Reuse of Food Waste: The Chemical Composition and Health Properties of Pomelo (Citrus maxima) Cultivar Essential Oils

The aim of the present study is to investigate the chemical profile, antioxidant activity, carbohydrate-hydrolysing enzyme inhibition, and hypolipidemic effect of essential oils (EOs) extracted from Sicilian Citrus maxima (pomelo) flavedo. Using gas-chromatography-mass spectrometry analysis (GC-MS) we analysed the Eos of five cultivars of C. maxima, namely, ‘Chadock’, ‘Maxima’, ‘Pyriformis’, ‘Terracciani’, and ‘Todarii’, and their blends. The antioxidant activity was performed by using a multi-target approach using 2,2′-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic acid (ABTS), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), ferric reducing ability power (FRAP), and β-carotene bleaching tests. The α-amylase, α-glucosidase, and lipase-inhibitory activities were also assessed. GC-MS analyses revealed D-limonene as the main monoterpene hydrocarbon in all cultivars, albeit with different percentages in the range of 21.72–71.13%. A good content of oxygenated monoterpenes was detected for all cultivars, especially for ‘Todarii’. The analysis of the principal components (PCA), and related clusters (HCA), was performed to find chemo-diversity among the analysed samples. EOs from ‘Chadock’ and ‘Maxima’ were statistically similar to each other, and they differed from P3 in the smaller amount of sesquiterpene hydrocarbons, while the oils from ‘Terracciani’ and ‘Todarii’ were found to be chemically and statistically different. ‘Chadock’ EO was the most active to scavenge radicals (IC50 values of 22.24 and 27.23 µg/mL in ABTS and DPPH tests, respectively). ‘Terracciani’ EO was the most active against both lipase and α-amylase, whereas the blends obtained by the combination (1:1 v/v) of C. maxima ‘Maxima’ + ‘Todarii’ were the most active against α-glucosidase. Generally, the blends did not exert a unique behaviour in potentiating or reducing the bioactivity of the pomelo EOs.


Introduction
Some of the key words in the field of Green Chemistry are 'recirculation, reuse', aimed at highlighting environmental sustainability through the use, or rather the reuse, of food waste, chemical by-products, and refuse as result of industrial processes. However, it acids into various organs. Recent studies may indicate that excess body fat is enough to increase oxidative stress, which suggests that free radicals can play an important role in the aetiology and development of comorbidity related to obesity, such as hyperglycaemia, leading to type 2 diabetes [36]. Hence, there is the need to identify the functional ingredients from the dual activity. In our continuous search of the potential reuse of Citrus-industry waste, we investigate, in the present study, the chemical composition of the EOs of five different cultivars of pomelo (C. maxima), 'Chadock' (P1), 'Maxima' (P2), 'Pyriformis' (P3), 'Terracciani' (P4), and 'Todarii' (P5) (Figure 1), cultivated in Palermo, Sicily alone and in combination (1:1 v/v) to assess the potential effect of the synergism or antagonism of action. Moreover, pomelo EOs have been evaluated for their antioxidant potential using different in vitro assays (ABTS, DPPH, FRAP, and the β-carotene bleaching test).
The α-amylase-, α-glucosidase-, and lipase-inhibitory activities were also performed. The inhibition of α-amylase and α-glucosidase, enzymes that are involved in the digestion of carbohydrates, can considerably reduce the post-prandial increase in blood glucose, and can therefore be a good strategy for the management of blood glucose levels in type 2 diabetic and borderline patients. Instead, the inhibition of pancreatic lipase is the most widely investigated mechanism for the identification of potential anti-obesity agents.

Composition of EOs and Their Blends
Flavedo hydro-distillation produced yellow oils. Overall, forty-two compounds were identified and listed in Table 1 according to their retention indices on a DB-5MS non-polar column and classified into five classes: monoterpene hydrocarbons (MHs), oxygenated monoterpenes (OMs), sesquiterpene hydrocarbons (SHs), oxygenated sesquiterpenes (OSs), and other compounds (Os).
From an examination of the results, a possible division of the cultivars into three different classes emerged: a group, constituted of C. maxima P1, P2, and P3 EOs, rich in The α-amylase-, α-glucosidase-, and lipase-inhibitory activities were also performed. The inhibition of α-amylase and α-glucosidase, enzymes that are involved in the digestion of carbohydrates, can considerably reduce the post-prandial increase in blood glucose, and can therefore be a good strategy for the management of blood glucose levels in type 2 diabetic and borderline patients. Instead, the inhibition of pancreatic lipase is the most widely investigated mechanism for the identification of potential anti-obesity agents.

