A field experiment was conducted during the winter season at the Indian Council of Agricultural Research–Directorate of Onion and Garlic Research (ICAR–DOGR) farm in Pune, Maharashtra, India. The site, located at 18.32° N and 73.51° E, is 645 m above sea level and has a tropical dry humid climate with an average annual precipitation of 820 mm. The soil at the site is clay loam, with low available nitrogen and medium soil organic carbon. The field experiment, designed as a completely randomized block design, included eight onion cultivars: Bhima Dark Red (BDR), Bhima Raj (BRJ), Bhima Super (BSR), Bhima Red (BRD), Bhima Shakti (BSK), Bhima Kiran (BKN), Bhima Shweta (BSW), and Bhima Shubra (BSU), each replicated three times (Table 1). Onion seeds were sown in the nursery in the second week of October. Organic manure was applied in the field at 5 t ha⁻¹ before transplanting. On the day of transplanting, the pre-emergence herbicide oxyfluorfen was applied, followed by irrigation for weed control. Before transplanting, 100% of the required phosphorus, potassium, and sulfur and 20% of nitrogen were applied as basal fertilizers (10:26:26, muriate of potash, and bentonite sulfur). Forty-five-day-old seedlings were transplanted in the third week of December at 15 cm spacing between rows and 10 cm between plants, maintaining a plant density of 66 plants m² in 16.8 m² plots. The remaining 80% of nitrogen was applied through urea in 11 equal splits at 6-day intervals from 0 to 60 days after transplanting. Irrigation was provided via drip system, weeds were manually removed 45 days after transplanting, and other intercultural and plant protection practices followed ICAR-DOGR’s guidelines. Onion bulbs were harvested in the second week of April, once 50% of the plants had top fall. After a 3-day field curing, bulbs were separated from the foliage, leaving a 2.5 cm neck. Leaves were removed and cured in farm shade for 2 weeks. The outer two papery layers of onion were removed from bulb, dried in shade, and powdered using kitchen blender. One part was used for LC–MS characterization at National Referral Laboratory, ICAR-National Research Centre for Grapes, Pune, and the remaining sample were used for biochemical analysis.

Total monomeric anthocyanin was determined based on the principle of pH-dependent structural changes from colored oxonium ion to colorless hemiketal forms (Lee et al., 2005) with some modifications suggested by Krithika et al. (2020). Each extract (200 μL) was diluted separately with 800 μL of 0.025 M potassium chloride buffer (pH 1.0) and 0.4 M sodium acetate buffer (pH 4.5) and incubated for 15 min in dark. Absorbance for each dilution was taken using SpectroStar Nano plate (BMG Labtech) at 520 and 700 nm against blank made of distilled water. Total anthocyanin content was expressed as cyanidin-3-glucoside equivalent per 100 gram of dry matter and calculated using the following equation:

where MW is the molecular weight of cynidin-3-glucoside (449.2 g mol⁻¹), DF is the dilution factor (e.g., DF is 5 for an extract of 200 μL diluted to a final volume of 1,000 μL), ɛ is the molar extinction coefficient of cynidin-3-glucoside (26,900 LM⁻¹ cm⁻¹), T.E.V is the total extract volume, and W is the weight of sample (Lee et al., 2008).

The total phenol content was estimated using Folin–Ciocalteu reagent followed by Singleton and Rossi (1965) with some modifications suggested by Ateeq et al. (2023). Sample extraction (100 μL) was added to 200 μL of 10% (v/v) F-C reagent and thoroughly vortexed. To the vortexed mixture, 800 μL of 700 mM Na₂CO₃ was added and incubated for 2 h at room temperature. The reaction mixture (200 μL) was transferred in 96-well microplate, and the absorbance was taken at 765 nm against 95% methanol as blank on a SpectroStar Nano plate (BMG Labtech). Gallic acid was used as standard (100–1,000 μg/mL) for the preparation of calibration curve (R² = 0.9996), and the total phenol content was expressed as gallic acid equivalent (GAE) per 100 gram of onion peel.

Free radical scavenging assay was followed to assay the antioxidant activity of onion peel extract. The same extract was used for FRAP, DPPH, and ABTS assay.

