Chemicals and materials
Folin’s phenol reagent, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPB), D-catechin, gallic acid monohydrate and ethyl alcohol were prepared by MilliporeSigma (St. Louis, MO, USA). Nitric acid (Dongwoo Fine-Chem, Iksan, Korea) and standards of minerals (AccuStandard, New Haven, CT, USA), namely Ca, K, copper (Cu), iron (Fe), Mg, manganese (Mn), sodium (Na) and zinc (Zn), were used.
Samples
Pepper of 5 kg was prepared from mature peppercorns harvested in March 2017, by Ottogi Sesame Mills Co., Ltd (Eumseong, Choungcheonbuk-do, Korea). The peppercorns were separated from the stems after harvesting them, and then they were naturally sun-dried for 7–10 days to obtain a black colour. Finally, pericarp was mechanically peeled off from the pepper using a decorticating machine (Sinco Mechanical JSC, Vietnam). It was ground and sieved by 60-mesh and then it was stored at room temperature in darkness.
Sample preparation
The ground pericarp sample of 5 g was homogenised with 80% ethyl alcohol of 100 mL using KWG-150 of Sunway Electric Manufacture (Heshan, China). The mixture was incubated for a day in shaking incubator (a DH.WIS 02011, Daihan Scientific Co., Ltd., Daegu, Gyeongbuk, Korea), followed by centrifugation at 15,000 rpm for 10 min (Mega21R, Hanil Scientific, Inc., Gimpo, Gyenggi-do, Korea). The upper layer was taken and filtered by a Minisart filter (0.45 μm pore size, regenerated cellulose) and then concentrated to 20 mL on a rotary evaporator (N-1000, Eyela, Tokyo, Japan). The extract was stored at − 80 °C until analyzing chemical components and antioxidant activities.
Bioactive compounds and antioxidant activities of pepper pericarp
Total flavonoids
The total flavonoids were determined by a colorimetric assay [19, 20]. A 1 mL of sample extract and distilled water (4 mL) were placed in a 15-mL tube, then 0.3 mL of 5% NaNO2 was added to the tube, and the mixture was left to react at room temperature for 5 min. After adding 0.3 mL of 10% AlCl3, the mixture was left to react further at room temperature for 6 min before 2 mL of 1 N NaOH was added. Distilled water was added to adjust the total volume to 10 mL. The absorbance was recorded by a spectrophotometer (Optizen POP, Mecasys, Daejeon, Korea) at 510 nm. Catechin was used as the standard to establish the calibration curve. Total flavonoids were expressed as milligrams of catechin equivalents (CE)/100 g fresh weight (FW).
Total phenols
The total phenols were analysed by the Folin–Ciocalteu colorimetric method [19, 20]. Folin–Ciocalteu reagent (0.2 mL) was added to a 15 mL tube containing 0.2 mL of the sample extract and 2.6 mL of distilled water. The mixture was left at room temperature for 6 min, and then 0.2 mL of 7% Na2CO3 was added, followed by incubation at room temperature for 90 min in the dark. Spectrophotometric absorbance was determined at 750 nm. The total phenol contents were shown as milligrams of gallic acid equivalents (GAE)/100 g FW by evaluating a gallic acid calibration curve.
Piperine
Piperine was determined, as described by Santosh et al. with some modifications [21]. A 50 mL conical tube containing 0.1 g of the sample extract and 50 mL methanol was extracted by ultrasonication at 50 °C for 20 min. The extracted sample was cooled down at room temperature and filtered through a Minisart syringe filter (0.45 μm, regenerated cellulose). Piperine was detected using a high-performance liquid chromatography (HPLC) apparatus (Agilent, Santa Clara, CA, USA) equipped with a diode array detector (340 nm) and Eclipse C18 Plus column (4.6 × 150 mm, 5 μm; Agilent) maintained at 25 °C. Acetonitrile–1% citric acid (45:55, v/v) was used as the mobile phase in isocratic mode at the flow rate of 1 mL/min for 20 min. The injection volume was 10 μL. Peak of piperine and its spectra in the HPLC chromatogram was identified (Fig. 1).
