TYPE Original Research PUBLISHED 28 February 2023 DOI 10.3389/fnut.2023.1132228 Integrated metabolite analysis and OPEN ACCESS health-relevant in vitro EDITED BY Dejian Huang, functionality of white, red, and National University of Singapore, Singapore REVIEWED BY orange maize (Zea mays L.) from Beatriz Andrea Acosta-Estrada, Monterrey Institute of Technology and Higher Education (ITESM), Mexico the Peruvian Andean race Ivan Luzardo-Ocampo, National Autonomous University of Mexico, Mexico Cabanita at different maturity *CORRESPONDENCE Lena Gálvez Ranilla stages lgalvez@ucsm.edu.pe SPECIALTY SECTION 1,2 3 1 This article was submitted to Lena Gálvez Ranilla *, Gastón Zolla , Ana Afaray-Carazas , Food Chemistry, Miguel Vera-Vega 3, Hugo Huanuqueño 4, a section of the journal 5 6 7,8 Frontiers in Nutrition Huber Begazo-Gutiérrez , Rosana Chirinos , Romina Pedreschi RECEIVED 27 December 2022 and Kalidas Shetty 9 ACCEPTED 09 February 2023 PUBLISHED 28 February 2023 1 Laboratory of Research in Food Science, Universidad Catolica de Santa Maria, Arequipa, Perú, 2 Escuela Profesional de Ingeniería de Industria Alimentaria, Departamento de Ciencias e Ingenierías Biológicas y CITATION Químicas, Facultad de Ciencias e Ingenierías Biológicas y Químicas, Universidad Catolica de Santa Ranilla LG, Zolla G, Afaray-Carazas A, Maria, Arequipa, Perú, 3 Laboratorio de Fisiología Molecular de Plantas, PIPS de Cereales y Granos Vera-Vega M, Huanuqueño H, Nativos, Facultad de Agronomía, Universidad Nacional Agraria La Molina, Lima, Perú, 4 Programa de Begazo-Gutiérrez H, Chirinos R, Investigación y Proyección Social en Maíz, Facultad de Agronomía, Universidad Nacional Agraria La Pedreschi R and Shetty K (2023) Integrated Molina, Lima, Perú, 5 Estación Experimental Agraria Arequipa, Instituto Nacional de Innovación Agraria metabolite analysis and health-relevant in vitro (INIA), Arequipa, Perú, 6 Instituto de Biotecnología, Universidad Nacional Agraria La Molina, Lima, Perú, functionality of white, red, and orange maize 7 Escuela de Agronomía, Facultad de Ciencias Agronómicas y de los Alimentos, Pontificia Universidad (Zea mays L.) from the Peruvian Andean race Católica de Valparaíso, Valparaíso, Chile, 8 Millennium Institute Center for Genome Regulation (CRG), Cabanita at different maturity stages. Santiago, Chile, 9 Department of Plant Sciences, North Dakota State University, Fargo, ND, United States Front. Nutr. 10:1132228. doi: 10.3389/fnut.2023.1132228 COPYRIGHT The high maize (Zea mays L.) diversity in Peru has been recognized worldwide, © 2023 Ranilla, Zolla, Afaray-Carazas, Vera- but the investigation focused on its integral health-relevant and bioactive Vega, Huanuqueño, Begazo-Gutiérrez, Chirinos, Pedreschi and Shetty. This is an open- characterization is limited. Therefore, this research aimed at studying the access article distributed under the terms of variability of the primary and the secondary (free and dietary fiber-bound the Creative Commons Attribution License phenolic, and carotenoid compounds) metabolites of three maize types (CC BY). The use, distribution or reproduction in other forums is permitted, provided the (white, red, and orange) from the Peruvian Andean race Cabanita at different original author(s) and the copyright owner(s) maturity stages (milk-S1, dough-S2, and mature-S3) using targeted and are credited and that the original publication in untargeted methods. In addition, their antioxidant potential, and α-amylase this journal is cited, in accordance with accepted academic practice. No use, and α-glucosidase inhibitory activities relevant for hyperglycemia management distribution or reproduction is permitted which were investigated using in vitro models. Results revealed a high effect of the does not comply with these terms. maize type and the maturity stage. All maize types had hydroxybenzoic and hydroxycinnamic acids in their free phenolic fractions, whereas major bound phenolic compounds were ferulic acid, ferulic acid derivatives, and p-coumaric acid. Flavonoids such as luteolin derivatives and anthocyanins were specific in the orange and red maize, respectively. The orange and red groups showed higher phenolic ranges (free + bound) (223.9–274.4 mg/100 g DW, 193.4– 229.8 mg/100 g DW for the orange and red maize, respectively) than the white maize (162.2–225.0 mg/100 g DW). Xanthophylls (lutein, zeaxanthin, neoxanthin, and a lutein isomer) were detected in all maize types. However, the orange maize showed the highest total carotenoid contents (3.19–5.87 μg/g DW). Most phenolic and carotenoid compounds decreased with kernel maturity in all cases. In relation to the primary metabolites, all maize types had similar fatty acid contents (linoleic acid > oleic acid > palmitic acid > α-linolenic acid > Frontiers in Nutrition 01 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 stearic acid) which increased with kernel development. Simple sugars, alcohols, amino acids, free fatty acids, organic acids, amines, and phytosterols declined along with grain maturity and were overall more abundant in white maize at S1. The in vitro functionality was similar among Cabanita maize types, but it decreased with the grain development, and showed a high correlation with the hydrophilic free phenolic fraction. Current results suggest that the nutraceutical characteristics of orange and white Cabanita maize are better at S1 and S2 stages while the red maize would be more beneficial at S3. KEYWORDS Zea mays, Peruvian maize, Cabanita, primary metabolites, secondary metabolites, antioxidant capacity, hyperglycemia, biodiversity 1. Introduction The maize race Cabanita has been cultivated since the Pre-Inca period in the southern Andean region of Arequipa in Peru at around Maize (Zea mays L. ssp. mays) originated about 9,000 years ago in 3,000 meters of altitude (19). Ears of Cabanita race have a conic- Mexico, and Latin America is considered the center of its genetic cylindrical shape and exhibit variable kernel pigmentations with diversity and primary domestication (1–3). This cereal is staple food predominance of white and partially red colored-pericarps (19). In a in Mesoamerican and Latin American countries since it is the base of previous study, some Peruvian maize races including Arequipeño, many traditional preparations. It has been reported that the Cabanita, Kculli, Granada, and Coruca races were evaluated in relation conservation and sustainable use of Latin American maize landrace to their phenolic composition, in vitro anti-hyperglycemia, and anti- diversity is fundamental for worldwide food security (1). Hence, obesity potential (20). Cabanita kernels showed the second highest efforts at multiple levels should be focused on the characterization of total oxygen radical absorbance antioxidant capacity (ORAC) and the genetically heterogenous landrace material as the base for further hyperglycemia management-relevant α-amylase inhibition following breeding improvements relevant for food security and health among the purple-colored maize group (Kculli race) (20). More recently, indigenous food systems (1, 4). Cabanita maize from two different provinces in Arequipa (Peru) were The diversity of maize landrace populations is represented in evaluated in relation to their physical characteristics, bioactive races, which are identified according to their common botanical (phenolic and carotenoid) composition and in vitro antioxidant characteristics, geographical distribution, ecological adaptation, and capacity (19). Although Cabanita samples from both provinces showed cultural importance (uses and customs) (5–7). Mexico and Peru have a certain grade of similarity according to the multivariate PCA concentrated around 30 percent of the Latin American maize diversity (Principal Component Analysis), in general maize cultivated under including 59 and 52 races, respectively (7, 8). The Peruvian Andean Andean environments with naturally higher ecological stress factors region with its great variety of ecological features has the highest such as higher altitudes and lower temperatures showed higher maize phenotypic diversity worldwide (7, 9, 10). However, limited phenolic and antioxidant capacity ranges (19). scientific information exist about Andean maize diversity which is The intake of this traditional Andean maize is mostly in the compromising its adequate conservation and essential health mature dried form. Andean farmers still maintain the postharvest relevant uses. traditional practices along the Cabanita maize production chain. Whole cereal grains are valuable sources of carbohydrates, Once the maize ears have reached their highest length and a certain proteins, dietary fiber, minerals, and vitamins along with other moisture level which is subjectively measured based on the farmer’s critical bioactive metabolites with known health-promoting benefits experience, the plants are cut and dried in a piled form in the same (11, 12). The regular intake of whole grains has been inversely land. Thereafter, dried plants are transported to the farmer’s correlated with lower incidence of several chronic warehouses where maize ears are unshelled and exposed directly to non-communicable diseases including type 2 diabetes (13), the sun until complete drying (19). Dried grains are then consumed cardiovascular disease (14), and some types of cancer (15, 16). Maize roasted, or further milled and used as flour in different contains nutritionally relevant macro and micronutrients mainly culinary preparations. carbohydrates, lipids (with mono and polyunsaturated fatty acids), Several studies focused mostly on sweet and waxy maize improved vitamins, minerals, and resistant starch (17). In addition, biologically varieties have shown that bioactive compounds such as phenolic active functional compounds such as phenolic compounds, antioxidants and carotenoids vary depending on the kernel maturation carotenoids, tocopherols, and phytosterols have been reported in stage. Phenolic compounds such as anthocyanins from different Asian maize (17). In fact, unique phenolic and carotenoid profiles have colored waxy maize genotypes increased along the kernel maturation been reported in different maize landraces linked to variable from 20 to 35 days after pollination (DAP) (21). Similarly, Zhang et al. nutraceutical properties (18). Accordingly, more studies are needed (22) observed an increase of the total phenolic contents (TPC) in to fully characterize maize landraces, targeting those that are the mature kernels from a yellow maize variety (48 DAP). The increase of base of needs of food security and economy in many geographical carotenoids such as lutein, zeaxanthin, α-cryptoxanthin, areas such as the Andean region. β-cryptoxanthin has also been reported during the kernel maturation Frontiers in Nutrition 02 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 of some sweet maize varieties from China (23). On the contrary, the ears were obtained per pot). Additional supplementation with total phenolic and total carotenoids contents decreased at the end of commercial fertilizers (urea and NPK + micronutrients) was grain maturation in yellow maize bred in United States (116 DAS, days performed during the vegetative period of maize plants (from week 2 after seeding) (24). These discrepancies reveal that different factors to 11 after sowing) and the phytosanitary control was undertaken including genetic factors (variety), the time of harvest, and the using conventional practices for the cultivation of maize in agroecological conditions of maize cultivation may influence the combination with the use of ecological insect traps. Well water was bioactive composition during the maize kernel maturation. used for the irrigation which was carried out under field capacity in a Consequently, the research