Composition of EOs and Their Blends
Flavedo hydro-distillation produced yellow oils. Overall, forty-two compounds were identified and listed in Table 1 according to their retention indices on a DB-5MS non-polar column and classified into five classes: monoterpene hydrocarbons (MHs), oxygenated monoterpenes (OMs), sesquiterpene hydrocarbons (SHs), oxygenated sesquiterpenes (OSs), and other compounds (Os). From an examination of the results, a possible division of the cultivars into three different classes emerged: a group, constituted of C. maxima P1, P2, and P3 EOs, rich in monoterpene hydrocarbons (83.47-85.41%), dominated by the clear presence of D-limonene (58.66-71.13%), and a moderate quantity of oxygenated ones (11.01-14.72%) caused by the presence of compounds, such as cis-linalool oxide, β-linalool, α-terpineol, and α-citral. The P4sample, compared to the group described above, had a smaller quantity of monoterpene hydrocarbons (49.92%) and a greater quantity of oxygenated monoterpenes (26.27%).
The research, conducted using the major scientific systems (Scopus, SciFinder, Google Scholar), highlighted the absence of information on the composition and/or on the possible biological applications of EOs for the varieties 'Terracciani', 'Chadock', 'Pyriformis', and 'Todarii'. Instead, several scientific works reported the chemical composition of the EOs produced from the flavedo of C. maxima: the analysis conducted by Tao and Liu [37] showed, for this cultivar, an essential oil practically made up of D-limonene (89.96% ) and 4.46% of β-myrcene, but with very low abundances of SH and OS; pomelo of C. maxima, grown in Vietnam, confirmed the high presence of hydrocarbon monoterpenes, D-limonene among all, and also, in this case, small percentages of sesquiterpene compounds [38]. This confirmed the results of the performed analyses, finding both a high percentage of MH and low percentage (<1%) of SH and OS. The difference compared to our oil is the higher presence of OM.    Sample obtained by combination P1, P2, P3, P4, and P5 EOs (1:1 v/v). a Linear retention index obtained for DB-5MS non-polar column; b linear retention index obtained for DB-Wax column; c content is the peak volume percentage of compounds in the essential oil sample; d : 1 = retention index identical to bibliography; 2 = identification based on comparison of MS; 3 = retention time identical to authentic compounds. Compounds are classified in order of linear retention time of non-polar column. e Sign: Significance at ** p < 0.05. Results followed by different letters in the same line are significantly different (p < 0.05) by Tukey's multiple-range test. For the EOs of the different C. maxima cultivars and their blends, as shown in the loading graph (Figure 2), all variables influenced PC1 and PC2. The PCA of C. maxima EO presented a total variance of 87.2% of the original data. In fact, PC1 (51.5% of the total significant contribution) was mainly represented by oxygenated monoterpenes and sesquiterpenes (OMs and OSs, respectively), and by other compounds (O), in the negative score, by an MH eigenvalue with a positive score, while the SH was statistically irrelevant; meanwhile, PC2 (35.7%) was represented by a positive score of OM, but to a greater level by MH, SH, OS, and O compounds.

