The samples of DE in vitro experiments were analyzed using an HPLC system consisting of a system controller (SCL-10 ATVP; Shimadzu, Japan), a binary pump (LC-10 ATVP, Shimadzu), a UV-VIS detector (SPD-10 AVP, Shimadzu), a column oven, and an autoinjector (SIL-10A, Shimadzu). The separation method was under the following conditions: C18 reversed phase analytical column (4.6 × 150 mm2, 5 μm, Shim-pack VP-ODS). The mobile phase was 60 : 40 (v/v) methanol-ammonium acetate buffer (0.05 M, pH 4.0), column temperature of 40°C, UV detective wavelength of 257 nm, flow rate of 1.0 mL/min, and injection volume of 10 μL. The data were acquired and analyzed by Shimadzu Class-VP chromatography software. There was no interference from skin and a well-separated peak was detected at the retention time of 9.1 ± 0.1 min with the sensitivity of 0.02 μg/mL. The peak area correlated linearly with DE concentration in the range from 1 to 500 μg/mL.
Scl 10at vp
Manufactured by Shimadzu
Sourced in Japan
The SCL 10AT VP is a liquid chromatography (LC) system controller module manufactured by Shimadzu. It is a core component responsible for controlling the operation and synchronization of other LC system modules, such as pumps, detectors, and autosamplers.
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3 protocols using scl 10at vp
1
HPLC Analysis of DE In Vitro Samples
2
HPLC Analysis of Beta-carotene
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Beta-carotene was measured according to the procedure described by [17, (link)18] (link) with some modifications. For HPLC determination, 0.2 g of freeze-dried material for each cultivar sample was used and carotenoids were extracted with 20 mL of tetrahydrofuran (THF). The mixture was mechanically stirred for 30 s and the extract was filtrated with syringe filters in regenerated cellulose 0.45 µm. The samples were injected into an HPLC model Shimadzu liquid chromatography system (model SCL 10AT VP) equipped with a high-pressure pump (model LC-10AT VP), automatic loop injector (50 µL; model SIL-10AF). Column: Tracer Extrasil ODS2 (250 × 45 mm, 5 µm) using the mobile phase methanol:THF:water (67:27:6, v/v/v). The flow-rate of the mobile phase was 1 mL min -1 and the absorbance was measured at 470 nm.
3
Quantification of 3-Deoxyanthocyanins in Samples
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The 3-deoxyanthocyanins: luteolinidin (LUT), apigeninidin (API), 5-methoxyluteolinidin (5-MeO LUT) and 7-methoxyapigeninidin (7-MeO API) contents were determined according to a method proposed by Yang, Allred, Geera, Allred, and Awika (2012) , modified by Cardoso et al. (2014) . The compounds were extracted from 1 g of sample with 10 mL of 1% HCl in methanol. Analyzes were performed in an HPLC system (Shimadzu, SCL 10AT VP, Japan) equipped with diode array detector (Shimadzu, SPD-M10A, Japan), high pressure pump (Shimadzu, LC-10AT VP, Japan), autosampler with loop of 500 mL (Shimadzu, SIL-10AF, Japan), and helium degassing system using the chromatographic conditions described by Cardoso et al. (2014) .
Identification was performed by correlating the retention time and the absorption spectrum of peaks of the standards and samples, analyzed under the same conditions. The quantification of each compound was performed by comparison of peak areas with those of standard curves constructed through injection, in duplicate, of six different standard concentrations (R 2 ranged from 0.9939 to 0.9992). The 5-MeO-LUT and 7-MeO-API contents were quantified using standards of luteolinidin and apigeninidin, respectively, as well as with the appropriate molecular weight correction factor (Dykes, Seitz, Rooney, & Rooney, 2009) . Results were expressed in lg/g dry matter.
Identification was performed by correlating the retention time and the absorption spectrum of peaks of the standards and samples, analyzed under the same conditions. The quantification of each compound was performed by comparison of peak areas with those of standard curves constructed through injection, in duplicate, of six different standard concentrations (R 2 ranged from 0.9939 to 0.9992). The 5-MeO-LUT and 7-MeO-API contents were quantified using standards of luteolinidin and apigeninidin, respectively, as well as with the appropriate molecular weight correction factor (Dykes, Seitz, Rooney, & Rooney, 2009) . Results were expressed in lg/g dry matter.
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