on this topic should be performed case by similar way as in field cultivation. case, according to specific ecological environments. During the plant reproductive stage, the female inflorescences were As a second stage follow up studies of previous advances to promptly protected with a plastic bag until the emergence of the styles characterize the Peruvian Andean maize Cabanita (19), the objective (silks). The pollination was manually developed using composite of current research was to study the primary (polar compounds and pollen collected from mature tassels (male inflorescence) of plants fatty acids) and secondary metabolite composition (free and bound from the same maize type. Once pollinated, each ear was protected phenolics and carotenoid compounds), and the in vitro health-relevant with paper bags until physiological maturity. This procedure avoided functional properties of three selected Cabanita types (white, red, and the cross-pollination among different maize types. Ears were collected orange pigmented kernels) harvested at different maturity stages, using at three different grain maturity stages according to the grain physical targeted and untargeted metabolomic platforms. The in vitro model appearance and moisture contents (25, 26). The milky stage (S1) is based functionality of Cabanita maize was evaluated in relation to its characterized by the starch accumulation and the observation of a antioxidant potential and inhibitory activity against key digestive milky white fluid upon finger pressure (25). In the dough stage (S2), enzymes (α-amylase and α-glucosidase) relevant for hyperglycemia the grain is still soft and humid, with intermediate humidity, whereas modulation. Results from this study will contribute with important the physiological mature stage (S3) corresponds to the completion of biochemical and metabolomic information for the characterization, kernel development, and a black layer is formed at the base of the and conservation of the maize race Cabanita. In addition, information kernel (25, 26). In case of the white maize type, S1 corresponded to 28 from this research would be important to diversify the consumption DAP and 79% moisture, S2 to 39 DAP and 68% moisture, and S3 to 75 options of this Andean maize beyond the traditional mature form. This DAP and 45% moisture. For the red type maize, S1 was at 33 DAP and would likely lead to potential beneficial effects of food crops at health 74% moisture, S2 at 36 DAP and 68% moisture, and S3 was at 77 DAP and economical levels among indigenous communities in the future. and 45% moisture. In the orange type, S1 corresponded to 32 DAP and 75% moisture, S2 to 43 DAP and 64% moisture, and S3 to 76 DAP and 46% moisture. 2. Materials and methods After harvest, ear samples were immediately stored under refrigeration (5–8°C) and transported to the laboratory. Husks were 2.1. Cultivation of Cabanita maize and eliminated, and samples (ear and kernels) were evaluated in relation sampling to their physical characteristics as will be described in next section. Kernels were separated, pooled per biological replicate, and frozen The germplasm of Cabanita maize (Zea mays L.) collected in a (−20°C). This process was developed within the 24 h after harvest. previous study was used (19). Maize with sample codes CAW, CCR, Afterwards, samples were freeze-dried in a FreeZone benchtop freeze COM representing white, red, and orange kernels were selected for dryer (Labconco, Kansas, MO, United States) for 60 h, at –40°C, and current study considering the pigmentation diversity found in 0.008 mbar of vacuum pressure. Then, dried kernels were milled in a Cabanita maize race (19). These maize samples were obtained from A11 Basic analytical mill (IKA, Germany) with liquid nitrogen to a the province of Caylloma (Cabanaconde district) located in the powdered flour, packed in 50 ml polypropylene tubes protected from southern Andean region of Arequipa in Peru and were stored under light, and stored at –20°C until analysis. refrigeration (2–5°C) (19). The field experiment was performed in the nursery garden Santa Maria at the Universidad Catolica de Santa Maria located in the Sachaca district (S: 16° 41′ 93.9″; W: 071° 56′ 2.2. Enzymes and reagents 34.0″; 2,240 meters of altitude), province of Arequipa (Arequipa, Peru). Cabanita maize was cultivated in 20 L pots under open air and sun Baker yeast α-glucosidase (EC 3.2.1.20), and porcine pancreas light exposure from 25 November 2020 to 28 June 2021. Commercial α-amylase (EC 3.2.1.1) were from Sigma-Aldrich (St. Louis, MO, prepared soil (containing humus, field soil, and manure) was used and United States). Phenolic standards (gallic acid, vanillic acid, caffeic its physico-chemical characteristics are shown in acid, ferulic acid, p-coumaric acid, cyanidin chloride, and quercetin Supplementary Table S1. The meteorological conditions during the aglycone), carotenoid standards (lutein, zeaxanthin, β-cryptoxanthin), maize plant development until sample harvest are shown in and the Folin–Ciocalteu reagent were from Sigma-Aldrich. The Supplementary Table S2. Groups of 6 pots were sown in three (±)-6-hydroxy-2,5,7,8-tetramethyl-chromane-2-carboxilic acid consecutive weeks (total 18 pots per maize type) and 5 Cabanita seeds (Trolox), and the 2,2-diphenyl-1-picrylhydrazyl (DPPH˙), and were sown in each pot (sowing dates: 25 November, 2 December, and 2–2′-azino-bis(3ethylbenothiazoline-6-sulfonic acid) (ABTS·+) 9 December 2020). This procedure was applied to ensure the number radicals were purchased from Sigma-Aldrich. Pyridine, phenyl-β-d- of biological replicates (four) at three maturity stages per type of maize glucopyranoside, methoxyamine hydrochloride, N,O- (white, red, orange) for the current study. The group of ears harvested bis(trimethylsilyl)trifluoroacetamide (BSTFA), and methyl from a single pot was considered a biological replicate (from 1 to 4 undecanoate were from Sigma-Aldrich. Frontiers in Nutrition 03 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 2.3. Physical measurements and moisture Calibration curves with external standards were used for the determination quantification of phenolic compounds (r2 ≥ 0.9990). Hydroxybenzoic phenolic acids (HBA) (unidentified 1 and 2) were quantified at 280 nm Relevant physical descriptors were evaluated in fresh harvested and expressed as vanillic acid. Vanillic acid derivatives (with similar ears and kernels (per maturity stage and maize type) per replicate UV–VIS spectra as that of vanillic acid but with different retention according to the International Board for Plant Genetic Resources (27). times) were expressed also as vanillic acid. Hydroxycinnamic phenolic The weight (g), length (cm), and central diameter (cm) were measured acids (HCA) including ferulic, p-coumaric, and caffeic acid derivatives in ears whereas the length (mm), width (mm), and thickness (mm) were quantified at 320 nm and expressed as ferulic, p-coumaric, and were determined in kernels. The kernel moisture was monitored caffeic acids, respectively. Flavonoids such as luteolin derivatives (with periodically to characterize the maturity stage for harvest and was similar UV–VIS spectra as that of luteolin, but with different retention determined by a gravimetric method at 105°C (28). times), and anthocyanins were detected at 360 and 525 nm, and quantified using quercetin aglycone and cyanidin chloride external standards, respectively. All results were expressed as mg per 100 g DW 2.4. Extraction of phenolic and carotenoid (dried weight). compounds from maize samples 2.5.2. Analysis of carotenoid compounds by 2.4.1. Free and bound phenolic fractions UHPLC The extraction of phenolic compounds from the lyophilized maize The analysis of carotenoid compounds was performed with a samples were performed according to Ranilla et al. (29) with some YMC carotenoid C30 reverse-phase analytical column (150 × 4.6 mm modifications. An amount of 1 g of maize sample was mixed with 4 ml i.d., 3 μm) coupled to a YMC C30 guard column (10 × 4.0 mm, 3 μm) of 0.1% HCl methanol/acetone/water (45, 45, 10, v/v/v) for the (YMC CO., LTD, Japan) using the same UHPLC system as previously extraction of the free phenolic fraction. The bound phenolic described for the phenolic compound analyses. Filtered carotenoid compounds were released from the insoluble free-phenolic residue by extracts (0.22 μl, PVDF filter) were injected at 1.7 ml/min flow rate and alkaline hydrolysis with 3 N NaOH following same procedure as monitored at 450 nm. A ternary gradient elution was used (methanol, Ranilla et al. (29). Final extracts were reconstituted in milliQ water dichloromethane, acetonitrile) and same reverse-phase and stored at-20°C until analysis. chromatographic conditions as those reported by Fuentes-Cardenas et  al. (19) were applied. The retention time and UV–VIS spectra 2.4.2. Carotenoids characteristics of external carotenoid standards and the library data The procedure of Fuentes-Cardenas et al. (19) was followed. A were used for the identification of carotenoid compounds in evaluated saponification process was first applied with 80% KOH (w/v) and samples. In addition, the information of carotenoid analyses in other methanol:ethyl acetate (6, 4, v/v) solvent was used for the carotenoid maize samples from reported literature was also useful for the extraction until a clear final extract was obtained. Carotenoids were identification of carotenoid isomers. Calibration curves made with extracted under light and oxygen protection and analyzed by ultra external standards were used for the quantification of carotenoids high-performance liquid chromatography (UHPLC) after the (r2 ≥ 0.9900) and results were presented as μg per g sample DW. Lutein extraction process the same day. and zeaxanthin compounds and their isomers were quantified as lutein and zeaxanthin, respectively. Unidentified carotenoid compounds, neoxanthin, and violaxanthin isomers were expressed as 2.5. Targeted metabolomic analysis lutein. β-cryptoxanthin isomers were expressed as β-cryptoxanthin. 2.5.1. Analysis of phenolic compounds by ultra 2.5.3. Analysis of the total phenolic contents high-performance liquid chromatography The TPC in the free and bound phenolic extracts were evaluated Free and bound phenolic extracts were filtered using a according to Singleton and Rossi (31) using the Folin–Ciocalteu polyvinylidene difluoride filters (PVDF, 0.22 μm) and the separation method. Results were presented as mg of gallic acid equivalents (GAE) was carried out in a Kinetex C18 reverse-phase analytical column per 100 g DW. (100 × 2.1 mm i.d., 1.7 μm) with a Kinetex C18 guard column (5 × 2.1 mm i.d., 1.7 μm) (Phenomenex Inc., Torrance, CA, 2.5.4. Fatty acids profiles by gas-chromatography United States). The injection volume was 5 μl and samples were injected with flame ionization detector at 0.2 ml/min flow rate in an Ultimate 3,000 RS UHPLC system The analysis was adapted from Uarrota et al. (32). The fatty acid (Thermo Fisher Scientific, Waltham, MA, United States) with a diode methyl ester synthesis (FAME) was obtained by combining ~55 mg of array detector, a quaternary pump, an autosampler, and column oven. lyophilized maize sample with 70 μl 10 N KOH (prepared in HPLC Acetonitrile and 0.1% formic acid in water were used as mobile phases water) and 530 μl of HPLC grade methanol in a reaction tube. The mix and the same gradient and chromatographic parameters reported by was incubated in a water bath at 55–60°C for 1.5 h with periodic Ranilla et al. (29) and Vargas-Yana et al. (30) were applied. Eluates were agitation every 30 min. Tubes were then cooled down to room monitored from 200 to 600 nm. The Chromeleon SR4 software version temperature and drops of fuming H2SO4 (24 N) were carefully added. 