Violaxanthin

Carotenoids were extracted following the method described by Liu et al. with slight modifications (Liu et al., 2007 (link)). Carotenoids were determined on a reverse phase Analytical YMC Carotenoid Column C30 (150 × 4.6 mm i.d., 3 μm, Wilmington, NC, USA) using a Waters HPLC system with a photodiode array detector (Waters, Milford, MA). Operation was conducted under subdued light to avoid carotenoid degradation. Identification of carotenoids was performed by comparison with standard spectra. Quantification was performed using the calibration curve generated with commercially available lycopene, β-carotene, β-cryptoxanthin, lutein, and violaxanthin standards (Sigma-Aldrich).

The methanol, hexane, acetone, petroleum ether, dichloromethane, and hydrochloric acid were of analytical grade and were purchased from Labscan (Dublin, Ireland). The HPLC-grade methanol, HPLC-grade acetonitrile, HPLC–grade ethyl acetate, formic acid, sodium chloride, and potassium hydroxide were obtained from Panreac (Barcelona, Spain). The β-Carotene, all-trans-β-apo-8′-carotenal, α-carotene, phytoene, violaxanthin, lutein, β-cryptoxanthin, and lycopene were purchased from Sigma-Aldrich (Taufkirchen, Germany) and the antheraxanthin from DHI (Hørsholm, Denmark). The lutein epoxide, luteoxanthin, zeinoxanthin, 9-cis-antheraxanthin, 9-cis-violaxanthin, 13-cis-violaxanthin, and 9-cis-lutein were obtained as described elsewhere [23 (link),24 (link),25 (link),26 (link),27 (link)]. The gallic acid, p-hydroxybenzoic acid, syringic acid, caffeic acid, m-coumaric, p-coumaric, chlorogenic acid, ferulic acid, naringin, naringenin, ethyl galate, quercetin, kaempferol, crisin, vanillic acid, and myricetin were purchased from Sigma-Aldrich (Madrid, Spain). The quercitrin was obtained from Extrasynthese (Genay, France). All the aqueous solutions were prepared with purified water in a NANOpure Dlamond^TM system (Barnsted Inc., Dubuque, IO, USA).

Pigment standards including chlorophyll a, violaxanthin, vaucheriaxanthin, and β-carotene were purchased from Sigma-Aldrich Chemical Co. (Shanghai, China; http://www.sigmaaldrich.com). Silica gel (200–300 mesh) was obtained from Qing Dao Marine Chemical Co. (Qingdao, China). HPLC-grade solvents used for HPLC analysis were purchased from Guangzhou Runhao Biotech Co. (Guangzhou, China), such as methanol, acetonitrile, acetic ether, and dichloromethane. Other analytical solvents (n-hexane, methanol, and acetone) used in extraction and isolation of violaxanthin were purchased from Guangzhou Runhao Biotech Co. (Guangzhou, China). Deionized water was prepared by a Milli-Q water purification system (Millipore Corp., Bedford, MA, USA).
Chemicals used for their antioxidant activity including 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium persulfate, ferrous chloride, potassium ferricyanide, trichloroacetic acid, hydrogen peroxide, ascorbic acid, sodium dihydrogen phosphate, and disodium hydrogen phosphate were obtained from Sangon Biotech Co. (Shanghai, China).

Carotenoids were extracted using methods specific to the material being examined. For the pericarp tissues, the extraction and saponification were conducted as described by Kim et al. (2016) (link). For the E. coli transformants, cell pellets were obtained using centrifugation, from which the carotenoids were extracted with 600 μl HPLC-grade acetone (Honeywell, Charlotte, USA), as described by López-Emparán et al. (2014) (link).
For the extracts derived from both the pericarps and E. coli cell pellets, the carotenoids were analysed using ultra-performance liquid chromatography (UPLC) on an Acquity UPLC-H-Class system (Waters). The compounds were separated using an Acquity UPLC HSS T3 column (2.1×100, 1.8 μm) at 35 °C. The mobile phase was a binary solvent system consisting of phase A (acetonitrile/methanol/methylene chloride, 65/25/10, v/v/v) and phase B (distilled water). The gradients were programmed according to the method described by Kim et al. (2016) (link). For the qualitative and quantitative analyses, 11 carotenoid standards were purchased from Sigma-Aldrich, namely antheraxanthin, capsanthin, capsorubin, lutein, neoxanthin, violaxanthin, zeaxanthin, α-carotene, α-cryptoxanthin, β-carotene, and β-cryptoxanthin.

Methyl tert-butyl ether (MTBE) and Methanol (MeOH) of HPLC grade were obtained from Thermo Fisher Scientific (Leicestershire, UK). Ptyalize, trypsin (porcine pancreas), pepsin (porcine gastric mucosa), and bile salts to be used in vitro were purchased from Sigma-Aldrich (St. Louis, MO, USA). The β-carotene standards were provided by Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Violaxanthin and antheraxanthin were obtained from Sigma (St. Louis, MO, USA). All other chemicals of analytical grade were provided by Sinopharm chemical reagent Co., Ltd. (Shanghai, China).

Carotenoid were extracted from fresh petals at the flowering stage and detected following the methods of Cao et al.^{51 (link)}. Carotenoid analysis was performed using LC-2010AHT HPLC (Shimadzu, Kyoto, Japan) with C30 column (YMC, Kyoto, Japan). Carotenoids were identified by the typical retention time of the standard compounds, including violaxanthin (Sigma-Aldrich, Saint Louis, America), lutein (Solarbio, Beijing, China), α-carotene and β-carotene (Wako, Osaka, Japan). The identification of prolycopene was performed based on reported the typical retention time and relative order of carotenoid compound peaks^{22 (link),43 (link),51 (link)}. Carotenoid content was quantified according to Morris’ method^{52 (link)}. The total carotenoid content was the sum of all the detected carotenoid compound contents. Three biological replicates were used for all analyses and the calculation of means and standard deviations were conducted. The significant difference between 92S105 and 15S1040 was analyzed by t-test.

Standards Chlorophyll a, analytical standard, PN: 96145, BN: BCCF9581, Violaxanthin, analytical standard, PN: 91904, BN: BCCG6377, Lutein, analytical standard, PN: 07168, BN: BCCG1715, 9-cis-Anteraxanthin, analytical standard, PN: 47999, BN: BCCG6378, Echinenone, analytical standard, PN: 73341, BN: BCCF6266, Zeaxanthin, analytical standard, PN: 14681, BN: BCCD7729, Neoxanthin, analytical standard, PN: 72994, BN: BCCC9416, β-Carotene, analytical standard, PN: 22040, BN: 100919781 were purchased from Sigma Aldrich (Saint Louis, MO, USA).