DPPH radical scavenging activity
DPPH radical scavenging activity was analyzed by the method reported by Brand-Williams et al. with some modifications [22]. DPPH solution (100 μM) was diluted with 80% methanol to have an absorbance of 0.65 ± 0.02 at a wavelength of 517 nm. The extracted sample (50 μL) was mixed with 2950 μL of DPPH diluted from 0.1 mM of DPPH solution and reacted for 30 min. The absorbance at 517 nm (Optizen POP spectrophotometer, Mecasys) was shown as milligrams of vitamin C equivalents (VCE)/100 g FW.
ABTS radical scavenging activity
The ABTS radical scavenging activity was measured with an analytical method described by Floegel et al. [23]. ABTS (137.175 mg) and AAPH (27.117 mg) were prepared in 100 mL of phosphate-buffer, followed by reaction in a water bath at 70 °C for 40 min to produce ABTS radicals, and then cooled down at room temperature. The working solutions were diluted with the ABTS radical solution by phosphate-buffered saline to obtain an optical density at 734 nm of between 0.63 and 0.67. Finally, the sample (20 μL) and working solution (980 μL) were mixed and reacted at 37 °C for 10 min. The absorbance of the solution at 734 nm was determined by spectrophotometer (Optizen POP, Mecasys). The antioxidant activity of the solution was expressed as milligrams of VCE/100 g FW.
Flavour compounds by gas chromatography/mass spectrometry (GC/MS)
Two grams of sample was added into an amber vial of 20 mL with PTFE/silicone septa, and it was equilibrated at 80 °C for 30 min with the headspace sampler under agitation at 300 rpm. GC/MS analysis was performed using a 6890 GC/MS system (Agilent Co., Santa Clara, CA, USA) installed with an HP-INNOWax column (60 m × 0.25 mm × 0.25 μm; Agilent Co.) [24]. The sample (1 mL) was injected by a syringe in split injection mode (20:1) at 230 °C, with helium (> 99.9% purity) as the carrier gas at a flow rate of 1 mL/min. The oven was initially maintained at 40 °C for 2 min, then heated at a rate of 5 °C/min to 230 °C and maintained at this temperature for 10 min. The flavours were identified by comparison of the obtained spectra to library (NIST 2005, John Wiley & Sons, New York, NY, USA). Figure 2 is the GC/MS chromatogram of the peaks of flavours in the black pepper pericarp.
Flavour compounds by electronic nose (e-nose)
A GC-type e-nose (Heracles II, Alpha MOS, Toulouse, France) with dual column (non-polar MXT-5 and slightly polar MXT-1701, 10 m length × 180 μm diameter; Restek, Lisses, France) and dual flame ionisation detector (FID) was used for flavour component profile analysis. The oven temperature was increased from 50 °C (maintained for 2 s) to 260 °C at 1 °C/s. The sample (0.8 g) was transferred to a 20-mL headspace vial and incubated at 50 °C for 5 min, with agitation at 500 rpm. Then, 1000 μL was injected at a flow rate of 1 mL/min and measured five times. For peak identification and principal component analysis (PCA), AroChemBase (NIST retention index database) and AlphaSoft (version 17.0; Alpha MOS) were used.
Minerals
Ca, Cu, Fe, K, Mg, Mn, Na and Zn were identified by the procedure developed by Jung et al. [24]. Aliquot of the sample of 0.6 g was prepared in a Teflon digestion vessel with concentrated nitric acid of 7 mL, and it was digested for 40 min under incrementally increasing pressure heating at 1000 W. The digested sample was diluted with water in flasks of 50 mL for analysis of the minerals using an inductively coupled plasma atomic emission spectrometer (ACTIVA-M, HORIBA Jobin–Yvon, Longjumeau, France) with a charge-coupled device. Argon gas plasma was used (flow rate of 13 L/min, sheath flow of 1.5 L/min). The absorption wavelengths were 317.933, 324.754, 238.207, 766.490, 285.213, 275.610, 589.592 and 213.857 nm for Ca, Cu, Fe, K, Mg, Mn, Na and Zn, respectively. A multi-element standard solution was diluted for calibration. The determined concentrations were shown as milligrams/100 g FW.
Statistical analysis
All experiments were conducted triplicately. Data were presented as mean ± standard deviation using Microsoft Office Excel 2010 (Microsoft Corporation, Redmond, WA, USA). Analysis of variance was performed using SAS version 9.4 (SAS Institute, Inc., Cary, NC, USA) for the statistical analysis of each experiment. Significance was determined by Duncan’s multiple range test with p < 0.05.