7.2 (Thermo Fisher Scientific) was used for chromatograms and data An incubation step was repeated as previously described. Samples processing. The identification of phenolic compounds was based on were cooled, then 500 μl of hexane and 10 μl of internal standard their retention times and ultraviolet–visible spectra characteristics (methyl undecanoate, 26.16 mg/ml) were added. The tubes were compared with those of the library data and external standards. vortexed for 2 min and centrifuged at 17,000 g for 10 min at 4°C. The Frontiers in Nutrition 04 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 upper layer was transferred to a vial with an insert and 1 μl was Trolox calibration curves prepared in methanol (20–160 μM), and injected in an Agilent 7890B gas chromatography system coupled to dichloromethane (10–120 μM) for the hydrophilic and lipophilic a flame ionization detector (FID) (Agilent Technologies, Santa Clara, fractions, respectively. CA, United  States). A 2560 capillary gas-chromatography (GC) column (100 m × 250 μm × 0.2 μm) (Supelco, Bellefonte, PA, 2.7.3. Antioxidant capacity by the 2.2′-azino-bis United States). The injector temperature was set at 220°C, the FID (3-ethylbenzothiazoline-6-sulfonic acid) radical detector at 225°C, air flow (400 ml/min), hydrogen flow (35 ml/min), cation (ABTS·+) scavenging assay helium flow (1.6 ml/min), using an injection with a split ratio of 50:1. The hydrophilic and lipophilic extracts were evaluated according The chromatographic run was set up at 80°C (initial temperature) and to Fuentealba et  al. (35) using a Biotek Synergy HTX microplate increased to 225°C with a heating ramp at a rate of 25°C per min, and reader (Agilent Technologies). Results were expressed as μmol Trolox held for 25 min. The retention times of detected peaks from samples equivalents per 100 g DW based on calibration curves built with were compared with those of external standards for fatty acid Trolox standard in methanol and dichloromethane for the evaluation identification. Calibration curves (r2 ≥ 0.9900) with palmitic, stearic, of hydrophilic and lipophilic fractions, respectively. The Trolox oleic, linoleic, and α-linolenic acids were used for the quantification concentration ranges used in calibration curves were the same as those of fatty acids in maize samples and results are presented as mg shown for the DPPH method. per g sample DW. 2.7.4. α-Glucosidase and α-amylase inhibitory activity 2.6. Untargeted metabolomic analysis of The hydrophilic and lipophilic fractions used for the polar compounds by gas chromatography determination of the α-amylase inhibitory activity were obtained mass spectrometry similarly as Fuentes-Cardenas et al. (19) but using a different sample and solvent ratio for the extraction (0.5 g sample in 12.5 ml 80% The extraction of polar metabolites from maize samples, the methanol). Final extracts (hydrophilic and lipophilic) were vacuum- derivatization process, and the instrumental parameters for the gas evaporated to dryness at 45°C and reconstituted in 2 ml 0.02 M NaPO4 chromatography mass spectrometry (GC–MS) analysis were the same buffer (pH 6.9) (35). In case of the α-glucosidase inhibition analysis, as reported by Fuentealba et  al. (33). An Agilent 7890B gas same extraction conditions were assayed as Fuentes-Cardenas et al. chromatography system equipped with a 5977A single quadrupole (19), and final hydrophilic and lipophilic extracts were also vacuum- MS, a PAL3 autosampler, an electron impact ionization source was evaporated to dryness but resuspended in 1 ml of 0.1 M KPO4 buffer used (Agilent Technologies). A HP-5 ms Ultra Inert column (pH 6.9) (35). The inhibitory activity against α-amylase and (30 m × 0.25 mm × 0.25 μm) (Agilent) was used for the separation of α-glucosidase enzymes were determined with the same methodology polar compounds. The Mass Hunter Quantitative software (Agilent reported by Gonzalez-Muñoz et  al. (36). The percentage (%) of Technologies) was used for the deconvolution and data processing. inhibition at different sample amounts was reported. For peak identification, their retention times and mass spectra were compared with data from NIST14 and a home library (obtained with commercial standards). Results are shown as the relative response of 2.8. Statistical analysis each compound calculated considering their respective sample weight, an internal standard (phenyl-β-d-glucopyranoside), and a quality Results (from four independent biological replicates) were control (QC) composite sample from all maize samples (33). expressed as means ± standard deviation. A two-way analysis of variance (ANOVA) with the LSD test were carried out to determine significant differences between the means (p < 0.05) using the software 2.7. In vitro functionality of Cabanita maize Infostat1 (accessed from October to November, 2022). Pearson samples correlations among all data were explored using the Statgraphics Centurion XVI software (StatPoint Inc., Rockville, MD, United States). 2.7.1. Extraction of hydrophilic and lipophilic All data (from the targeted and untargeted metabolite analyses, the fractions physical characteristics, and the functionality assays) were evaluated The soluble hydrophilic and lipophilic fractions from lyophilized through the multivariate principal component analysis (PCA) using maize samples were considered for the in vitro assays. The hydrophilic the Metaboanalyst software version 5.02 (accessed on 2 October, and lipophilic fractions were extracted with 80% methanol and 2022). For the PCA, data were first mean-centered and divided by the dichloromethane; respectively, following same extraction parameters standard deviation of each variable. Afterward, an ANOVA with the described by Fuentes-Cardenas et al. (19). Tukey’s HSD post hoc analysis (p < 0.01) was carried out to identify significant variables. The heat map or cluster analysis was performed 2.7.2. Antioxidant capacity by the using the Euclidean distance and the Ward algorithm in Metaboanalyst 2,2-diphenyl-1-picrylhydrazyl radical scavenging with the top significant metabolites or variables (p < 0.01). assay The method of Duarte-Almeida et al. (34) adapted to a microplate reader (Biotek Synergy HTX, Agilent Technologies) with modifications reported by Fuentes-Cardenas et al. (19) was applied. 1 https://www.infostat.com.ar/ Results are shown as μmol Trolox equivalents per 100 g DW using 2 https://www.metaboanalyst.ca/MetaboAnalyst/home.xhtml Frontiers in Nutrition 05 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 A A B B FIGURE 1 FIGURE 3 Changes of ear (A) and kernel (B) physical characteristics of white Changes of ear (A) and kernel (B) physical characteristics of orange Cabanita maize at different maturity stages (S1, S2, S3, from left to Cabanita maize at different maturity stages (S1, S2, S3, from left to right). right). and the red maize (16 weeks after sowing). This may be important for A the adequate planning of maize cultivation periods since Andean farmers traditionally sow maize mixing different Cabanita types in the same land. The physical changes of ears and kernels from the three types of Cabanita maize along the maturity stages are shown in Figures 1–3. The development of all maize types was characterized by changes in the pericarp color. The white maize type varied from light-white to B white-yellow at S3 which may be related to the accumulation of dry matter with maturity (Figure  1) (37). In case of the red type, the pigmentation appeared in the S2 stage as a small spot at the stigma-end of the kernel that then extended toward almost the half of the grain at S3 (Figure 2). Hong et al. (38) observed a similar trend in a purple- pericarp sweet corn; however, the purple pigment fully spread until the base of the kernel at the highest maturity phase (32 DAP). The orange maize varied from light-yellow at S1 to orange at S3 and this FIGURE 2 pigmentation only reached the middle of the kernels similarly as in Changes of ear (A) and kernel (B) physical characteristics of red the red case. Cabanita maize at different maturity stages (S1, S2, S3, from left to Table 1 shows the physical characteristics evaluated in kernels right). sampled at different developmental stages. No significant interaction between the maize type (M) and the maturity stage (S) factors was found in any of the measured physical parameters. The variation of 3. Results and discussion the ear weight and diameter, and the kernel width along the maturation period was similar in all maize types. Nevertheless, the ear 3.1. Physical changes of Cabanita maize length, kernel length, and thickness were influenced by the maize type. types at different maturity stages The ear length was higher in the white and red maize than in the orange type, but this latter showed higher kernel length ranges than The maturity stages of Cabanita maize characterized by the DAP the former. The maturity stage factor (S) was significant in all the and moisture levels were similar among evaluated maize types. physical characteristics except the ear length which remained almost However, their vegetative periods (from the plant emergence stage to similar during the maturation. The ear weight, and diameter along the end of the tasseling time) were somewhat different (26). This with the kernel length, width, and thickness increased with period occurred at around 9, 10, and 11 weeks after the sowing stage maturation. Overall, the yield-relevant physical parameter (ear in case of the orange, white, and red maize types, respectively. weight) was similar among all maize classes; however, some Consequently, the start of the reproductive period (emergence of the morphological differences have been observed among the kernel types silk or the female inflorescence) was earlier in the orange maize type (Figures 1–3). Fuentes-Cardenas et al. (19) also studied the Peruvian (13 weeks after sowing), followed by the white (15 weeks after sowing), maize race Cabanita and reported no differences in the quantitative Frontiers in Nutrition 06 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 TABLE 1 Physical characteristics of Cabanita maize kernels at different pigmentations and maturity stages. Maize Stage Ear (cm) Kernel (mm) type Weight Length Diameter Length Width Thickness White S1 70.4 ± 19.9c 9.1 ± 0.7ab 4.2 ± 0.4f 0.80 ± 0.04e 0.79 ± 0.07d 0.59 ± 0.04e S2 150.0 ± 72.9a 9.9 ± 1.2a 5.3 ± 0.2abcd 1.11 ± 0.06 cd 0.80 ± 0.05 cd 0.61 ± 0.02de S3 138.4 ± 51.7ab 9.6 ± 0.7ab 5.6 ± 0.7abc 1.44 ± 0.10b 0.97 ± 0.10ab 0.71 ± 0.10abcd Red S1 88.8 ± 21.5bc 8.5 ± 0.9abc 4.6 ± 0.5def 0.95 ± 0.15de 0.83 ± 0.17bcd 0.63 ± 0.08cde S2 106.9 ± 36.6abc 9.9 ± 1.8a 5.1 ± 0.3 cde 0.93 ± 0.12e 0.89 ± 0.11abcd 0.74 ± 0.08ab S3 144.1 ± 64.6ab 9.3 ± 0.4ab 5.9 ± 0.8a 1.43 ± 0.15b 0.95 ± 0.14abc 0.73 ± 0.07ab Orange S1 77.0 ± 22.8c 8.1 ± 1.2bc 4.5 ± 0.6ef 1.19 ± 0.06c 0.82 ± 0.06bcd 0.66 ± 0.04bcde S2 76.0 ± 16.8c 7.4 ± 0.9c 5.1 ± 0.3bcde 1.21 ± 0.10c 0.82 ± 0.07 cd 0.72 ± 0.01abc S3 121.1 ± 18.3abc 8.6 ± 0.7abc 5.8 ± 0.5ab 1.65 ± 0.19a 1.03 ± 0.11a 0.77 ± 0.10a F value Maize (M) 1.49ns 7.23** 0.16ns 17.32**** 0.46ns 4.40* Stage (S) 5.75** 0.99ns 19.68**** 66.24**** 9.29*** 7.75** M × S 1.09ns 1.42ns 0.50ns 2.32ns 0.60ns 0.66ns Different letters in the same column indicate significant statistical differences (*p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001); ns, no significant. physical characteristics between the CAW (white), CCR (red), and disappeared with the grain development of waxy maize from 86–109 COM (orange) maize samples (which are the parental seeds of the to 110–138 DAS (37). Besides vanillic acid, syringic and white, red, and orange maize types evaluated in the current study). p-hydroxybenzoic acids have been also reported in maize (40, 41). However, mature, and dried ears and