0.00 ± 0.00 | 0.86 ± 0.01LMNP | 2.55 ± 0.01 |
Seleta | SLD | 0.79 ± 0.01A | 39.39 ± 0.12 | 0.00 ± 0.00 | 2.07 ± 0.01KOQ | 0.80 ± 0.01Vc |
Hexane extraction | ||||||
Baia | LBH | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.53 ± 0.01 |
Lima | LLH | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.06 ± 0.00 |
Lima-of-persian | LIPH | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.25 ± 0.01Y |
Morcote | MOH | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 1.67 ± 0.02 |
Morcote (dried) | MSH | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 2.42 ± 0.07 |
Pera | PLH | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.50 ± 0.01TU |
Ponkan | PKH | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 2.85 ± 0.01 |
Seleta | SLH | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.39 ± 0.00 |
Methanol extraction | ||||||
Baia | LBM | 2.98 ± 0.02 | 37.84 ± 0.13 | 0.86 ± 0.01 | 4.74 ± 0.01 | 1.09 ± 0.02 |
Lima | LLM | 0.00 ± 0.00 | 24.93 ± 0.13 | 0.00 ± 0.00 | 0.82 ± 0.01 | 0.44 ± 0.01 |
Lima-of-persian | LIPM | 0.00 ± 0.00 | 3.30 ± 0.01 | 1.21 ± 0.01I | 0.00 ± 0.01 | 0.17 ± 0.02 |
Morcote | MOM | 1.64 ± 0.01 | 3.60 ± 0.11G | 0.00 ± 0.00 | 0.00 ± 0.00 | 2.97 ± 0.09 |
Morcote (dried) | MSM | 2.23 ± 0.06 | 4.69 ± 0.01 | 0.00 ± 0.00 | 4.73 ± 0.06J | 3.79 ± 0.01 |
Pera | LPM | 0.00 ± 0.00 | 10.66 ± 0.09 | 0.00 ± 0.00 | 0.00 ± 0.00 | 1.17 ± 0.02S |
Ponkan | PKM | 0.47 ± 0.01ABC | 29.10 ± 0.04F | 0.00 ± 0.00 | 0.75 ± 0.01LMN | 0.84 ± 0.01V |
Seleta | SLM | 2.12 ± 0.04E | 36.36 ± 0.02 | 0.00 ± 0.00 | 1.96 ± 0.02KO | 0.19 ± 0.03YZa |
*Abbreviation used in multivariate analysis; **Results in triplicates (± SD). Detection limit of 0.3 ppm or 0.003 mg/g. Data are means ± S.D. of three independent determinations. Means within a column of Naringin sharing the letter A are significantly reduced (p < 0.05) by One-Way ANOVA compared to LBM, B compared to LBD, C compared to MSM, E is significantly increased by One-Way ANOVA compared to PKM. Means within a column of Hesperidin sharing the letter F are not significantly different (p > 0.05) by One-Way ANOVA compared to LLD, G compared to LIPM and H compared to MOD. Means within a column of Hesperetin sharing the letter I are significantly increased (p < 0.05) by Student unpaired t-test comparing LBM to LIPM. Means within a column of Naringenin sharing the letter J are not significantly different (p > 0.05) by One-Way ANOVA compared to LBM, L compared to LLM, M compared to LLD, N compared to MOD and P compared to PKM. Means within a column of Tangeretin sharing the letter S are not significantly different (p > 0.05) by One-Way ANOVA compared to LBM, T compared to LBH, U compared to LLM, V compared to LLD, W compared to LPM, Y compared to LIPM, Z compared to LIPH, a compared to LIPD, b compared to MOH and c compared to PKM.