PCA and HCA Analyses of the EOs and Their Mixes
The statistical analyses were conducted considering the different chemical classes in which the different chemical compounds identified on the GC-MS were releveled, according to the loading plot obtained by principal component analysis (PCA) for monoterpene hydrocarbons (MH), oxygenated monoterpenes (OM), sesquiterpene hydrocarbons (SH), oxygenated sesquiterpenes (OS), and other compounds (O).
For the EOs of the different C. maxima cultivars and their blends, as shown in the loading graph (Figure 2), all variables influenced PC1 and PC2. The PCA of C. maxima EO presented a total variance of 87.2% of the original data. In fact, PC1 (51.5% of the total significant contribution) was mainly represented by oxygenated monoterpenes and sesquiterpenes (OMs and OSs, respectively), and by other compounds (O), in the negative score, by an MH eigenvalue with a positive score, while the SH was statistically irrelevant; meanwhile, PC2 (35.7%) was represented by a positive score of OM, but to a greater level by MH, SH, OS, and O compounds. HCA based on the Euclidean distance between groups indicated a solution with four clusters (A, B, C, and D), with a distance < 0.8 (Figure 3), which was mainly due to the variation along the major axis in the PCA analysis. These clusters formed separate groups in the PCA biplot ( Figure 2). HCA based on the Euclidean distance between groups indicated a solution with four clusters (A, B, C, and D), with a distance < 0.8 (Figure 3), which was mainly due to the variation along the major axis in the PCA analysis. These clusters formed separate groups in the PCA biplot (Figure 2).
With a dissimilarity < 0.8 (red line), four clusters existed. The first one, group A, was represented by P1P5 and P2P5 EOs mixes, characterised by a high percentage of MH With a dissimilarity < 0.8 (red line), four clusters existed. The first one, group A, was represented by P1P5 and P2P5 EOs mixes, characterised by a high percentage of MH (65.62 and 64.74%, respectively) and a moderate content of OM (27.50 and 29.53%). Group B included samples P1, P2, and P1P2, represented by an identical quantity of compounds divisible into the two monoterpene classes and slightly different for the percentages of sesquiterpenes. Mixes P1P3 and P2P3 formed the cluster C: both mixtures had identical quantities of all the chemical classes taken into consideration, even of the compounds belonging to the O class (0.55 and 0.50%, respectively). Finally, group D consisted of two samples, P1P4 and P2P4, rich in MH compounds, but unlike the other clusters, they presented not insignificant quantities of SO (2.94-2.64%, respectively) and O compounds (4.10-4.02%).
Increasing the level of dissimilarity (cut-off = 1.6, Figure 3, green line), the macroclusters became three. Cluster A', which, in addition to the inclusion of cluster A, also incorporated samples P3P5 and P1 + P2 + P3 + P4 + P5, which was slightly different in comparison to P1P5 and P2P5, in the content of SH and O constituents. Sample P3 could be contained within the C cluster, forming the C' group, although it did not have oxygenated sesquiterpenes and had a higher percentage of other compounds. Finally, cluster D was enlarged to D' by incorporating the P3P4 sample not previously included due to the slightly higher content of SH compounds.
The samples P4, P5, and P4P5, due to the dissimilar percentages of OM and O compared to the other mixes, could not be included in any possible cluster. As is already evident from Tables 1 and 2, their composition was distinctly different from all other EOs and related blends, and the statistical analysis confirmed this evidence.

Radical Scavenging and Antioxidant Activities of Pomelo EOs and Their Blends
Different in vitro assays were performed to evaluate, with a multi-target approach, the antioxidant potential of pomelo EOs and their blends obtained by the flavedo of different cultivars of C. maxima. The concentration-dependent effects were observed for all investigated samples, except for the FRAP assay ( Table 3). The P1 EO was the most active Increasing the level of dissimilarity (cut-off = 1.6, Figure 3, green line), the macroclusters became three. Cluster A', which, in addition to the inclusion of cluster A, also incorporated samples P3P5 and P1 + P2 + P3 + P4 + P5, which was slightly different in comparison to P1P5 and P2P5, in the content of SH and O constituents. Sample P3 could be contained within the C cluster, forming the C' group, although it did not have oxygenated sesquiterpenes and had a higher percentage of other compounds. Finally, cluster D was enlarged to D' by incorporating the P3P4 sample not previously included due to the slightly higher content of SH compounds.
The samples P4, P5, and P4P5, due to the dissimilar percentages of OM and O compared to the other mixes, could not be included in any possible cluster. As is already evident from Tables 1 and 2, their composition was distinctly different from all other EOs and related blends, and the statistical analysis confirmed this evidence.