kernels were evaluated in Total free HBA ranges from current study (4.8–44.2 mg/100 g DW) such study. were comparable to levels found in US yellow and Indian specialty maize kernels (~33.7 and 2.7–38 mg/100 g DW, respectively) (40, 41). Other cereals such as barley, wheat, and oat have shown lower free 3.2. Phenolic contents and profiles of HBA concentrations (~15.5, 12.5, and 4.6 mg/100 g, respectively) (40). Cabanita maize types at different maturity A variable trend was observed in case of the HCA group (Table 2 stages and Supplementary Figures S4–S6). All Cabanita maize types contained p-coumaric, and ferulic acid derivatives whereas caffeic acid The phenolic profiles and contents determined in the free and derivatives were detected at some maturity stages. These HCA bound phenolic fractions of Cabanita maize samples are shown in derivatives may be  soluble conjugated phenolic acids such as Table  2. In case of the free phenolic fraction, all maize samples hydroxycinnamic acid amides (HCAAs) as was previously reported in contained phenolic acids such as hydroxybenzoic (HBA) and different cereals (42, 43). Several HCAAs derived mostly from hydroxycinnamic acids (HCA), but specific flavonoid types such as p-coumaric, ferulic and caffeic acids (N,N-di-p-coumaroylspermine, anthocyanin and luteolin derivatives were detected only in red and N-p-coumaroyl-N-feruloylputrescine, caffeoylputrescine) have been orange maize types, respectively. previously reported in the free phenolic fraction of maize from For the HBA group, the contents were more influenced by the different origins (44, 45). Hence, further studies are necessary to better maturity stage (S) than by the maize type (M). The interaction of both identify the HCA derivatives found in current research. The maturity factors (M × S) was significant on the vanillic acid derivatives and the (S) and maize type (M) showed an important effect on p-coumaric total HBA contents. The highest total HBA contents were observed at and ferulic acid derivatives, but the interaction of both factors was stage S1, and white and orange maize had higher levels than red maize significant only on the ferulic acid derivatives contents. p-Coumaric (44.2, 39.2, and 34.1 mg/100 g DW, for the white, orange, and red acid and caffeic acid derivatives increased with kernel development. maize, respectively). With the kernel maturation, the total HBA The increase of p-coumaric acid derivatives levels from S1 to S3 was concentrations decreased around 80–90% in all cases (from S1 to S3). on average 2.6-fold, and white and orange maize types exhibited At least 3 classes of HBA have been detected in all Cabanita samples higher ranges than the red maize (0.8–2.2, 0.6–1.6, and (Supplementary Figures S1–S3). Major HBA at S1 was HBA-1 0.4–1.1 mg/100 g DW for white, orange, and red maize, respectively). (λmax = 279 nm), followed by vanillic acid derivatives (λmax = 249, Conversely, the concentrations of ferulic acid derivatives declined by 289 nm). Contents of both HBA then decreased with maturity to reach 32–48% from S1 to S3  in all maize groups. Different studies have similar concentrations at S3. A minor HBA compound (HBA-2) was shown variable tendencies of the HCA compounds with kernel only found at S1 in white and orange maize types. Giordano et al. (39) maturity. The free ferulic and chlorogenic acid contents reduced with reported that the free vanillic acid levels found in open-pollinated kernel maturation in several Italian maize varieties, and the same maize varieties from Italy with variable kernel pigmentations trend was observed in Chinese waxy pigmented maize samples with decreased from 1.8–15 to 0–0.08 mg/100 g DW when maturation ferulic and p-coumaric acids (37, 39). Recently, Hu et al. (46) observed stages varied from 5 to 76 DAS, respectively. Similarly, the contents of an overall increment of ferulic and p-coumaric acids during kernel vanillic and protocatechuic acids significantly decreased or maturation of sweet maize from China, whereas chlorogenic acid Frontiers in Nutrition 07 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 Frontiers in Nutrition 08 frontiersin.org TABLE 2 Phenolic profiles and contents by UHPLC (mg/100 g DW) in Cabanita maize kernels of different pigmentations and maturity stages. Fraction Compound White Red Orange F-value S1 S2 S3 S1 S2 S3 S1 S2 S3 Maize Stage M × S (M) (S) Free HBA-1 30.6 ± 11.2a 16.6 ± 2.6c 2.8 ± 1.0d 23.2 ± 4.7abc 22.2 ± 1.6bc 2.5 ± 1.8d 27.6 ± 9.5ab 17.4 ± 1.3c 5.9 ± 1.8d 0.10ns 58.83**** 1.78ns HBA-2 0.06 ± 0.05a ND1 ND ND ND ND 0.11 ± 0.08a ND ND Vanillic acid 13.6 ± 1.4a 5.6 ± 1.2c 2.0 ± 1.1d 10.8 ± 3.5ab 9.6 ± 1.2b 2.5 ± 0.5 cd 11.4 ± 4.8ab 5.2 ± 1.2c 2.5 ± 0.5 cd 0.98ns 58.05**** 2.88* derivatives2 Total HBA 44.2 ± 11.6a 22.2 ± 3.1c 4.8 ± 2.0d 34.1 ± 7.8b 31.8 ± 1.9bc 5.1 ± 2.2d 39.2 ± 14.2ab 22.6 ± 2.1c 8.4 ± 1.8d 0.01ns 69.29**** 2.47* p-Coumaric acid 0.8 ± 0.3 cd 0.6 ± 0.2 cd 2.2 ± 0.9a 0.4 ± 0.1 cd 0.3 ± 0.1d 1.1 ± 0.4bc 0.6 ± 0.2 cd 0.53 ± 0.04 cd 1.6 ± 0.8ab 5.39* 22.38**** 1.02ns derivatives3 Ferulic acid 3.3 ± 0.5a 1.4 ± 0.2c 1.7 ± 0.5c 2.5 ± 0.5b 2.8 ± 0.5ab 1.7 ± 0.8c 2.5 ± 0.5b 1.2 ± 0.3c 1.7 ± 0.4c 3.96* 18.79**** 6.44*** derivatives4 Caffeic acid ND 0.2 ± 0.1bc 1.2 ± 0.6a ND ND 1.5 ± 0.6a 0.03 ± 0.00bc 0.02 ± 0.00c 0.6 ± 0.6Bb derivatives5 Total HCA 4.1 ± 0.8abc 2.1 ± 0.2de 5.1 ± 1.3a 3.0 ± 0.6 cde 3.1 ± 0.5bcd 4.3 ± 1.0ab 3.1 ± 0.6bcd 1.7 ± 0.3e 3.8 ± 1.7bc 3.07ns 17.15**** 1.78ns Luteolin ND ND ND ND ND ND 22.7 ± 12.5a 11.3 ± 4.9ab 5.1 ± 4.4b derivatives6 Total ND ND ND 0.6 ± 0.2a 1.4 ± 1.3a 14.5 ± 18.7a ND ND ND anthocyanins7 Total flavonoids ND ND ND 0.6 ± 0.2b 1.4 ± 1.3b 14.5 ± 18.7ab 22.7 ± 12.5a 11.3 ± 4.9ab 5.1 ± 4.4b Total UHPLC free 48.3 ± 11.6b 24.3 ± 3.2 cde 10.0 ± 3.3f 37.6 ± 8.2bc 36.2 ± 3.5bcd 23.8 ± 17.7de 65.0 ± 14.1a 35.6 ± 4.8bcd 17.3 ± 3.9ef 4.80* 38.22**** 4.11* Free – TPC8 57.9 ± 17.3ab 27.0 ± 3.2c 32.3 ± 3.3c 38.0 ± 6.5bc 44.6 ± 6.5abc 53.6 ± 26.1ab 53.0 ± 11.4ab 37.6 ± 6.2bc 58.7 ± 23.5a 1.72ns 3.13ns 2.93* Bound p-Coumaric acid 4.9 ± 1.2bc 5.2 ± 1.1bc 7.5 ± 1.9ab 6.4 ± 4.3abc 4.1 ± 1.1c 9.4 ± 2.6a 5.9 ± 0.5bc 7.8 ± 2.4ab 7.2 ± 2.2abc 0.77ns 4.28* 1.98ns Ferulic acid 163.3 ± 7.4abc 150.1 ± 14.9bc 133.2 ± 21.2c 161.9 ± 42.8abc 142.5 ± 17.7c 177.2 ± 12.0ab 190.3 ± 11.8a 179.3 ± 23.5ab 177.5 ± 29.2ab 6.84** 1.29ns 1.64ns Ferulic acid 8.5 ± 0.7d 10.8 ± 0.5d 11.6 ± 6.0d 12.0 ± 3.0 cd 10.6 ± 4.1d 19.4 ± 2.4ab 13.1 ± 1.2 cd 16.7 ± 6.2bc 22.0 ± 1.9a 11.92*** 11.08*** 1.75ns derivatives4 Total UHPLC 176.7 ± 7.9abc 166.1 ± 16.4bc 152.3 ± 28.4c 180.3 ± 49.8abc 157.1 ± 21.9c 206.0 ± 12.4a 209.4 ± 13.2a 203.8 ± 29.4ab 206.6 ± 29.7a 7.68** 0.97ns 1.72ns bound Bound – TPC8 155.8 ± 4.5bc 144.3 ± 10.1c 142.2 ± 38.0c 146.0 ± 23.4c 122.6 ± 35.1c 187.0 ± 7.0ab 181.3 ± 14.2ab 188.1 ± 29.9ab 197.1 ± 21.8a 11.23*** 3.12ns 2.72ns Total UHPLC TPC 225.0 ± 9.2bcd 190.4 ± 14.2de 162.2 ± 29.2e 217.9 ± 44.4bcd 193.4 ± 21.3cde 229.8 ± 22.4bc 274.4 ± 26.8a 239.4 ± 27.4ab 223.9 ± 26.8bcd 12.45*** 6.11** 2.70ns (free + bound) TPC8 213.7 ± 18.8bcd 171.4 ± 8.7e 174.5 ± 35.8de 184.0 ± 19.5cde 167.2 ± 37.6e 240.7 ± 29.1ab 234.3 ± 23.2ab 225.7 ± 32.1abc 255.8 ± 39.4a 10.90*** 4.67* 3.14* Different letters in the same row indicate significant statistical differences (*p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001); ns, no significant. F values were calculated only in complete dataset per variable. HBA, hydroxybenzoic acids (HBA 1 and 2 indicate different unidentified HBA with different UV–VIS spectra). HCA, hydroxycinnamic acids. S1, S2, S3 indicate maturity stages. 1Non-detected. 2Expressed as vanillic acid. 3Expressed as p-coumaric acid. 4Expressed as ferulic acid. 5Expressed as caffeic acid. 6Expressed as quercetin aglycon. 7Expressed as cyanidin chloride. 8Folin–Ciocalteu TPC expressed as mg GAE/100 g DW. Ranilla et al. 10.3389/fnut.2023.1132228 declined (from 15 to 30 DAP) after an initial increase (from 10 to 15 phenolic contents were stable with kernel maturation from 15 to 48 DAP). In the current study, the total HCA contents first decreased DAP (22). Nonetheless, the levels of bound ferulic and p-coumaric from S1 to S2 in white and orange maize types, then increased at S3 in acids significantly reduced with kernel development (from 5 to 76 all cases. The origin, maize type (genetic factors), and the harvesting DAS) in several Italian maize samples whereas in other research same time may explain differences found in this study. bound HCA showed a variable tendency depending on the genotype Anthocyanins were present only in red maize, showing an increase (39, 46). from 0.6 mg/100 g DW at S1 to 14.5 mg/100 g DW by the end of kernel On the whole, these results suggest differences in the metabolism maturity. Other flavonoids such as luteolin derivatives were specific of phenolic compounds during kernel development among the three for orange maize samples and significantly decreased by 80% from S1 types of Cabanita maize. A possible metabolic flux of precursors of to S3 (from 22.7 to 5.1 mg/100 g DW). No flavonoids were detected in hydroxybenzoic acids such as some intermediates of the shikimate or white Cabanita. Hong et al. (38) observed a continuous anthocyanin the phenylpropanoid pathways toward the biosynthesis of HCA accumulation from 105 mg/100 g DW at 20 DAP to 179 mg/100 g DW derivatives may occur in case of the white and orange grains (52). at 36 DAP in purple-pericarp “supersweet” sweet maize. The increase HCA may be used as precursors for the biosynthesis of anthocyanins of the total monomeric anthocyanin contents with kernel ripening has in case of the red maize. Enzymes involved in the biosynthesis of cell been also confirmed by different studies (21, 37, 47). The flavone wall-relevant phenolic compounds may have been upregulated toward luteolin has been reported in Indian Himalayan pigmented maize the flavone pathway in the orange maize explaining its overall higher accessions and some Chinese maize hybrids (48, 49). In addition, a ranges of bound phenolic compounds through the kernel growing C-glycosylflavone known as maysin (a luteolin derivative) has also process (53, 54). been found in mature maize seeds (49). The luteolin derivatives found Ultra high-performance liquid chromatography total phenolic in the current study (λmax = 256, 270, 349 nm) may be maysin or contents (free+bound) declined with kernel maturity in white and similar compounds that should be  confirmed in future studies. orange grains whereas in red maize the contents first decreased from However, higher concentrations of these flavones were found in the S1 to S2, and then increased at S3. Concentrations at the physiological orange Cabanita maize at all maturity stages compared to levels maturity stage were higher in the case of the orange and red maize obtained by Zhang et al. (49) (1.13 ng/g DW of maysin in mature type (223.9 and 229.8 mg/100 g DW, for the orange and red maize, seeds). C-glycosylflavones have shown potential neuroprotective respectively) than results obtained by Fuentes-Cardenas et al. (19) in properties relevant for Alzheimer’s disease prevention (50, 51). Cabanita race (134.3 and 190.9 mg/100 g DW, for the orange and red The total free phenolic fraction decreased from S1 to S3, and its maize, respectively). However, above authors reported higher total composition was variable depending on the kernel stage and maize phenolic contents in the white maize type (206 mg/100 g DW) than in type. HBA were the most important compounds in white and red the current research (162.2 mg/100 g DW). Differences in the maize at S1 and S2. In case of the orange group, HBA and luteolin postharvest treatments and the agroecological conditions for the derivatives highly contributed to the total free phenolic fraction at S1 growth of Cabanita maize may explain such