In the PCA of
and higher levels of hesperidin are observed in one of the other groups. The presence and the quantity of naringenin, hesperetin, hesperidin and naringin in the methanol and DMSO extracts, have clustered these samples into two groups. The hierarchical clustering analysis (HCA), using Ward`s method, clustered the samples in two groups with a relative distance lower than 5.5 (
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 (
An attempt to increase the flavonones extraction was made using DMSO, after
extracting seleta peels with hexane obtaining the following concentrations (in mg∙g−1): naringin from 0.792 ± 0.002 to 2.301 ± 0.077, hesperidin from 39.388 ± 0.119 to 47.339 ± 1.128, naringenin from 2.074 ± 0.006 to 2.318 ± 0.023 (small change). This study shows that this consecutive extraction was more efficient in the extraction of naringin, hesperidin and naringenin, than when using DMSO alone (
This selective extraction of flavonoids is a step forward to separate glycosides flavanones (hesperidin and naringin) from their aglycones (hesperetin and narigenin) and polymethoxylated flavones (tangeretin) (
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 [
Molina-Calle et al. [
It was reported that capillary electrophoresis (CE) coupled to mass spectrometry (MS) is a good technique to identify and quantify flavonoids in bitter and sweet orange peel samples. The authors found 5.1 ± 0.2 and 7.9 ± 0.7 mg∙g−1 of naringin and neohesperidin in bitter orange peel and 26.9 ± 2.1 and 35.2 ± 3.6 mg∙g−1 of narirutin and hesperidin in sweet orange peel, respectively. In this study the amounts found were between 0.47 ± 0.01 mg∙g−1 (PKM) to 2.98 ± 0.02 mg∙g−1 (LBM) of naringin and between 2.10 ± 0.29 mg/g (MOD) to 41.17 ± 0.08 mg∙g−1 (LBD) of hesperidin, showing a similar amount of the latter as reported in the literature [
Liu et al. [
Chen et al. [
Orange peels from other citrus species were extracted only with DMSO: cravo (Citrus reticulata Blanco) (LCRD), kinkan (Fortunella margarita) (LRD) [
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 (
Flavonoids** | |||||
---|---|---|---|---|---|
Material* | Naringin | Hesperidin | Hesperetin | Naringenin | Tangeretin |
BCGD | 1.414 ± 0.004 | 22.631 ± 0.008 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.056 ± 0.001 |
BCD | 3.197 ± 0.009A | 42.728 ± 0.075C | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.136 ± 0.001E |
BRD | 5.072 ± 0.023A | 44.236 ± 0.016C | 0.000 ± 0.000 | 0.000 ± 0.000 | 1.930 ± 0.002EF |
PUD | 0.956 ± 0.011B | 11.496 ± 0.007D | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 |
CRD | 5.807 ± 0.040A | 34.206 ± 0.013C | 2.149 ± 0.004 | 0.000 ± 0.000 | 0.000 ± 0.000 |
*Results in triplicates (± SD). Legend: BCGD, “Bagacilio”; BCD, Bagasse; BRD, Bagasse for animal food; PUD, Pulp WEUE and CRD, CORE. Means within a column of Narigin sharing the letter A are significantly increased (p > 0.05) by One-Way ANOVA compared to BCGD, B are significantly reduced (p > 0.05) by One-Way ANOVA compared to BCGD. Means within a column of Hesperidin sharing the letter C are significantly increased (p > 0.05) by One-Way ANOVA compared to BCGD, D are significantly reduced (p > 0.05) by One-Way ANOVA compared to BCGD. Means within a column of Tangeretin sharing the letter E are significantly increased (p > 0.05) by One-Way ANOVA compared to BCGD, F are significantly increased (p > 0.05) by One-Way ANOVA compared to BCD.
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 [
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.
We are grateful to Lincoln A.Kurihara for your technical support and for Citrovita Agroindustrial Ltda. for providing the industry processing juice waste samples.
Pereira, R.M.S., López, B.G.-C., Diniz, S.N., Antunes, A.A., Garcia, D.M., Oliveira, C.R. and Marcucci, M.C. (2017) Quantification of Flavonoids in Brazilian Orange Peels and Industrial Orange Juice Processing Wastes. Agricultural Sciences, 8, 631-644. https://doi.org/10.4236/as.2017.87048