The results of TAC, TPC, FRAP, DPPH, and ABTS assay were presented as mean ± standard deviation of three replicates (biological replications). Significance of difference between samples was evaluated through analysis of variance (ANOVA) using SAS base 9.2 (Tukey’s test, p < 0.05). Multivariate analysis, that is, principal component analysis (PCA), cluster analysis, and correlation coefficients were carried out with JMP 9.0.0. Biplot between PCA 1 and PCA 2 was drawn using MS excel. Chemical structure was drawn on ChemDraw 19.1.

OPE of eight onion varieties was taken for high-resolution UHPLC-Orbitrap Mass Spectrometric analysis, and compound identification was performed against an in-house updated high-resolution accurate mass (HRAM) database, which was originally received from Thermo Fisher Scientific. The automated analyte identification and confirmation were based on the HRAM measurement of the precursor and its characteristic productions, each within ±5 ppm of mass error and retention time tolerance of ±0.1 min. Matching of the isotopic pattern (by >90%) was considered as an additional filter. For example, quercetin glucoside was identified based on the precursor ion (m/z = 465.1030) with a mass error of 0.5 ppm (as observed in MS spectra) (Figure 1). It was above the threshold intensity of 5,000. In addition, the detection of characteristic fragment (m/z = 303.0501) supported its identification (as observed in MS/MS spectra). We identified 49 polyphenolic compounds including 33 anthocyanin (Table 2, compounds 14–46), 8 flavanol (6–13), 4 flavones (1–4), and 1 each of pyranoanthocyanin (47), chalcone (5), phenolic acid (48), and ellagitannins (49). Recoveries of reference standard quercetin and pelargonidin-3-O-glucoside were used for relative quantification of identified compounds. Since reference standards of all compounds were not available, pelargonidin-3-O-glucoside was used for quantitation of all anthocyanin, and quercetin was used for quantitation of all flavonoids.

The relative distribution of major classes of putatively identified polyphenolic compounds in differentially pigmented onion varieties is presented in Figure 3. Based on the absolute area response of the identified peak, it was noted that out of 49 identified phenolic compounds, maximum of 43 were detected in var. BSR which include nine flavanol, four flavones, and thirty anthocyanin glycosides. In white onion varieties BSU, only nine compounds were identified including five flavanol and eleven anthocyanin.

Flavanols are hydrophilic compound with β-glycosidic linkages which makes it a most bioavailable antioxidant in human diet (Price and Rhodes, 1997). Onion bulbs are reported to have higher flavanol content than anthocyanin (Metrani et al., 2020; Khandagale and Gawande, 2019) and are reportedly only 10% of the total polyphenols (Rodrigues et al., 2017) in bulb. In onion peel extract (OPE) of differentially pigmented onion varieties, anthocyanin was the most abundant polyphenolic compounds than flavanol. Albishi et al. (2013) also reported higher flavonoid content in red onion skin than bulb. Inner scales of onion bulbs have lower flavonoid level than outer scales (Patil and Pike, 1995). Among flavanol, quercetin glucoside was most abundant flavanol observed across pigmented onion varieties followed by isorhamnetin, kaempferol, and myricetin. Glucose is the exclusive moieties in quercetin attached to 3, 7, or 4′ position of aglycone. Quercetin 4′-glucoside and quercetin 3,4′-glucosides are widely reported to be the most abundant flavanol in onion bulbs (Pucciarini et al., 2019). The abundance of all the flavanol was very low in white onion cultivars in comparison with others. Price and Rhodes (1997) also reported quercetin and its glycosides as most predominant flavanol, and it was higher in red onion varieties Red baron, Rose, and Rijnsburger than white variety albino (Price and Rhodes, 1997).

Flavones are 2-phenyl-1-benzopyran-4-one, having additional double bond between C2 and C3 of flavonoid skeleton and no hydroxyl group at C3 position. It is reported to have lower antioxidant potential and poor absorption in human intestine but has significant role in biotic defense against insect and microbes (Hostetler et al., 2017). In OPE of differentially pigmented varieties, flavone was most abundant in light red varieties (BSR), less abundant in red varieties, and absent in white onions. A chalcone, butein, was observed only in var. BRD and protocatechuic acid in var. BSK. Trigalloyl levoglucosan was observed in all red varieties but found absent in white varieties (Table 2).