Radical Scavenging and Antioxidant Activities of Pomelo EOs and Their Blends
Different in vitro assays were performed to evaluate, with a multi-target approach, the antioxidant potential of pomelo EOs and their blends obtained by the flavedo of different cultivars of C. maxima. The concentration-dependent effects were observed for all investigated samples, except for the FRAP assay ( Table 3). The P1 EO was the most active in both tests applied to verify the radical scavenging potential of samples with IC 50 values of 22.24 and 27.23 µg/mL for the ABTS and DPPH assays, respectively. A slightly lower radical scavenging potential was observed when the EOs were combined with IC 50 values from 24.28 to 35.27 µg/mL for P1 + P2 + P3 + P4 + P5 and P4P5, respectively, in the ABTS test, and from 26.82 to 35.24 µg/mL for P1 + P2 + P3 + P4 + P5 and P2P3, respectively, in the DPPH test. It is interesting to note that, except for the blend obtained by mixing equal volumes of P2 and P3, all blends had better FRAP values than those obtained with BHT used as a positive control. A great variability was observed in the protection from lipid peroxidation. In this assay, P4 was the most active, with an IC 50 value of 25.56 µg/mL. Among the blends, the EOs with the greatest protective power for lipid peroxidation were P1P2 and P1P3 (IC 50 values of 26.28 and 21.87 µg/mL, respectively). Based on the Relative Antioxidant Capacity Index (RACI), which creates a ranking clustering of the antioxidant capacity for different samples, P1 showed the highest antioxidant potential with an RACI value of −0.55, followed by the P1P5 blend (RACI value of −0.38) (Figure 4). Sign. ** ** ** ** Blend obtained by combination P1, P2, P3, P4, and P5 EOs (1:1 v/v). Data are expressed as means ± standard deviation (SD). The following positive controls were used: ascorbic acid in 2,2 -Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic acid (ABTS) (IC 50 value of 1.72 ± 0.09 µg/mL) test and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (IC 50 value of 5.03 ± 0.79 µg/mL); butylated hydroxytoluene (BHT) in ferric reducing ability power (FRAP) (FRAP value 63.27 ± 4.48 µM Fe(II)/g), and propyl gallate in β-carotene bleaching test (IC 50 value of 0.09 ± 0.004 µg/mL); Sign: significance at ** p < 0.05. Results followed by different letters in the same line are significantly different according to Tukey's multiple-range test.
Our results are better than those reported by Lan-Phi et al. [39], which evaluated the DPPH radical scavenging activity of different pomelo varieties, namely, DaXanh, Duong-Cam, Nam Roi, and Buoi Long, and found IC 50 values in the range of 43.8-63.1 mg/mL. On the contrary, a better radical scavenging activity was observed for the Indian C. maxima EO with an IC 50 value of 8.84 µg/mL [40], and also for pomelo from Malaysia [14].
A comparison between the antioxidant activity of pomelo distilled essential oil (DEO) and cold-pressed essential oil (CPEO) revealed that CPEO was more potent than DEO in the total reducing power and radical scavenging activity [41].
A comparison between the antioxidant activity of pomelo distilled essential oil (DEO) and cold-pressed essential oil (CPEO) revealed that CPEO was more potent than DEO in the total reducing power and radical scavenging activity [41].

