Ascorbic acid standard

All chemicals and reagents, including a 99.7% purity ascorbic acid standard, were purchased from Sigma-Aldrich S.p.a. (Milan, Italy). Analytical grade solvents were bought from VWR International S.r.l. (Milan, Italy).

The effectiveness of the vacuum impregnation was assessed on the basis of ascorbic acid content (AAC) and impregnation degree (ID). Ascorbic acid extraction was carried out with 1 % m-HPO₃[19] (link). The samples were analyzed using an LC Agilent Technologies 1200 Rapid Resolution (Waldbronn, Germany) system, equipped with a Zorbax SB-C18 (4.6 mm × 150 mm; 5 µm) (Agilent Technologies, Santa Clara, CA, USA). Mobile phases used methanol (phase A) and 0.005 mol/L KH₂PO₄ solutions (phase B). To detect the ascorbic acid, a gradient of methanol (solvent A) (Sigma Aldrich, Steinheim, Germany) per 0.005 mol/L KH₂PO₄ and pH 2.6 (solvent B) (Sigma Aldrich, Tokyo, Japan) was used according to the following program: linear increment starting with 5 % A to 22 % in 6 min., then a return to the initial conditions within the next 9 min., with a flow rate of 0.7 mL/min [20] (link). The eluate was detected using a UV–vis detector set to 245 nm. The ascorbic acid was identified by comparing its retention time with that of an ascorbic acid standard (Sigma Aldrich, St. Louis, MO, USA).
The impregnation degree (ID) was the relative change in the batch mass determined by the relationship: $\mathit{ID}=\frac{m_i-m_0}{m_0}·100\%$ where m₀ and m_i are the masses of the material before and after impregnation, respectively.

Ascorbic acid was extracted from 1 g of tomato peel dispersed in 2 mL of distilled water followed by homogenization with Ultra-Turrax (IKA^®) and filtration through a 0.45 µm membrane filter [66 (link)]. For the HPLC method, an RP-C18 column (SUPELCO Kromasil 100A-5u-C18 4.6 mm × 250 mm) was used. The mobile phase consisted of 0.01 mol/L KH₂PO₄ buffer solution (pH = 2.6 with o-phosphoric acid), with a flow rate of 0.5 mL/min and a detection absorbance set at 250 nm. For quantification, a standard calibration curve consisting of five points at the increasing concentrations of 6.25, 12.5, 25, 50 and 100 µg/mL of ascorbic acid standard (Sigma Chemical, St. Louis, MI, USA) was used.

All chemicals used in the current study were of analytical grade and procured from SRL Chemical Pvt. Ltd., Bangalore, India. Sodium nitrite, standard ascorbic acid, and carotenoids were purchased from Sigma Chemicals, St. Louis, MO, USA. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), hippuryl-histidyl-leucine (HHL), captopril, and angiotensin-converting enzyme (ACE) were also were purchased from Sigma Chemical Co. All the organic and inorganic solvents used in the study were of analytical grade and were procured from SRL Chemical Pvt. Ltd., Bangalore.