Among anthocyanin, delphinidin was the most predominant class of anthocyanin followed by cyanidin > petunidin > peonidin > malvidin > pelargonidin in most of the cultivars (Figure 4). As presented in Table 3, relative abundance of cyanidin and its glycosides observed maximum in dark red var. BDR and BRJ and gradually lower level in red var. BSR, BRD, BSK, and light red var. BKN, while it was minimum in white var. BSU. Contrary of cyanidin, percentage abundance of delphinidin and its glycoside was observed minimum in dark red verities, slightly higher in red and light red var. and maximum in white onions. Relative abundance of cyanidin and delphinidin might be the key factor associated with differential pigmentation of onion bulb. Cyanidin glycosides were observed with most acylation followed by pelargonidin. Acylation is predominant in dark red and red onion cultivars than light red and white. Cyanidin-3-(6-malonylglucoside), delphinidin, and delphinidin-3-galactoside were the predominant pigment in dark red var. BDR and BRJ, and its abundance suggests a key role in differential pigmentation pattern. Downes et al. (2009) also reported predominant presence of cyanidin-3-(6-malonylglucoside) in dark red onion. Similar reports were made by Fossen et al. (1996) and Donner et al. (1997). Most abundant aglycone across all the varieties was delphinidin followed by petunidin and aglycone form of both are more abundant in colored cultivars than white. In cyanidin glycoside, aglycone form is more abundant in white than colored varieties. As presented in Figure 5, third carbon position in benzopyrylium ring was the most preferred substitution followed by fifth carbon position. Glucose (glc), galactose (gal), rhamnose (rham), sophoroside (soph), xylose (xyl), arabinose (ara), and rutinoside (rut) were the observed glycoside substitution, and among these, glucoside and galactoside were the most preferred.

PCA was performed on the relative abundance data obtained for the putatively assigned anthocyanin compounds (14–47). Principal component score of eight varieties varying on PC1 (66.72%) and PC2 (14.75%) is presented in Figure 6. Eight distinctly pigmented varieties under investigation grouped in four groups. White varieties BSU and BSW grouped together having negative correlation with both PC1 and PC2. Varieties BSK, BRD, and BKN grouped together and showed very low variation with PC1 and considerable variation on PC2. Red var. BSR and dark red var. BRJ showed very positive correlation with PC1 and PC2. Dark red variety BRD showed very high positive correlation with PC1 and negative correlation with PC2. Examination of component pattern distribution (Supplementary Figure 1) on principle component axis revealed that substituted and acylated anthocyanin are strongly correlated with PC1 and show very less variation, whereas it is well distributed along PC2. Cyanidin (14–23) exhibited exclusive distribution on negative axis of PC2 and positive axis of PC1, whereas pelargonidin (36–38) on positive axis of PC1 and PC2. Among the five most abundant anthocyanin cyanidine-3-(6-malonylglucoside), delphinidin-3galactoside, delphinidin, petunidin 3-glucoside, and petunidin, all are positively correlated with PC2 except cyanidine-3-(6-malonylglucoside). This suggests that preceding four anthocyanin compounds might be the key determinant of red color var. BSR, BRJ, and BRD while cyanidine-3-(6-malonylglucoside) in dark red var. BDR.

Varieties were well classified by cluster analysis supporting the color distribution pattern (Figure 7). At partial R square value 0.1, we observed two cluster, one with light red var. BKN and red var. BSK while other having red var. BSR and BRD and dark red var. BRJ. Both clades separate with dark red var. BDR at R square value of 2.8. White onion var. BSU and BSW formed a separated clade at R square value >0.4. PCA and cluster analysis revealed that red var. BSK and light red var. BKN have similar anthocyanin distribution pattern. Similarly, dark red var. BRJ exhibited similar distribution of anthocyanin that of red onion var. BSR and BRD.