Inhibition of Enzymes Involved in Metabolic Syndrome and Obesity by Pomelo EOs and Their Blends
The inhibition of lipase, as well as carbohydrate-hydrolysing enzymes, α-glucosidase, and α-amylase, was evident in a concentration-dependent manner (Table 4). 'Terracciani' and 'Todarii' EOs exhibited the highest lipase-inhibitory activity with IC50 values of 23.22 and 24.23 µg/mL, respectively. The combinations of the EOs (1:1 v/v) did not significantly improve the lipase-inhibitory activity (IC50 values from 25.22 to 39.12 µg/mL for P2P4 for P1P4, respectively).
Regarding the carbohydrate-hydrolysing enzymes, the P4 EO was the most active against α-amylase (IC50 value of 25.23 µg/mL), whereas P2 was the most active against αglucosidase (IC50 value of 25.67 µg/mL). Regarding blends data, the P1P4 sample was the most active against α-amylase (IC50 value of 21.43 µg/mL). A combination of the EOs increased the α-glucosidase-inhibitory activity, where the blend obtained by mixing equal volumes of 'Maxima' (P2) and Todarii' (P5) was the most active with an IC50 value of 21.67 µg/mL, followed by the P1P2 blend. Our results are in agreement with those reported for the lemon flavedo EO by Oboh et al. [42], which obtained IC50 values of 8.16 and 7.56 μg/mL against α-amylase and α-glucosidase, respectively. A promising activity was also observed for the EO obtained by orange flavedo (IC50 values of 11.51 and 11.53 µg/mL, respectively).
Recently, Itoh et al. [43] analysed different Citrus by-products as possible lipase inhibitors and found IC50 values of 43 and 44 µg/mL for leaves and flower methanol extract, respectively. Values in the range of 135.51-282.65 µg/mL were obtained for EOs obtained from C. × clementina leaves collected in Rosarno and Cetraro, respectively, against α-amylase and α-glucosidase, whereas values in the range of 185.43-287.91 µg/mL were obtained for EOs obtained from flavedo of fruits of the same species collected in Corigliano Calabro and Rosarno, respectively [44,45]. The EO from C. × clementina flavedo exhibited lipase-inhibitory activity, although with lower potency than the pomelo EOs. [44]. Dang
Regarding the carbohydrate-hydrolysing enzymes, the P4 EO was the most active against α-amylase (IC 50 value of 25.23 µg/mL), whereas P2 was the most active against α-glucosidase (IC 50 value of 25.67 µg/mL). Regarding blends data, the P1P4 sample was the most active against α-amylase (IC 50 value of 21.43 µg/mL). A combination of the EOs increased the α-glucosidase-inhibitory activity, where the blend obtained by mixing equal volumes of 'Maxima' (P2) and Todarii' (P5) was the most active with an IC 50 value of 21.67 µg/mL, followed by the P1P2 blend. Our results are in agreement with those reported for the lemon flavedo EO by Oboh et al. [42], which obtained IC 50 values of 8.16 and 7.56 µg/mL against α-amylase and α-glucosidase, respectively. A promising activity was also observed for the EO obtained by orange flavedo (IC 50 values of 11.51 and 11.53 µg/mL, respectively).
Recently, Itoh et al. [43] analysed different Citrus by-products as possible lipase inhibitors and found IC 50 values of 43 and 44 µg/mL for leaves and flower methanol extract, respectively. Values in the range of 135.51-282.65 µg/mL were obtained for EOs obtained from C. × clementina leaves collected in Rosarno and Cetraro, respectively, against αamylase and α-glucosidase, whereas values in the range of 185.43-287.91 µg/mL were obtained for EOs obtained from flavedo of fruits of the same species collected in Corigliano Calabro and Rosarno, respectively [44,45]. The EO from C. × clementina flavedo exhibited lipase-inhibitory activity, although with lower potency than the pomelo EOs. [44]. Dang [46] did not obtain a-glucosidase-inhibitory activity for pomelo EO from Vietnam. According to Dang et al. [46], D-limonene was the main abundant compound in pomelo oils; although this is presented in the literature as an antidiabetic phytochemical, it is not responsible of the carbohydrate-hydrolysing enzyme-inhibitory activity [47,48]. On the contrary, α-pinene, β-pinene 1,8-cineole, 4-terpineol, and α-terpineol resulted in being able to inhibit α-amylase [49], and the activity of the EO is often the result of an antagonistic or additive effect between the constituents of the oil.

Plant Material
The five C. maxima samples analysed, cultivated in the Botanical Garden of Palermo (38 • 06 48.39" N; 13 • 22 21.68" E), Sicily, were collected in February 2020. In the present work, fruits belonging to the following five cultivars were examined: C. maxima 'Chadock' (P1), C. maxima 'Maxima' (P2), C. maxima 'Pyriformis' (P3), C. maxima 'Terracciani' (P4), and C. maxima 'Todarii' (P5). The samples, identified by Prof. Rosario Schicchi and Prof. Anna Geraci, were kept in the "Herbarium Mediterraneum" of the Botanical Garden of the University of Palermo (PAL). The number of the voucher was reported for each cultivar. C. maxima 'Chadock' (P1) (Voucher No. 109746) is characterised by large, flattened, turbinate fruits, which are slightly umbilicate at the apex. The leaves are ovate-oblong, with a winged petiole, especially for those at the ends of twigs. C. maxima 'Maxima' (P2) (Voucher No. 109743) is characterised by very large fruits, cylindrical in shape, and slightly narrowed at the insertion of the peduncle. The leaves are ovate-oblong, quite curled, with a slightly winged petiole. C. maxima 'Pyriformis' (P3) (Voucher No. 109742) is characterised by small-medium-sized pyriform fruits, tapering at the insertion of the peduncle, and with a small protuberance at the apex. The leaves are ovate-oblong with a slightly winged petiole. C. maxima 'Terracciani' (P4) (Voucher No. 109744) is characterised by small, ovalshaped fruits with a persistent style and stigma. The leaves are ovate-oblong, with a slightly winged petiole. C. maxima 'Todarii' (P5) (Voucher No. 109745) is characterised by medium-large, mostly globular, fruits with a slightly depressed base. The leaves are ovate-oblong, with a slightly winged petiole.