variations. Generally, and S2. This maize showed the highest total free phenolic contents at phenolic contents measured with the Folin–Ciocalteu method showed S1 among all samples (65 mg/100 g DW). The red maize was rich in the same trend as those analyzed with the UHPLC method. However, HBA at S1, and S2 whereas anthocyanins were the major contributors the lack of specificity of the Folin–Ciocalteu method may be associated to the free phenolic fraction at S3. with differences observed specially in results from the free phenolic Major compound in the bound phenolic fraction was ferulic acid, fraction (55). followed by ferulic acid derivatives, and p-coumaric acid (Supplementary Figures S7–S9). The M × S interaction was not significant for any of the bound phenolic compounds; however, the 3.3. Carotenoid contents and profiles of maize type showed an important effect on the ferulic acid, ferulic acid Cabanita maize types at different maturity derivatives, and the total bound UHPLC phenolic contents (Table 2). stages Orange and red maize types had higher ranges of ferulic acid (177.5– 190.3 mg/100 g DW, 142.5–177.0 mg/100 g DW, and 133.2– Cabanita maize types at different maturity stages were also 163.3 mg/100 g DW, for the orange, red, and white maize, respectively), evaluated in terms of their carotenoid composition (Table  3, and ferulic acid derivatives than the white group (13.1–22.0 mg/100 g Supplementary Figures S10–S12). In contrast to the variable effect of DW, 10.6–19.4 mg/100 g DW, and 8.5–11.6 mg/100 g DW, for the studied factors (M and S) on phenolic compounds, carotenoid orange, red, and white maize, respectively). Consequently, higher total contents were highly influenced by the maize type. Only xanthophylls bound phenolic levels determined by UHPLC were found in orange were found in all Cabanita samples while no carotenes were detected. and red maize, specially at S3 than in the white type. Kernel maturity Moreover, different profiles were observed among studied Cabanita highly influenced the p-coumaric acid and ferulic acid derivatives maize groups. White and red maize had similar profiles and all-trans- contents. Both compounds showed an increase of around 1.2–1.7-fold neoxanthin, neoxanthin isomer (~13-cis-neoxanthin), all-trans- from S1 to S3. Ferulic acid remained almost stable from S1 to S3 in zeaxanthin, and a lutein isomer (~13-cis-lutein) were the major white and orange maize samples, and a similar trend was observed in carotenoids. All-trans-lutein and all-trans-zeaxanthin were the main their total UHPLC bound phenolic contents. In case of the red maize, compounds in the orange maize, followed by ~13-cis-lutein, and ferulic acid and the total bound phenolic compounds first decreased neoxanthin compounds. β-cryptoxanthin isomers along with some from S1 to S2, to further increase at S3. Similar results as those unidentified carotenoids (2, 3) were only detected in this maize type. obtained for white and orange Cabanita maize have been reported by The concentrations of all mentioned carotenoids in orange maize were Zhang et  al. (22) in yellow maize. In that study, the total bound higher than values found in white and red types. A violaxanthin Frontiers in Nutrition 09 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 Frontiers in Nutrition 10 frontiersin.org TABLE 3 Carotenoid profiles and contents (μg/g DW) determined by UHPLC in Cabanita maize kernels of different pigmentations and maturity stages. Compound White Red Orange F-value S1 S2 S3 S1 S2 S3 S1 S2 S3 Maize Stage M × S (M) (S) Neoxanthin isomer2 0.18 ± 0.02c 0.14 ± 0.04c 0.11 ± 0.02c 0.11 ± 0.04d 0.15 ± 0.05c 0.08 ± 0.01c 0.39 ± 0.07a 0.29 ± 0.13b 0.31 ± 0.08ab 38.69**** 2.42ns 1.24ns (~13-cis-neoxantin) All-trans- 0.19 ± 0.03b 0.15 ± 0.11b 0.22 ± 0.01b 0.16 ± 0.16b 0.17 ± 0.07b 0.18 ± 0.02b 0.17 ± 0.11b 0.22 ± 0.11b 0.37 ± 0.12a 2.79ns 2.76ns 1.21ns neoxanthin2 Unidentified 0.05 ± 0.02 ND1 ND ND ND ND ND ND ND carotenoid-12 Violaxanthin isomer2 0.06 ± 0.02b 0.04 ± 0.02b 0.05 ± 0.01b ND ND 0.04 ± 0.02b 0.13 ± 0.04a 0.14 ± 0.06a 0.13 ± 0.05ab (~9-cis-violaxanthin) Unidentified ND ND ND ND ND ND 0.14 ± 0.05b 0.26 ± 0.04a 0.17 ± 0.06b carotenoid-22 Lutein isomer2 0.10 ± 0.03c 0.14 ± 0.08c 0.14 ± 0.05c 0.12 ± 0.06c 0.18 ± 0.11c 0.14 ± 0.03c 0.64 ± 0.27a 0.75 ± 0.24a 0.41 ± 0.04b 48.72**** 2.69ns 2.40ns (~13-cis-lutein) Unidentified ND ND ND ND ND ND 0.12 ± 0.03a 0.11 ± 0.03a ND carotenoid-32 Zeaxanthin isomer3 ND ND ND ND 0.03 ± 0.01c ND 0.18 ± 0.05ab 0.21 ± 0.08a 0.12 ± 0.03b (~13-cis-zeaxanthin) All-trans-lutein 0.14 ± 0.04c 0.07 ± 0.06c ND 0.06 ± 0.03c 0.03 ± 0.02c ND 1.48 ± 0.62b 2.00 ± 0.40a 1.02 ± 0.34b All-trans-zeaxanthin 0.07 ± 0.04c 0.16 ± 0.04c 0.08 ± 0.03c 0.15 ± 0.07c 0.23 ± 0.06bc 0.09 ± 0.03c 1.44 ± 0.35a 1.35 ± 0.20a 0.54 ± 0.22b 155.08**** 17.18**** 11.56**** Lutein isomer2 (~9 ND ND ND ND ND ND ND ND 0.07 ± 0.03 or 9′-cis-lutein) β-cryptoxanthin ND ND ND ND ND ND 0.17 ± 0.06a 0.20 ± 0.09a 0.06 ± 0.01b isomer4 (~13 or 13′-cis-β- cryptoxanthin) β-cryptoxanthin ND ND ND ND ND ND 0.10 ± 0.04a 0.19 ± 0.12a ND isomer4 (~9 or 9′-cis- β-cryptoxanthin) Total carotenoids 0.77 ± 0.10d 0.69 ± 0.22d 0.62 ± 0.10d 0.66 ± 0.28d 0.84 ± 0.27d 0.56 ± 0.11d 4.97 ± 1.26b 5.87 ± 0.73a 3.19 ± 0.61c 209.22 **** 10.53*** 7.21*** Different letters in the same row indicate significant statistical differences (***p < 0.001; and ****p < 0.0001); ns, no significant. F-values were calculated only in complete dataset per variable. S1, S2, S3 indicate maturity stages. 1Non-detected. 2Expressed as lutein. 3Expressed as zeaxanthin. 4Expressed as β-cryptoxanthin. Ranilla et al. 10.3389/fnut.2023.1132228 isomer (~9-cis-violaxanthin) was detected in white and orange maize Carotenoids are highly sensitive to light, heat, and oxygen, therefore at all maturity stages, and only at S3  in the red grain. These postharvest practices applied by Andean farmers such as the xanthophylls diversity may be based on the fact that β-cryptoxanthin sun-drying of Cabanita ears first in the plant and later on the field for is the metabolic precursor of zeaxanthin which is further metabolized undetermined time may lead to the degradation of to violaxanthin and then to neoxanthin (56). Several studies have carotenoid compounds. confirmed that predominant carotenoid compounds in maize are Higher total carotenoid amounts than those from current research generally lutein, zeaxanthin, β-cryptoxanthin along with other minor have been reported mostly in yellow and sweet maize varieties. Xu xanthophylls such as zeinoxanthin, antheraxanthin, violaxanthin, et al. (24) found concentrations of 22.78–28.76 μg/g DW at different neoxanthin, and their isomers (23, 57–59). However, carotene maturity stages in yellow maize. Ranges of 0.55–43.23 μg/g DW and compounds such as α-carotene and β-carotene have been also 11.4–24.0 μg/g DW have been shown in sweet maize harvested at reported in comparable concentrations in yellow maize varieties (44, maturity stages from 10 to 32 DAP (23, 63). Floury maize types 60). Liu et al. (23) reported that two genotypes of Chinese sweet maize generally show lower carotenoid contents than hard maize classes such showed variable carotenoid profiles and contents during the grain as pop, dent, or flint (64). Therefore, it is expected to find lower maturation from 10 to 30 DAP. This indicates an important influence carotenoid levels in the amylaceous floury Cabanita maize (19). of genetic factors and the kernel maturity stage (61). The maturity stage and the interaction of both factors (M × S) significantly influenced the all-trans-zeaxanthin, and the total 3.4. Fatty acid composition of Cabanita carotenoid contents. Neoxanthin isomer, all-trans-neoxanthin, maize types at different maturity stages violaxanthin isomer, and ~13-cis-lutein did not show significant changes with kernel development in white and red maize types, but The fatty acid composition of Cabanita maize is shown in Table 4. all-trans-lutein was not detected at S3. Overall, both maize types No significant effect was found by the maize type indicating similar showed similar total carotenoid concentrations which were somewhat fatty acid profiles and contents among all samples. Polyunsaturated stable along the kernel growth (ranges of 0.77–0.62 μg/g DW and fatty acids (PUFA) including linoleic and α-linolenic acids represented 0.84–0.56 μg/g DW, for the white and red maize, respectively). In the the major fatty acid fraction in all Cabanita maize types (55–59% of orange Cabanita, all-trans-lutein increased by ~35% from S1 to S2 the total fatty acid content). The monounsaturated oleic acid (1.48 and 2.0 μg/g DW at S1 and S2, respectively), but then decreased contributed with 21–28% of the total fatty acids, followed by saturated by 50% at S3 (1.02 μg/g DW). All-trans-zeaxanthin remained almost acids (palmitic and stearic acids, 18–21%). Among all detected fatty constant from S1 to S2 (1.44 and 1.35 μg/g DW, respectively). However, acids, linoleic and oleic acids were the most abundant compounds in it declined by 60% at S3 (0.54 μg/g DW). Similar carotenoid reductions maize kernels (50–54%, and 21–28% for linoleic and oleic acids, at S3 were observed in case of the 13-cis-lutein, 13-cis-zeaxanthin, respectively). β-cryptoxanthin isomers, and the other unidentified compounds. Comparable percentages of linoleic acid have been also reported All-trans-neoxanthin and its isomers showed a certain increase at S3 in Mexican subtropical maize populations (41–51%), sweet maize which indicates the downstream metabolic conversion of from the United States (50–63%), and maize varieties from Turkey β-cryptoxanthin, and zeaxanthin (56). The contents of lutein, (50–53%) (65–67). In the case of other Peruvian germplasm, similar zeaxanthin, α-cryptoxanthin, and β-cryptoxanthin have shown to fatty acids profiles and concentrations were observed in mature native steadily increase with kernel development from 10 to 30 DAP in sweet varieties such as Chullpi, Piscorunto, Giant Cuzco, Sacsa, and purple, corn (23). The increase of zeaxanthin and lutein with kernel with ranges of 18.3–25.2 mg/g DW and 9.8–14.6 mg/g DW for linoleic maturation from 16 to 24 DAP has been also reported in other sweet and oleic acids, respectively (68). However, α-linolenic acid maize hybrids (62). Variable zeaxanthin and lutein patterns were concentrations were almost 2 to 3.5-fold higher in Cabanita samples observed in some zeaxanthin-biofortified sweet maize depending on (1.3–1.4 mg/g DW, at S3 maturity stage) than in the other Peruvian the genotype and the kernel position on the cob (61). However, an varieties (0.4–0.7 mg/g DW) (68). Lower α-linolenic acid percentages overall lutein and zeaxanthin accumulation was reported in same have been also reported in sweet maize (1.7–2.1%) compared with study (61). In the current research, higher maturity stages were Cabanita maize (3.2–3.7%) (66). Furthermore, this fatty acid was not evaluated (from 28–32 to 75–77 DAP) which likely explains even detected in several Korean maize hybrids (69). The increase of contrasting results compared with previous studies. Xu et al. (24) α-linolenic acid from 0.61 to 4.93% along with the oil contents have evaluated a yellow maize variety during maturation from 74 to 116 been obtained after a long-term breeding process of Mexican maize DAS and found that the contents of zeaxanthin decreased at the end (65). The contribution of this fatty acid in relation to the total fatty of kernel maturity, whereas lutein increased from 74 to 98 DAS to acid content was higher at early maturity stages in Cabanita samples finally decrease at 116 DAS. (5.1–5.8%). The orange maize exhibited higher total carotenoid contents Linoleic (ω-6) and α-linolenic acids (ω-3) are essential fatty acids (3.19–5.87 μg/g DW) than white and red