Anthocyanin is a subclass of flavonoid compounds imparting diverse color to onion bulbs and possess various nutraceutical properties. As presented in Table 4, the mean total anthocyanin content in dark red varieties was maximum followed by red, light red, and white varieties. Within the dark red varieties, the anthocyanin content in BDR (28.23 mg/100 g DW) was significantly high than BRJ (5.29 mg/100 g DW) with p ≤ 0.0001 level. Anthocyanin content was observed at par in red varieties BRD, BSK, and BSR and in light red var. BKN. In white varieties, very low level of anthocyanin was observed, and both were not significantly different. Anthocyanin content in dark red var. BDR is ~3–5 times higher than red onion var. and ~ 7–8 times higher than light red varieties. Zhang et al. (2016) reported range of 0.75 ± 0.40 mg/100 g FW anthocyanin content in white, 9.64 ± 0.30 mg/100 g FW in yellow, and 29.99 ± 1.19 mg/100 g FW in red onion. Albishi et al. (2013) also reported more anthocyanin content in onion peel extract (OPE) than bulb.

Total phenol content (TPC) in OPE ranged from 1738.21 to 1757.76 mg GAE/100 g DW in dark red onion, 1306.58 to 1646.73 mg GAE/100 g DW in red onion, and 78.77 to 85.5 mg GAE/100 g DW in white varieties. As presented in Table 4, TPC in all category of OPE extract was significantly different among each other with probability level at p ≤ 0.0001 level. Among red onion, TPC was maximum in var. BSR followed by var. BSK and BRD. Although OPE of light red onion BKN exhibited lower anthocyanin content than red onion, TPC was 1441.13 mg GAE/100 g DW which is higher than red onion var. BRD. High level of polyphenols in onion skin with similar range was reported in literature (Sagar and Pareek, 2020b; Lee et al., 2015; Sagar et al., 2020).

Antioxidant activity has been used widely to characterize various food matrix for the ability to scavenge or neutralize free radicals (Pyrzynska and Pękal, 2013). However, there is no single versatile method to determine antioxidant activity accurately. Here, we followed three methods, viz., FRAP, DPPH, and ABTS, to estimate total antioxidant activity (TAA). The results were compared with water-soluble tocopherol analog Trolox and expressed as micromole per gram. In all the three methods (FRAP, ABTS, and DPPH), the mean antioxidant activity was more in dark red varieties followed by light red and white varieties. The antioxidant activity ranged from 4.71 to 79.80 μmol/g DW, 22.71 to 286.7 μmol/g DW, and 8.72 to 156.89 μmol/g DW in FRAP, ABTS, and DPPH methods, respectively. Total antioxidant activity was maximum in dark red variety BDR and minimum in white variety BSU. Among dark red varieties, BDR showed significantly high antioxidant activity than BRJ in all the three methods (Table 4). In red varieties, the antioxidant activity by FRAP method showed the significantly high value in BSR and significantly low in BRD. BSR and BSK showed similar level of antioxidant activity. All the three red varieties (BSR, BSK, and BRD) were significantly different for ABTS and DPPH activities with maximum in BSR (DPPH method) and BSK (ABTS method). In both the methods (ABTS and DPPH), minimum was found in BRD variety. Within the white varieties, no significant difference was observed in BSW and BSU for FRAP and DPPH. However, significantly high antioxidant value was observed in BSW (42 μmol/g DW) compared to BSU (22.71 μmol/g DW) in ABTS method owing to the better level of polyphenol content.

In all the methods, dark red varieties exhibited highest antioxidant activity, and white varieties had lowest which is evident from the corresponding anthocyanin and polyphenol content. To investigate the profound factor contributing to antioxidant activity of onion peel extract, we performed a correlation analysis among total phenols, total anthocyanin, and three antioxidant methods. As presented in Figure 8, the coefficient of correlation (R²) of antioxidant activity was more in all three methods for TPC than anthocyanin content (TAC). The coefficient of correlation (R²) for TAA and TPC ranged from 0.8105 to 0.9637, while it was considerably low with TAC (0.3271–0.6211). Lee et al. (2015) also observed poor correlation of total antioxidant capacity and anthocyanin content in white, yellow, and red onion. Zhang et al. (2016) reported high correlation between total polyphenols and antioxidant activity, but contrary to our finding, they also reported highly positive correlation between total anthocyanin content and total antioxidant content. In bulb of two red onion varieties, a strong correlation between total flavanol content and antioxidant activity was also observed but reported a poor correlation between total phenol and anthocyanin (Metrani et al., 2020).