Essential Oil Extractions and Their Blend Preparation
The extraction of EOs was performed according to Basile et al. [50]. Variable quantities of flavedos of P1, P2, P3, P4, and P5 (47-121 g) were subjected to hydro-distillation for 3 h using Clevenger's apparatus [51] to obtain the oils with yields of 0.73%, 1.03%, 0.62%, 0.35%, and 0.47% (v/w), respectively. A combination of EOs was obtained by mixing oils in a 1:1 v/v ratio. EOs and their blends were stored in the freezer at −20 • C, until the time of analysis.

Pomelo EOs' Volatile Profiles
The analyses of EOs and their blends were performed according to the procedure reported by Catinella et al. [52]. Chromatograms of all EOs are reported in Supplementary  Materials (Figures S1-S5). Linear retention indices (LRIs) were determined by using the retention times of n-alkanes (C 8 -C 40 ), and the peaks were identified by comparison with mass spectra and by comparison to their relative retention indices with WILEY275 (Wiley), NIST 17 (NIST, The National Institute of Standards and Technology, Gaithersburg, MD, USA), ADAMS [53], and FFNSC2 (Shimadzu, Kyoto, Japan) libraries.
Briefly, a solution of ABTS + radical, opportunely prepared, was diluted with EtOH to reach an absorbance of 0.70 at 734 nm. EOs were added to a diluted ABTS + solution to test the concentration from 400 to 1 µg/mL and, following incubation, the absorbance was read at 734 nm. The procedure to test the DPPH radical scavenging potential of samples was assessed, as previously described [13]. Briefly, the EOs (1-1000 µg/mL) were mixed with DPPH· radical solution and, following the incubation time, the absorbance was read at 517 nm. Ascorbic acid was used as a positive control in both tests.

Ferric Reducing Ability Power (FRAP Assay)
The Ferric Reducing Ability Power (FRAP) test was used, as previously reported [13]. The EOs (100 µL) were mixed with water and FRAP reagent. After 30 min of incubation, the absorbance was read at 595 nm. The FRAP value was expressed as µM Fe(II)/g. Butylated hydroxytoluene (BHT) was used as a positive control.

β-Carotene Bleaching Test
The protection from lipid peroxidation by pomelo EOs and their combination was assessed using the β-carotene bleaching test [13]. Briefly, β-carotene, Tween 20, and linoleic acid were mixed. The resulting emulsion was added to a 96-well microplate containing EOs in concentrations ranging from 100 to 2.5 µg/mL. Following incubation, the absorbance was read at 470 nm. Propyl gallate was used as a positive control.

Lipase-Inhibitory Assay
To assess the potential anti-obesity effects of pomelo EOs and their mixes, the activity of porcine pancreatic lipase (type II) (EC 3.1.1.3) activity was measured using p-nitrophenyl octanoate as a substrate [54]. The EOs were mixed with the enzyme solution (1 mg/mL in water), 0.005 M of p-nitrophenyl octanoate solution, and Tris-HCl buffer (pH = 8.5). Following incubation at room temperature, the absorbance was read at 412 nm. Orlistat was used as a positive control.

Carbohydrate-Hydrolysing Enzyme-Inhibition Assay
Both α-amylase and α-glucosidase are involved in carbohydrate digestion and have been recognised as targets for postprandial hyperglycaemia modulation [55]. For the α-amylase-inhibitory assay, a starch solution of α-amylase enzyme (EC 3.2.1.1) and colorimetric reagent were prepared [54]. Pomelo oils were added to the starch solution and left to react with the enzyme. The absorbance was read at 540 nm. In the α-glucosidaseinhibitory-activity assay, α-glucosidase (EC 3.2.1.20) was mixed with a maltose solution and O-dianisidine [54]. The pomelo oils were added at different concentrations and were left to incubate at 37 • C for 30 min. Subsequently, perchloric acid was added and the mixture was centrifuged. The supernatant was collected and mixed with DIAN, PGO, and left to incubate at 37 • C for 30 min. Then, the absorbance was read at 500 nm.