maize (0.77–0.62 μg/g DW that cannot be  synthesized by humans (70). After the ingestion, and 0.84–0.56 μg/g DW, for the white and red maize, respectively). α-linolenic acid is transformed into long-chain ω-3 PUFAs such as Nevertheless, carotenoids significantly decreased by ~50% at S3 (from docosahexaenoic and eicosapentaenoic acids which play important 5.87 to 3.19, at S2 and S3, respectively). Fuentes-Cardenas et al. (19) roles within the organism (71). Dietary α-linolenic acid, and its ω-3 found lower total carotenoid values in the orange Cabanita maize type metabolic derivatives have been reported to show antioxidant and at physiological maturity (1.95 μg/g DW, COM code) than in the anti-inflammatory properties with potential for the prevention of present study. In addition, no carotenoids were detected in the brain malfunction, and cardiovascular disease (72–74). Moreover, corresponding red and white parental seeds (CCR, CAW) (19). diets with ω-6:ω-3 ratios close to 1:1 have been associated with less Frontiers in Nutrition 11 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 TABLE 4 Contents and profiles of fatty acids (mg/g DW) in Cabanita maize kernels of different pigmentations and maturity stages. Maize type Stage Saturated fatty acids Unsaturated fatty acids Total fatty acids Palmitic acid Estearic acid Oleic acid Linoleic acid α-Linolenic acid White S1 5.1 ± 0.9ab 0.9 ± 0.1bcd 5.9 ± 1.3d 14.7 ± 3.2c 1.6 ± 0.1a 28.0 ± 5.3c S2 5.1 ± 0.6ab 0.8 ± 0.1 cd 6.6 ± 1.4 cd 15.6 ± 2.7bc 1.3 ± 0.1de 29.3 ± 4.7bc S3 5.9 ± 0.9ab 1.1 ± 0.2a 11.0 ± 2.2a 20.5 ± 4.1a 1.3 ± 0.1e 39.8 ± 7.2a Red S1 5.3 ± 0.8ab 0.8 ± 0.1 cd 6.6 ± 1.9 cd 15.3 ± 3.3c 1.5 ± 0.1b 29.5 ± 6.1bc S2 5.2 ± 0.6ab 0.8 ± 0.1 cd 7.5 ± 0.9bcd 15.2 ± 2.3c 1.4 ± 0.1 cde 30.1 ± 3.8bc S3 6.3 ± 1.3a 1.0 ± 0.2ab 9.8 ± 2.5ab 21.1 ± 4.4a 1.30 ± 0.03e 39.5 ± 8.2a Orange S1 4.9 ± 0.6b 0.7 ± 0.1d 5.7 ± 0.9d 14.5 ± 1.5c 1.5 ± 0.1b 27.2 ± 2.8c S2 6.0 ± 1.0ab 0.9 ± 0.2bc 9.0 ± 1.9abc 20.4 ± 4.5ab 1.4 ± 0.1bcd 37.6 ± 7.6ab S3 5.9 ± 0.6ab 0.9 ± 0.1abc 9.6 ± 1.6ab 21.2 ± 2.9a 1.4 ± 0.1bc 39.0 ± 4.8a F-value Maize (M) 0.35ns 0.62ns 0.06ns 0.97ns 2.84ns 0.48ns Stage (S) 3.91* 11.72*** 17.63**** 10.29**** 20.45**** 11.19*** M × S 0.81ns 2.01ns 1.55ns 1.08ns 4.24** 1.08ns Different letters in the same column indicate significant statistical differences (*p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001); ns: no significant differences. S1, S2, S3 indicate maturity stages. incidence of chronic diseases including diabetes and cardiovascular lipophilic fractions of Cabanita maize samples (Table  5). The diseases (70). Higher ω-6:ω-3 ratios have been found in unbred maize interaction of maize type (M) and the maturity stage (S) was not cultivars from Mexico (59–80:1), sweet maize from US (29–33:1), and significant in all cases; however, all variables were influenced by S. The Peruvian germplasm from other races (36–50:1) in comparison with DPPH hydrophilic antioxidant capacity (DPPH-HF) declined with ratios found in evaluated Cabanita maize at physiological maturity grain growth and there were differences depending on the maize type stage (15–16:1) (65, 66, 68). Genetic factors may play a role on (M significant). Orange Cabanita showed higher values (422.3– observed differences. In addition, differences in the agroecological 821.7 μmol TE/100 g DW) than white and red types (310.7–490.6 and and post-harvest management conditions may also be involved. Based 308.9–520.2 μmol TE/100 g DW, for the white and red maize, on the current results, Cabanita maize shows potential as a dietary respectively). The DPPH-HF decreased from S1 to S3 by 37, 41 and source of health relevant PUFAs. 49% in the white, red, and orange maize, respectively. The opposite The maturity stage had a strong influence on fatty acid variability in trend was observed in case of the DPPH lipophilic antioxidant all Cabanita types (Table 4). Saturated acids slightly increased at S3, but capacity (DPPH-LF). Values increased from S1 to S3 around 3.4 and their proportions with respect to the total fatty acid contents decreased in 4.8-fold in the orange and red maize, respectively. In the white maize, all cases from 18 to 15% on average. Contents and percentages of stearic the DPPH-LF was not detected at S1, but then it increased to 7.8 and acid almost remained constant with maturation. Oleic acid increased 20.8 μmol TE/100 g DW with kernel development at S2 and S3, from 21 to 22% at S1 to 25–28% at S3. Linoleic acid concentrations also respectively. increased and were high at S3, but their percentages in relation to the total The ABTS hydrophilic antioxidant capacity (ABTS-HF) also fatty acid contents showed almost no variation with kernel growth (from decreased with kernel maturity in a range of 36–51%, similarly as in 52 to 51%, from 52 to 53%, and from 53 to 54% for white, red and orange the case of the DPPH-HF. However, the maize type did not show an maize, respectively). The α-linolenic acid concentrations and percentages important effect. Ranges were almost comparable among all Cabanita decreased from S1 to S3 (5.1–5.8% to 3.2–3.7%). Palmitoyl-CoA is a types at all maturity stages (1009.0–2065.2 μmol TE/100 g DW, metabolic precursor of palmitic acid, and of stearoyl-CoA which in turn 1297.5–2012.3 μmol TE/100 g DW, and 1085.2–2194.4 μmol TE/100 g serves as a precursor of oleic, linoleic, and linolenic acids biosynthesis via DW, for the white, red, and orange maize, respectively). In addition, several desaturase enzymes (56). A possible metabolic change toward the the ABTS lipophilic antioxidant capacity (ABTS-LF) increased biosynthesis of oleic and linoleic acids instead of palmitic acid may 1.6–2.0-fold from S1 to S3 as also was noticed in case of the explain its percentage decrease with maturity. It is noteworthy that DPPH-LF. The orange maize exhibited the highest value at S3 ω-6:ω-3 ratios are lower at S1 stage (9.2–10.4:1) than at S3 (15–16:1) (94.2 μmol TE/100 g DW) among samples (M significant). indicating better PUFAs balance when Cabanita maize is at milk stage. The hydrophilic extracts strongly inhibited both free radicals more than lipophilic fractions indicating higher contents of hydrophilic antioxidants such as soluble polyphenols. In fact, higher 3.5. In vitro health-relevant functionality of concentrations of total free phenolic compounds have been Cabanita maize types at different maturity determined in this study in comparison with the total carotenoid stages contents (ranges of 10–65 mg/100 g DW and 0.56–5.87 μg/g DW, for the total free phenolic and total carotenoid contents, respectively). The 3.5.1. DPPH and ABTS antioxidant capacity UHPLC total free phenolic contents highly correlated with the The antioxidant capacity was evaluated with two different in vitro antioxidant capacity (r = 0.7709 and r = 0.7863, p < 0.05 for the methods and only in the bioavailable-relevant soluble hydrophilic and DPPH-HF and ABTS-HF, respectively). Hydroxybenzoic acid Frontiers in Nutrition 12 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 TABLE 5 In vitro antioxidant capacity (μmol TE/100 g DW) in Cabanita maize kernels of different pigmentations and maturity stages. Maize type Stage Inhibition of DPPH Inhibition of ABTS HF LF HF LF White S1 490.6 ± 57.3bcd ND 2065.2 ± 98.7ab 36.4 ± 5.0f S2 313.5 ± 79.2 cd 7.8 ± 4.6 cd 1959.7 ± 278.1ab 45.1 ± 7.0ef S3 310.7 ± 20.7d 20.8 ± 3.5ab 1009.0 ± 86.0d 70.0 ± 3.7bc Red S1 520.2 ± 329.6bc 4.1 ± 3.6d 2012.3 ± 121.8ab 40.6 ± 3.0ef S2 507.5 ± 106.0bcd 14.3 ± 7.7bc 1819.8 ± 138.6b 48.4 ± 12.9de S3 308.9 ± 150.8d 19.9 ± 12.5ab 1297.5 ± 353.1c 81.0 ± 15.0b Orange S1 821.7 ± 114.5a 8.0 ± 3.4 cd 2194.4 ± 121.5a 58.9 ± 5.5 cd S2 582.2 ± 135.8b 12.7 ± 4.5bcd 1949.6 ± 185.2ab 71.7 ± 1.6b S3 422.3 ± 53.1bcd 27.5 ± 4.1a 1085.2 ± 45.9 cd 94.2 ± 6.4a F-value Maize (M) 8.51** 0.38ns 30.91**** Stage (S) 10.05*** 92.36**** 68.17**** M × S 1.24ns 2.01ns 0.50ns Different letters in the same column indicate significant statistical differences (*p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001); ns, no significant. F-values were calculated only in complete dataset per variable. HF, hydrophilic fraction; LF, lipophilic fraction. S1, S2, S3 indicate maturity stages. compounds such as vanillic acid derivatives showed a positive respectively) than in the current research (28–77 DAP) (22, 79). correlation with this property (r = 0.5740 and r = 0.7502, p < 0.05 for Furthermore, Liu et al. (23) observed that the lipophilic oxygen radical the DPPH-HF and ABTS-HF, respectively). Likewise, the HBA-1 absorbance antioxidant capacity (ORAC) increased with grain compound, and the total HBA contents were correlated with the maturity (10–30 DAP) in sweet maize showing correlation with the antioxidant capacity measured with both methods (r = 0.5790 and total and individual carotenoids such as lutein and zeaxanthin. 0.7962, p < 0.05 for the DPPH-HF and ABTS-HF, respectively in case Differences in the current results from those of above studies indicates of the HBA-1; and r = 0.5900 and r = 0.7987, p < 0.05 for the DPPH-HF an important influence of genetic factors, the maturity stage, and the and ABTS-HF, respectively in case of the total HBA contents). origin of maize. Moreover, free luteolin derivatives also contributed to the antioxidant In a previous study with Peruvian white, red, and orange Cabanita capacity in the orange maize group (r = 0.6246, p < 0.05 for the DPPH- maize, Fuentes-Cardenas et  al. (19) pointed out that hydrophilic HF). Consistent with the current study, soluble phenolic compounds compounds strongly contributed to the in vitro antioxidant capacity were correlated with high antioxidant capacity evaluated with the (DPPH and ABTS methods) than lipophilic fractions like this study. ferric reducing antioxidant power (FRAP) and DPPH methods in Nevertheless, lower ABTS-HF was reported by above authors (566.3– several Italian maize landraces (75). Flavonoids including 685.4 μmol TE/100 g DW) than in this research (1009.0–1297.5 μmol anthocyanins have shown to highly contribute to the free radical TE/100 g DW) at physiological maturity stage. This may suggest that antioxidant capacity in Mexican red and purple-pigmented maize (76, postharvest management also plays a role in the observed bioactive 77). In the current study, no correlation was found between the total variability and associated functional quality. anthocyanin contents and the antioxidant capacity in Cabanita red maize which may be due to its lower anthocyanin ranges compared to 3.5.2. Inhibitory activity against α-amylase and HBA concentrations specially at S1 and S2 stages. α-glucosidase enzymes In case of the lipophilic antioxidant capacity, a moderate The intake of natural inhibitors of key intestinal carbohydrate- correlation was found between this functional quality and the total hydrolyzing enzymes such as α-amylase and α-glucosidase may carotenoid contents (r = 0.5354, p < 0.05 with the DPPH method). represent an important dietary strategy for hyperglycemia Other lipophilic compounds such as tocopherols and tocotrienols management relevant for the type-2 diabetes prevention (80–82). common in the germ of maize grains but not analyzed in the current Table 6 shows the potential in vitro inhibitory activity of the soluble study may also play a role (17). Some tocopherol compounds from hydrophilic and lipophilic fractions from Cabanita maize samples spelt grain (Triticum spelta) have shown significant correlation with against α-amylase and α-glucosidase enzymes. the antioxidant capacity measured with the DPPH method (78). All hydrophilic (HF) and lipophilic (LF) maize extracts inhibited A continuous decrease of the DPPH and FRAP antioxidant the α-glucosidase enzyme in a sample dose dependent manner capacity was observed in the soluble phenolic