Screening and Characterization of Phenolic Compounds and Their Antioxidant Capacity in Different Fruit Peels

Fruit peels have a diverse range of phytochemicals including carotenoids, vitamins, dietary fibres, and phenolic compounds, some with remarkable antioxidant properties. Nevertheless, the comprehensive screening and characterization of the complex array of phenolic compounds in different fruit peels is limited. This study aimed to determine the polyphenol content and their antioxidant potential in twenty different fruit peel samples in an ethanolic extraction, including their comprehensive characterization and quantification using the LC-MS/MS and HPLC. The obtained results showed that the mango peel exhibited the highest phenolic content for TPC (27.51 ± 0.63 mg GAE/g) and TFC (1.75 ± 0.08 mg QE/g), while the TTC (9.01 ± 0.20 mg CE/g) was slightly higher in the avocado peel than mango peel (8.99 ± 0.13 mg CE/g). In terms of antioxidant potential, the grapefruit peel had the highest radical scavenging capacities for the DPPH (9.17 ± 0.19 mg AAE/g), ABTS (10.79 ± 0.56 mg AAE/g), ferric reducing capacity in FRAP (9.22 ± 0.25 mg AA/g), and total antioxidant capacity, TAC (8.77 ± 0.34 mg AAE/g) compared to other fruit peel samples. The application of LC-ESI-QTOF-MS/MS tentatively identified and characterized a total of 176 phenolics, including phenolic acids (49), flavonoids (86), lignans (11), stilbene (5) and other polyphenols (25) in all twenty peel samples. From HPLC-PDA quantification, the mango peel sample showed significantly higher phenolic content, particularly for phenolic acids (gallic acid, 14.5 ± 0.4 mg/g) and flavonoids (quercetin, 11.9 ± 0.4 mg/g), as compared to other fruit peel samples. These results highlight the importance of fruit peels as a potential source of polyphenols. This study provides supportive information for the utilization of different phenolic rich fruit peels as ingredients in food, feed, and nutraceutical products.


Extraction of Phenolic Compounds
To extract the phenolic compounds, 2.0 ± 0.5 g of each fruit peel powder was mixed with 20 mL 70% ethanol. The samples were homogenized at 10, 000 rpm for 30 s using the IKA Ultra-Turrax T25 homogenizer (Rawang, Selangor, Malaysia) and subjected to shaking incubator (ZWYR-240, Labwit, Ashwood, VIC, Australia) at 120 rpm for 12 h (4 °C ). After incubation, the extracts were centrifuged with Hettich Refrigerated Centrifuge (ROTINA380R, Tuttlingen, Baden-Württemberg, Germany) at 5, 000 rpm for 15 min. The supernatants were collected and stored at − 20 °C for 2 weeks for antioxidant analysis. For HPLC and LC-MS analysis, the extracts were filtrated through a 0.45 μm syringe filter (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Estimation of Polyphenols and Antioxidant Potential
For polyphenol estimation in selected fruit peel samples, TPC, TFC, and TTC assays were performed while for measuring their antioxidant potential, four different types of antioxidant assays including DPPH, ABTS, FRAP and TAC were performed by adopting our previously published methods of Tang, et al. [18]. The data was determined using a Multiskan® Go microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA).

Determination of Total Phenolic Content (TPC)
For the TPC, 25 μL extracts of each peel extract, 200 μL of water and 25 μL of Folin-Ciocalteu reagent solution (1:3 v/v), diluted with water was added to 96 well plate (Corning Inc., Midland, NC, USA) followed by incubation at 25 °C for 5 minutes. After that, 25 µ L 10% (w:w) sodium carbonate was added and incubated for 1 h at 25 °C followed by the measurement of absorbance at 765 nm by a spectrophotometer plate reader (Thermo Fisher Scientific, Waltham, MA, USA). The quantification of total phenolic content was based on a standard curve generated from gallic acid with the concentrations from 0 -200 µ g/mL and results were expressed as mass (mg) of gallic acid equivalents (GAE) per weight of sample.

Determination of Total Flavonoids Content (TFC)
For the TFC, 80 μL of each peel extract, 80 μL of 2% (w/v) aluminum chloride solution and 120 μL of 50 g/L sodium acetate solution were added in a 96-well plate followed by incubation at 25 °C for 2.5 h and absorbance was measured at 440 nm. For quantification, a standard curve was made with quercetin (0 -50 μg/mL) and results were expressed as mass (mg) of quercetin equivalents (QE) per weight of sample.