Statistical Analysis
All analyses were performed in triplicate. Data are expressed as the means ± standard deviation (S.D.). The concentration-response curve was obtained by plotting the percentage inhibition versus concentration. The concentration that yielded 50% inhibition (IC 50 ) was calculated by nonlinear regression, with the use of GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). Differences within and between the groups for antioxidant assays were evaluated by one-way ANOVA followed Tukey's test, and was applied in order to determine any significant differences among the investigated samples (** p < 0.05). To obtain a ranking of the EOs and their mixes, the antioxidant capacity Relative Antioxidant Capacity Index (RACI) was calculated following the procedure described by Todorovic et al. [56]. This statistical application was generated from the perspective of statistics by integrating the antioxidant-capacity values generated from different in vitro methods.
Principal component analysis was performed according to the procedure reported by Badalamenti et al. [13]. The different chemical classes used to describe the composition of individual essential oils and their mixes were considered as original variables and subjected, after normalisation, to cluster analysis (CA) and to principal component analysis (PCA). The statistical analyses were performed using PRIMER 6 (Massey University Eastbourne, Albany, New Zealand) with two principal component (PC) variables, and the number of clusters were determined by using the rescaled distances in the dendrogram, using a cut-off point (Euclidean distance = 0.8) that allowed for the attainment of consistent clusters. The Principal Components Analysis (PCA) and the Hierarchical Cluster Analysis (HCA) were used to comprehend the similarity among the essential oils in relation to the contents of their chemical constituents. We tested two different cut-off similarity levels (cut-off levels 0.8 and 1.6) that were chosen based on the mean distance between the cluster's measure and the similarities-differences between the samples belonging to the same cluster. Since the HCA analysis is a function of variables and observations, the highest correspondence between PCA and HCA was produced when we applied a cut-off of 0.8. The statistical analysis of the absence/presence was conducted using the cluster method of the PRIMER 6 software (Massey University Eastbourne, Albany, New Zealand).

Conclusions
EOs obtained by the flavedo of C. maxima cultivars 'Chadock', 'Maxima', 'Pyriformis', 'Terracciani', and 'Todarii', collected from the Palermo Botanical Garden, were chemically and biologically investigated, alone and as combinations. D-Limonene was the main abundant monoterpene hydrocarbon in all cases. Chemical and statistical analyses (PCA and HCA) can provide chemodiversity information on the investigated samples. A division of the cultivars into four classes emerged: the first one, composed of P1P5 and P2P5 samples, was characterised by a high percentage of MH and a moderate content of OM. Group B included samples characterised by an equal content of monoterpenic compounds. The P1P3 and P2P3 mixes, chemically similar in all classes, formed the third group. The last group, cluster D, was, instead, made up of samples with the same content of monoterpenic compounds, oxygenated sesquiterpenes, and other metabolites. On the other hand, more detailed PCA and HCA analyses showed the clustering of EOs into three groups: cluster A' can incorporate samples P3P5 and P1P2P3P4P5, which were slightly different, compared to group A, in the content of SH and O constituents. Cluster C' was formed by inclusion in group C of sample P3, containing a higher percentage of other compounds. Finally, cluster D was enlarged to D' by incorporating the P3P4 sample, not previously included due to the slightly higher content of SH compounds.
All tested Sicilian Pomelo EOs and their blends exhibited a promising antioxidant activity trough different mechanisms of action. Among them, pomelo from 'Chadock' was the most active, followed by the blend obtained by an equal-volume combination of 'Chadock' and 'Todarii' cultivars. Regarding the inhibitory activity against the enzymes involved in metabolic syndrome, C. maxima 'Terracciani' and 'Todarii' EOs exhibited the highest lipase-inhibitory activity. Moreover, all EOs were able to inhibit carbohydratehydrolysing enzymes without any significant differences between the EO tested alone and as a blend.
According to our previous data, the bioactivity was not strictly related to the main compound: D-limonene. This was probably due to the common effect observed for natural products when "1 + 1 does not equal 2" [57]. In fact, minor compounds can significantly contribute to the functional properties of EOs, in which they sometimes act synergically.
This study evidenced, for the first time, the chemical and biological properties of Sicilian pomelo essential oils. However, further studies are necessary to identify the possible application of pomelo oils as functional ingredients for the development of functional food or nutraceutical products, alone or as a blend.