fractions from yellow (3–10 mg). However, HF extracts showed higher inhibition than LF maize at different developmental stages (from 74 to 116 DAS) (24). Hu fractions. The type of maize (M) was not significant on the and Xu (37) and Giordano et al. (39) reported that the free radical α-glucosidase inhibitory activity of both HF and LF fractions at all inhibitory activity along the kernel growth stages was highly variable evaluated sample doses. This indicates similar inhibitory potential depending on the maize variety. An increase of the antioxidant among all Cabanita maize types. No effect of the M x S interaction was response with kernel maturity were reported in sweet and yellow found on results from HF and LF fractions, except at 10 mg (HF). In maize in maturity periods shorter (17–25 DAP and 15–48 DAP, this case, HF fractions from white and orange maize exhibited greater Frontiers in Nutrition 13 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 inhibition than red maize at S1 (40.0, 41.0, and 32.3%, for the white, orange, and red maize, respectively). Nonetheless, the inhibitory activity of red maize was higher than white and orange at S2 (31.8, 20.2, 22.7%, for the red, white, and orange samples, respectively), whereas results of all HF fractions were almost similar at S3. The maturity stage (S) had a significant influence on the HF inhibitory activity at all sample doses. At 10 mg, this property decreased with kernel development (from 28–33 to 75–76 DAP) by 63, 37 and 45% in the white, red, and orange group, respectively. This reduction occurred at all sample doses, indicating that hydrophilic inhibitors may be related to the soluble phenolic fraction which also declined with kernel maturity as previously stated. This in vitro functional quality had high correlation with the free UHPLC soluble phenolic compounds at all sample doses (r = 0.7386, r = 0.8064, and r = 0.8545, p < 0.05 at 3, 6, and 10 mg sample dose, respectively). Specific phenolic compounds such as the vanillic acid derivatives, HBA-1, and the total HBA contents positively correlated with the inhibitory potential of Cabanita maize samples (r = 0.7962, r = 0.7728, and r = 0.7961, p < 0.05, respectively, 10 mg sample dose). Among free HCA compounds, ferulic acid derivatives also showed a significant correlation (r = 0.6597, p < 0.05, 10 mg sample dose). In case of LF fractions, results were not influenced by S, showing comparable α-glucosidase inhibition during the grain growth. However, a certain increase (from 5.7 to 8.7% and from 8.6 to 15.1% at 6, and 10 mg of sample dose, respectively) was observed in the orange maize. No significant correlations were found between the LF α-glucosidase inhibitory activity, and any metabolites measured in the current study. The α-amylase enzyme, relevant for the hydrolysis of α-1,4-glucan polysaccharides into maltose and maltooligosaccharides (83), was inhibited only by HF maize fractions in a dose-dependent manner (Table 6). All values decreased with kernel maturity (S significant) similarly as in the case of the α-glucosidase inhibitory activity. When the maturity stage changed from S1 to S3, the white maize (125 mg dose) showed the highest loss of the inhibitory potential (around 85%), followed by the red, and orange maize (~54 and 58% in red and orange maize, respectively). Both M × S interaction and M factors greatly influenced the α-amylase inhibition. White maize samples had higher α-amylase inhibition at S1 among Cabanita maize types. The α-amylase inhibitory potential positively correlated with the free UHPLC total phenolic contents (r = 0.6358 and r = 0.6574, p < 0.05, at 62 and 125 mg of sample dose, respectively). Furthermore, all HBA compounds and free ferulic acid derivatives were correlated with this in vitro functional property (r = 0.5278–0.6471 at all sample doses, and r = 0.5340–0.5599 at 62–125 mg, respectively). Different studies have highlighted the role of phenolic compounds for hyperglycemia prevention and countering associated oxidative complications through several mechanisms including the modulation of gastric enzymes at intestinal level (84–87). Cereal-derived phenolic acids including several HBA, and HCA compounds have shown inhibitory potential against the intestinal α-glucosidase enzyme which was highly dependent on the number of hydroxyl and methoxy groups in their structure (88). HBA derivatives such as methyl vanillate (a vanillic acid derivative), syringic acid, and vanillic acid from Thai colored rice showed higher inhibition of α-glucosidase than on α-amylase with a mixed-type inhibition mode against α-glucosidase (89). In same study, in silico analysis revealed that the inhibition involved the molecular interaction between HBA and the binding sites of digestive enzymes through 3–4 hydrogen bonds depending on the Frontiers in Nutrition 14 frontiersin.org TABLE 6 In vitro inhibitory activity against α-amylase and α-glucosidase in Cabanita maize kernels of different pigmentations and maturity stages. Maize Stage α-Glucosidase inhibitory activity (%) α-Amylase inhibitory activity (%) type 3 mg1 6 mg 10 mg 25 mg 62 mg 125 mg HF LF HF LF HF LF HF LF HF LF HF LF White S1 17.2 ± 3.5a 6.1 ± 4.3a 31.3 ± 4.0a 7.5 ± 2.6ab 40.0 ± 6.3a 8.9 ± 2.0b 7.0 ± 3.0a ND2 18.6 ± 8.3a ND 55.0 ± 25.4a 4.3 ± 5.9 S2 10.7 ± 4.2 cd 4.7 ± 4.9a 16.6 ± 4.8de 7.1 ± 1.7ab 20.2 ± 4.9 cd 11.9 ± 3.3ab 1.4 ± 1.6b ND 4.1 ± 3.0c ND 13.5 ± 4.4b ND S3 6.5 ± 1.4d 5.6 ± 1.6a 10.9 ± 2.9e 7.3 ± 0.3ab 14.8 ± 2.8d 8.6 ± 0.7b ND ND 1.8 ± 2.7c ND 8.1 ± 1.7b ND Red S1 18.4 ± 3.3a 3.4 ± 2.7a 28.0 ± 5.4ab 8.1 ± 4.2ab 32.3 ± 6.9b 12.2 ± 4.0ab 2.4 ± 3.4b ND 6.6 ± 5.9bc ND 18.2 ± 8.9b ND S2 16.1 ± 1.7ab 3.8 ± 2.9a 23.0 ± 4.3bc 8.7 ± 2.5ab 31.8 ± 2.8b 12.3 ± 2.8ab 2.0 ± 2.8b ND 8.0 ± 3.0bc ND 16.3 ± 3.2b ND S3 7.5 ± 3.8 cd 5.5 ± 4.6a 15.2 ± 6.8de 9.7 ± 3.0a 20.2 ± 7.6 cd 12.2 ± 3.5ab 2.1 ± 2.6b ND 4.2 ± 4.5c ND 8.3 ± 2.9b ND Orange S1 20.5 ± 2.3a 4.9 ± 5.8a 30.0 ± 3.4a 5.7 ± 6.2ab 41.0 ± 2.1a 8.6 ± 5.8b 6.8 ± 1.8a ND 12.8 ± 4.4ab ND 19.0 ± 3.2b ND S2 10.8 ± 3.0 cd 1.9 ± 2.9a 17.4 ± 3.5 cd 4.6 ± 3.2b 22.7 ± 4.1c 10.2 ± 2.2ab ND ND 3.9 ± 1.6c ND 9.3 ± 4.3b ND S3 12.1 ± 4.2bc 3.4 ± 2.8a 18.3 ± 1.9 cd 8.7 ± 1.3ab 22.6 ± 4.1c 15.1 ± 5.3a ND ND 3.0 ± 2.0c ND 7.9 ± 0.5b ND F-value Maize (M) 3.02ns 0.88ns 1.22ns 1.86ns 1.99ns 1.39ns 0.66ns 7.11** Stage (S) 30.13**** 0.48ns 38.26**** 1.08ns 43.89**** 1.09ns 15.76**** 19.26**** M × S 2.60ns 0.29ns 2.48ns 0.50ns 5.12** 1.71ns 4.10* 6.78*** Different letters in the same column indicate significant statistical differences (*p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001); ns, no significant. F-values were calculated only in complete dataset per variable. HF, hydrophilic fraction; LF, lipophilic fraction. 1Sample dose. 2Non-detected. S1, S2, S3 indicate maturity stages. Ranilla et al. 10.3389/fnut.2023.1132228 phenolic compound and the enzyme (89). Conjugated 3.6. Primary polar metabolites analysis by hydroxycinnamic acids amides (HCCA) identified in maize and in GC–MS of Cabanita maize types at other grains from the Poaceae family grains such as N,N-di- different maturity stages and principal feruloylputrescine, N-p-coumaroyl-N-feruloylputrescine have also component analysis been targeted as α-glucosidase inhibitors (90, 91). Recently, another ferulic acid derivative named 6’-O-feruloylsucrose isolated from black The GC–MS analysis allowed to detect 63 polar metabolites rice bran showed high α-glucosidase inhibition when in vitro and in including sugars, amino acids, other nitrogen-containing compounds, silico studies were performed (92). free fatty acids, organic acids, sugar alcohols, and phytosterols. A PCA Based on above information, the free phenolic fraction including analysis was performed considering all data from the targeted, HBA, and some HCA derivatives likely involving ferulic acid untargeted metabolomic analyses, and the in vitro functional quality derivatives detected in the current research may explain the in vitro to reveal underlying relationships among all variables (Figure 4). The anti-hyperglycemia potential of Cabanita maize HF extracts. However, first two principal components from the PCA model explained 58.1% other polar compounds may also be  involved. Some compounds of the total dataset variability. Different groups were observed based detected with the untargeted GC–MS analysis as will be discussed in on the maize type and maturity stage which were explained by 98 next section, have shown direct correlation with results from both significant variables. The heat map considering the top 60 significant enzymatic assays. Alcohol sugars including d-sorbitol, meso-erythritol variables is shown in Figure 5. PC 1 (45.2% of explained variance) and phytosterols such as campesterol, and sitosterol positively separated (from top to the bottom) all maize types at S3 (OIII, RIII, correlated with the α-amylase (r = 0.7588, r = 0.6556, r = 0.6235, and and WIII, for the orange, red, and white maize at S3, respectively) r = 0.7850, p < 0.05, at 125 mg dose respectively) and α-glucosidase from samples at earlier maturity stages (I, II, for S1 and S2, inhibitory activities (r = 0.7275, r = 0.7062, r = 0.6965, and r = 0.7218, respectively). p < 0.05, at 10 mg, respectively). Some studies have pointed out the role Maize samples at S3 were characterized by overall lower of erythritol and triterpenoid compounds for the management of concentrations of secondary metabolites (phenolic and carotenoid postprandial blood glucose through the inhibition of α-glucosidase compounds), along with reduced in vitro antioxidant and anti- enzyme (93, 94). Stigmasterol among other phytochemicals identified hyperglycemia potential than maize at S1 and S2. In relation to the in maize silk have been indicated as potential inhibitors of both primary polar metabolites detected by GC–MS, free monosaccharides α-glucosidase and α-amylase enzymes (95). (glucose, fructose, galactose), disaccharides (sucrose) among other The observed LF α-glucosidase inhibitory activity may be ascribed unidentified sugar molecules decreased with kernel maturity in all to lipophilic compounds that increase in maize kernel with maturity maize types (Figure 5). Furthermore, several amino acids (glutamic time specially in case of the orange maize. The contents of acid, glycine, alanine, phenylalanine, proline, isoleucine, leucine, α-tocopherol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol had valine, and serine) also declined with grain maturity. Simple sugars increased with maturity in grains of Amaranthus cruentus (96). may have been used as carbon sources for cellular energy metabolism, Lipophilic extracts from Vicia fava L. seeds containing α-tocopherol and the biosynthesis of starch which is known to accumulate in and γ-tocopherol compounds exhibited high α-glucosidase inhibition mature cereal grains (101). The decrease of sugar contents with the (97). It is possible that the inhibitory activity of Cabanita maize concurrent increase of starch during grain development was observed extracts against digestive enzymes may be due to the synergistic action by Xu et al. (24) and Saikaew et al. (101) in yellow and purple waxy of HL and LF compounds as also was reported by Parizad et al. (98) maize, respectively. Amino acids may have been transformed into in several pigmented cereals. Future studies are necessary to reveal the proteins or used as metabolic precursors for the synthesis of secondary identity of HF and LF compounds from Cabanita maize and their metabolites (102). The increase of protein with kernel maturation has molecular mechanisms of inhibition against hyperglycemia- been reported in maize and rice kernels (101, 103). Among data from relevant enzymes. all maize types at S3, the orange maize clearly separated and stood out α-Amylase