Determination of Total Tannins Content (TTC)
For the TTC, 25 μL of extract, 150 μL 4% (w/v) vanillin solution and 25 μL of 32% (v/v) sulphuric acid were incubated at 25 °C for 15 min, absorbance was measured at 500 nm. For quantification, a standard curve was generated from catechin using the concentrations of 0 -1000 μg/mL and results were expressed as mass (mg) of catechin equivalents (CE) per weight of sample.
2.4.4. Determination of 2,2'-Diphenyl-2-picryl-hydrazyl (DPPH) Antioxidant Assay For the DDH assays, 40 μL of each fruit peel extract and 260 μL of 0.1 M DPPH radical methanol solution was added into 96-well plate and incubated at 25 °C for 30 min. The absorbance was measured at 517 nm using a microplate reader. A standard curve was generated using 0 -50 μg/mL ascorbic acid aqueous solution. The results were expressed as mass (mg) of ascorbic acid equivalents (AAE) per weight of sample.

Determination of Ferric Reducing Antioxidant Power (FRAP) Assay
To prepare the FRAP reagent, 300 mM sodium acetate buffer (pH 3.6), 10 mM TPTZ solution, and 20 mM ferric chloride in a ratio of 10:1:1 (v/v/v) was prepared freshly. A 20 μL of peel extracts and 280 μL of freshly prepared FRAP reagent were mixed in a 96 well plate followed by incubation at 37 °C for 10 min, absorbance was measured at 593 nm. A standard curve was achieved using concentrations of 0 -50 μg/mL ascorbic acid and results were expressed as mass (mg) of AAE per weight of sample.
2.4.6. Determination of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Assay The ABTS + dye was prepared with 5 mL of 7 mM of ABTS solution mixed with 88 μL of 140 mM potassium persulfate solution, incubated in the dark at room temperature for 16 h to generate an ABTS + free radical solution. Further, ABTS + stock solution was prepared by diluted with ethanol to gain absorbance of 0.70 at 734 nm. For the ABTS assay, 10 μL fruit peel extract and 290 μL of freshly prepared ABTS + solution were added in 96 well plate and incubated at 25 °C for 6 min. Subsequently, the absorbance was measured at 734 nm. A standard curve was achieved using concentrations of 0 -150 μg/mL ascorbic acid and the results were expressed as mass (mg) of AAE per weight of sample.

Determination of Total Antioxidant Capacity (TAC)
For the TAC, 40 μL of each fruit peel extract was added to 260 μL of phosphomolybdate reagent (0.6 M H2SO4, 0.028 M sodium phosphate and 0.004 M ammonium molybdate). The mixture was incubated at 95 °C for 10 min, cooled at room temperature and absorbance was measured at 695 nm. A standard curve was generated using concentrations of 0 -200 μg/mL ascorbic acid and the results were expressed as mass (mg) of AAE per weight of sample.

Characterization of Phenolic compounds using LC-ESI-QTOF-MS/MS Analysis
The phenolic compound characterization was performed on an Agilent 1200 HPLC with an Agilent 6520 Accurate-Mass Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA). The separation was conducted using a Synergi Hydro-RP 80 Å , reverse phase column (250 mm x 4.6 mm, 4 μm particle size) with protected C18 ODS (4.0 × 2.0 mm) guard column (Phenomenex, Lane Cove, NSW, Australia) by adopting our previously published method of Zhong,et al. [19]. In brief, the mobile phase consisted of water/acetic acid (98:2, v/v; eluent A) and acetonitrile/acetic acid/ water (50:0.5:49.5, v/v/v; eluent B). The gradient profile was described as follows: A 6 µ L of each peel extract was injected and the flow rate was set at 0.8 mL/min. Peaks were identified in both positive and negative ion modes with the capillary and nozzle voltage set to 3.5 kV and 500 V, respectively. Additionally, following conditions were maintained; i) nitrogen gas temperature at 300 °C, ii) sheath gas flow rate of 11 L/min at 250 °C, ii) nitrogen gas nebulisation at 45 psi. A complete mass scan ranging from m/z 50 to 1300 was used, MS/MS analyses were carried out in automatic mode with collision energy (10, 15 and 30 eV) for fragmentation. Peak identification was performed in both positive and negative modes while the instrument control, data acquisition and processing were performed using LC-ESI-QTOF-MS/MS MassHunter workstation software (Qualitative Analysis, version B.03.01, Agilent Technologies, Santa Clara, CA, USA).

Quantification of Phenolic compounds using HPLC-PDA
The quantitative measurement of targeted phenolic compounds present in different fruit peels samples was performed with an Agilent 1200 HPLC equipped with a photodiode array (PDA) detector by adopting our previously published protocol of Ma,et al. [20]. In brief, the same column and conditions were maintained as described above in LC-ESI-QTOF-MS/MS, except for a sample injection volume of 20 µ L. The twenty most abundant phenolic compounds present in the different fruit peels including 10 phenolic acids and 10 flavonoids, were selected for quantification purposes. The phenolic compounds were determined at three different wavelengths, including 280 nm, 320 nm, and 370 nm. The quantification of targeted polyphenols was based on the calibration standard curve and the result was expressed as mg/g of sample. Data collection and processing was performed using Agilent MassHunter workstation software (Agilent Technologies, Santa Clara, CA, USA).