Series 200 autosampler

The HPLC system (PerkinElmer, Inc., Shelton, CT 06484, USA) consisted of a Series 200 autosampler, Series 200 LC Pump, and a Flexar UV/Vis detector. The column was a LiChrospher RP-18 (5 um, 125 mm × 4 mm, 100A) (Phenomenex, Torrance, CA 90501, USA) with a corresponding guard column (4 × 4 mm, replaced after 50–60 injections). Individual boswellic acid standards of primary analytical grade (Acetyl-11-Keto-β-Boswellic Acid, 3-(P) (AKBA) and Keto-β-Boswellic Acid, 11-(P) (KBA)) were purchased from ChromaDex (Irvine, CA 92618, USA). All chemicals and reagents used in the study were of high purity. HPLC grade methanol (MeOH), acetonitrile (ACN) and 85%-ortho-phosphoric acid were purchased from Fisher Scientific (Suwanee, GA 30024, USA). Ultra-pure water was produced using a Purelab Classic system (ELGA LabWater, High Wycombe HP14 3BY, UK).

Garlic hydroalcoholic extract was weighed, dissolved in water and filtered before injection into a HPLC Perkin Elmer apparatus consisting of a Series 200 LC pump, a Series 200 DAD and a Series 200 autosampler, including TotalChrom Perkin Elmer software for the acquisition of data.
Chromatography was performed on an RP phenyl-hexyl column using a mobile phase consisting of acetonitrile and water acidified by 5% acetic acid, with a linear gradient from 98% to 50% aqueous phase in 33′, followed by a second step in isocratic mode, at a flow of 1 mL/min. Calibration curves of alliin (y = 8.96x + 41.84; R² = 0.9974), gallic acid (y = 41.24x + 67.40; R² = 0.9969), protocatechuic acid (y = 20.38x + 49.44; R² = 0.9998) and quercetin-3-galactoside (y = 49.689x + 34.98; R² = 0.9999) were used for the quantitative analyses. All the standards compounds were purchased from Merck Life Science S.R.L. (Milan, Italy).

Dried extracts were weighed, dissolved in DMSO or water and filtered before injection into an HPLC Perkin Elmer apparatus (Waltham, MA, USA) consisting of a Series 200 LC pump, a Series 200 DAD and a Series 200 autosampler, including a Totalchrom Perkin Elmer software for the data acquisition. Chromatography was performed on a Luna RP18 column (250 × 4.6 mm i.d., 5 μm) using a mobile phase made by acetonitrile and water acidified by 5% formic acid, in gradient with a flow rate of 1 mL/min, at 360 and 530 nm. Punicalagin anomers α and β and ellagic acid were quantified as previously described [7 (link)]. The multi-component phenolic fingerprint was also studied for the most promising pomegranate extracts (DC and Mz) according to a validated method as reported in the Supplementary Materials [36 (link)].

HPLC analysis was carried out by a Perkin–Elmer (Waltham, MA, USA) apparatus equipped with a series LC 200 pump, a series 200 diode array detector and a series 200 autosampler. Data acquisition and processing were carried out with a Perkin–Elmer Totalchrom software. The chromatographic separation was performed as previously described [22 (link)]. In brief, a Luna RP18 column (250 × 4.6 mm, i.d. 5 μm) and a mobile phase, consisting of acetonitrile (A) and acidic water solution (B) in gradient, were used. The detection wavelengths were set at 520 nm for the detection of anthocyanins, at 360 nm for the detection of other flavonoids and at 280 nm for phenolic acids, catechins and other polyphenolic components. The injection volume for each extract was 10 μL. Pelargonidin-3–glucoside, identified at 520 nm in each sample, was quantified by an external-matrix matched calibration method on the basis of the area ratios respect to the pure chemical standard (R² = 0.9984). The other anthocyanins were calculated as the sum of all the chromatographic peaks identified at 520 nm. Calibration curves were built and used for quantitation of polyphenols, using, at 280 nm, epicatechin (R² = 0.9878), caffeic acid (R² = 0.9984), p–coumaric acid (R² = 0.9879) and ferulic acid (R² = 0.9974) and, at 360 nm, rutin (R² = 0.9986) and quercetin-3-D-galactoside (R² = 0.9999) as reference standards.

Kiwi peels hydroalcoholic extracts were weighed, dissolved in methanol and analysed with a HPLC-DAD (Perkin Elmer, Milan, Italy), equipped with a Series 200 LC pump, a Series 200 DAD and a Series 200 autosampler, including a TotalChrom Perkin Elmer software for plotting data. The analyses were performed on a Luna RP-18, 3µ, with a linear gradient constituted by acetonitrile and water acidified by 5% formic acid, from 100% of aqueous phase to 35% in 55 min, at flow rate of 0.9 mL/min. Calibration curves were expressed in µg/mL and were constructed for catechin (y = 5.18 x–− 24.29; R² 0.9997), epicatechin (y = 5.01x + 21.6; R² 0.9995), caffeic acid (y = 26.03x + 20.37; R² 0.9974), sinapic acid (y = 11.37x + 9.92; R² 0.9984).