inhibitory activity results from this study are from the red and white groups, whereas some replicates from these comparable with those reported by Ranilla et  al. (20) in mature last maize types overlapped. This difference was mainly correlated Cabanita maize kernels from Peru (8.9–10.2%, at 125 mg sample with the highest carotenoid contents in the orange group than in red dose). However, higher α-glucosidase inhibitory activities were and white maize. obtained by above authors (34.9–40.8%, at 12.5 mg of sample dose) PC 2 (12.9% of explained data variability) separated orange maize which may be linked to the higher sample doses evaluated. On the (at all maturity stages) from white and red types (from left to right). other hand, lower inhibitory activities against α-amylase and The orange maize showed unique flavonoids such as luteolin α-glucosidase were found in the free phenolic fraction from Chinese derivatives and had the highest carotenoid concentrations (especially fresh waxy maize harvested at milk stage (~28–72% and ~32–48% for at S1 and S2) as previously stated. Interestingly, the white maize was the α-amylase and α-glucosidase inhibitory activities, respectively, at different from the other maize types specifically at the S1 stage 1,000 mg sample dose) than in current study at similar maturity stage (Figure  5). Primary metabolites including simple sugars (2.4–55.0% at 25–125 mg sample dose, and 17.2–41.0% at 3–10 mg (monosaccharides and sucrose), amino acids, free fatty acids (linoleic sample dose for the α-amylase and α-glucosidase inhibitory activities, and palmitic acids), organic acids (4-aminobutanoic acid or GABA, respectively) (99). Several studies have shown the anti-hyperglycemic fumaric acid), amines (putrescine, ethanolamine), myo-inositol and potential of maize from different origins (36, 98, 100). Nevertheless, xylitol were more abundant in the white maize at milk stage than in the impact of the maturity stage on this functional property in selected the other maize groups at same maturity period. samples from the Peruvian Cabanita maize diversity is shown for the Phytosterols such as campesterol and β-sitosterol were detected first time in this study. in all maize groups. The orange and red maize showed comparable Frontiers in Nutrition 15 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 FIGURE 4 Principal component analysis (PCA) score plot of all data from white (W), red (R), and orange (O) Cabanita race at different maturity stages (I: S1, II: S2, III: S3). campesterol contents, but the white maize exhibited the highest Other detected primary metabolites such as free fatty acids sitosterol abundance at S1. Phytosterols are bioactive compounds (palmitic acid, linoleic acid), organic acids (glyceric acid, fumaric acid, with potential for the prevention of cardiovascular diseases because ribonic acid, GABA), amines (ethanolamine, putrescine), of their cholesterol-lowering properties (104). Both detected myo-inositol, and alcohol sugars (erythritol, xylitol) were also reduced phytosterols decreased with grain maturity in all Cabanita types. The with grain maturity in all maize types. Free fatty acids may have been reduction of β-sitosterol has been also observed during Camellia metabolized for the synthesis of triacylglycerols (TAG) since the total chekiangoleosa Hu. seeds development (105). β-sitosterol is the key fatty acid contents (derived from the triacylglycerol saponification) precursor of sitosterol-β-glucoside which play a role on the increased with grain maturity in all Cabanita groups (Table 4). The biosynthesis of cellulose (106). The increase of dietary fibre, which increase of the total lipid contents during the grain development of is composed of cellulose, hemicellulose, lignin, among other yellow maize has been related to the late embryo formation (24). The polymers has been reported in Amaranthus cruentus with grain reduction of ethanolamine and glyceric acid may have been targeted maturity (96). Furthermore, the cell wall feruloylation along with the as precursors of glycerophospholids (components of the cell lignin-cross links of the wheat grain outer layers increased during membranes) during grain growth. Some types of kernel development (107). This likely explains the decrease of free phosphatidilethanolamines esterified with variable fatty acids have ferulic acid derivatives and the increase of some cell wall phenolics increased during wheat kernel filling (108). The reduction of (bound ferulic acid derivatives) specially in the red and orange intermediate metabolites involved in the tricarboxylic acid cycle maize at S3 maturity stage. (TCA) such as fumaric acid and GABA-derived succinic acid might Frontiers in Nutrition 16 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 FIGURE 5 Heat map considering 60 significant variables from white (W), red (R), and orange (O) Cabanita race at different maturity stages (I: S1, II: S2, III: S3). reflect a high mitochondrial activity for energy generation during the Cabanita showed variable primary (polar metabolites), and secondary maize grain development (109). Polyamines including putrescine have metabolite composition (phenolic and carotenoid compounds) and essential roles in many biochemical and physiological processes, in were greatly influenced by the grain maturity stage. All maize types particular stress and senescence responses during plant growth and showed free HBA and HCA phenolic compounds, but luteolin development (110). Putrescine content also decreased with kernel derivatives and anthocyanins were only detected in orange and red maturity in Cabanita maize as observed in other polar metabolites. maize, respectively. Major bound phenolic compounds were ferulic This polyamine may have been used for the biosynthesis of some acid, followed by ferulic acid derivatives, and p-coumaric acid in all HCAAs such as N-p-coumaroyl-N-feruloylputrescine and Cabanita groups. However, orange, and red types had higher bound caffeoylputrescine which have been detected in mature maize (44). In ferulic acid, and total phenolic contents (free + bound) (223.9– the current study, p-coumaric and caffeic acid derivatives (possibly 274.4 mg/100 g DW, 193.4–229.8 mg/100 g DW for the orange and red HCAAs) have increased with Cabanita kernel maturation. maize, respectively) than the white maize (162.2–225.0 mg/100 g DW). Xanthophylls including lutein, zeaxanthin, neoxanthin, lutein isomer (~13-cis-lutein) were detected in all maize types. The orange maize had 4. Conclusion the highest total carotenoid contents (3.19–5.87 μg/g DW) and contained specific carotenoids such as β-cryptoxanthin and zeaxanthin Maize with different kernel pigmentations (white, red, and orange) isomers. Most phenolic and carotenoid compounds decreased with and representing the diversity of the Peruvian Andean maize race kernel maturity in all Cabanita maize types. With respect to the primary Frontiers in Nutrition 17 frontiersin.org Ranilla et al. 10.3389/fnut.2023.1132228 metabolites, all maize types showed similar fatty acid contents (linoleic Funding acid > oleic acid > palmitic acid > α-linolenic acid > stearic acid) which increased with kernel development. Other primary metabolites such as This research was supported by PROCIENCIA-CONCYTEC simple sugars, alcohols, amino acids, free fatty acids, organic acids, (Peru) under the Basic Research Program E041-2018-01, Contract amines, and phytosterols declined with grain maturity and were overall N°114-2018-FONDECYT. more abundant in white maize at S1. The in vitro antioxidant potential and the inhibitory activity against digestive enzymes (α-amylase and α-glucosidase) were high in the hydrophilic fractions and correlated Acknowledgments with the free phenolic fraction. In general, all Cabanita maize types had similar in vitro health-relevant functionality which significantly We thank Eng. Humberto Jose Stretz-Chavez, and Nestor Zeballos decreased with grain development. Based on above results, for the technical assistance during the preliminary experiments and recommended harvesting periods for the consumption of the orange the final Cabanita maize cultivation, respectively. We also thank to and white Cabanita would be at S1 and S2 stages due to their higher Abraham Mamani, and Excequel Ponce Guequen for their support phenolic, carotenoid contents (in case of the orange type), in vitro during the UHPLC-DAD, and GC–MS/FID analysis, respectively. In functional qualities, phytosterol concentrations, and better ω-6:ω-3 addition, we thank Jose Villanueva for his support during the use of PUFAs balance. The red Cabanita maize would be more valuable at S3 the freeze drier. Evaluated Cabanita maize samples are from Peru and because of its higher total anthocyanin, and phenolic contents. have been accessed under the contract N° 002-2021-MIDAGRI-INIA/ Nevertheless, the potential changes on Cabanita technological and DGIA (Peru) with Resolución Directoral N°0006-2021-INIA- processing characteristics at lower maturity stages should be further DGIA. Information about Cabanita maize is shown in Tables 1–6 and evaluated. Current study provides relevant metabolomic and Figures  1–5 of current manuscript. Access and Benefit-Sharing biochemical information that contribute to the characterization of the Clearing-House (ABSCH) ABSCH-IRCC-PE-256874-1. Andean Cabanita maize diversity. Insights from this research would Internationally recognized certificate of compliance constituted from be important for promoting its consumption beyond its mature form as information on the permit, or its equivalent made available to the it is currently applied, and for future improvements at postharvest level. Access and Benefit-sharing Clearing-House of the Convention on Studies at transcriptomic level would help to reveal the mechanisms Biological Diversity (CBD) (https://absch.cbd.int/en/database/ involved in the metabolic changes related to the secondary and primary ABSCH-IRCC-PE-256874). metabolites during Cabanita maize maturation. Next level studies should focus on improving the nutraceutical and nutritional properties of Cabanita maize with its sustainability and consumer-relevant yield Conflict of interest characteristics within the context of Andean food systems. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could Data availability statement be construed as a potential conflict of interest. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Publisher’s note All claims expressed in this article are solely those of the authors Author contributions and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product LR and GZ conceived and designed the study. LR directed the that may be evaluated in this article, or claim that may be made by its research and wrote the manuscript. AA-C performed the experiments. manufacturer, is not guaranteed or endorsed by the publisher. RC helped with the experimental work. HH contributed with the direction of crop management and pollination control. MV-V helped with the statistical analysis. HB-G coordinated and helped with the Supplementary material original sample collection. RP developed the untargeted metabolomic and free fatty acid analysis. GZ, RC, RP, HH, and KS critically reviewed The Supplementary material for this article can be found online the manuscript. All authors contributed to the article and approved at: https://www.frontiersin.org/articles/10.3389/fnut.2023.1132228/ the submitted version. full#supplementary-material References 1. Guzzon, F, Arandia Rios, LW, Caviedes Cepeda, GM, Céspedes Polo, M, Chavez 3. Matsuoka, Y, Vigouroux, Y, Goodman, MM, Sanchez, J, Buckler, E, and Cabrera, A, Muriel Figueroa, J, et al. Conservation and use of Latin American maize Doebley, J. 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