4.1. Total Antioxidant Capacity (TAC) and Total Phenolic Content (TPC)
Three antioxidant assays (FRAP, TEAC and DPPH) based on single electron transfer and one based on hydrogen atom transfer (ORAC) were applied to evaluate antioxidant activities. This compound approach was adopted because no universal assay can accurately reflect all of the antioxidants in a complex system [22
Among the vegetables assayed, the moringa leaves presented the highest TAC and TPC values for all the methods and extracts assayed, the lower water content of moringa might explain its greater antioxidant capacity. Very high values of TAC and TPC were obtained for red chicory, lollo rosso lettuce, and oak leaf lettuce in both extracts (ME and HE). The lowest TAC and TPC values were obtained for iceberg lettuce, green cabbage, and roman lettuce plants in ME and for watercress, green sprout lettuce, and Chinese cabbage in HE.
In the lipophilic extract (LE), MO also presented the highest values of TAC and TPC, by all methods, followed by beet and the mix of rocket salad, watercress, lamb’s lettuce, and lollo rosso lettuce. The lowest values were obtained for endive, iceberg lettuce and roman lettuce, which also had the highest water content. The correlation between water content and TAC was negative (r = 0.85–0.90), even when the moringa value was excluded (r = 0.52–0.67).
The average TAC of the hydrophilic fraction (HE) of the total extract (HE + LE) ranged from 58.2% for TEAC to 86–87% for the ORAC and DPPH methods. Three of the 29 samples presented higher antioxidant activity in the lipophilic than the hydrophilic extracts for the ORAC and DPPH methods, and also in nine of the 29 samples for the TEAC and FRAP methods. The highest activity in the lipophilic extract was obtained for curly green cabbage (green leaves), green sprout lettuce, and watercress, in all methods assayed.
With respect to other vegetables, the TAC and TPC has been examined in many studies. For example, Carlsen et al. [23
] examined the antioxidant capacity of 3100 foods (including 303 vegetables), generating a large database. According to these authors, the antioxidant content of vegetables measured by FRAP in water/methanol ranges from 0.01 mmol Fe2+
/100g in lettuce to 48.07 mmol Fe2+
/100g in leaves from the African baobab tree. To compare the latter with our results, we examined the values for ten samples. The values obtained by these authors were statistically different from ours, but there was a very high correlation between them (r = 0.768, p
Pellegrini et al. [10
] studied the antioxidant capacity of 104 foods (34 vegetables). For comparison with our results, we examined the reported values for six samples extracted in water/acetone and analyzed by FRAP and TEAC. The values of the samples compared ranged from 494 µmol Fe2+
/100 g FW in green lettuce to 2694 µmol Fe2+
/100 g FW in spinach by FRAP and from 0.1 mmol TE/100g FW to 0.8 mmol TE/100g FW by TEAC for the same samples. These results differed significantly from ours and there was no correlation between them.
The United States Department of Agriculture Agricultural Research Service Database [24
] includes the TAC (determined by ORAC) and the TPC (determined by Folin–Ciocalteu) of 326 foods, 98 of which are vegetables. The values of the nine samples compared ranged from 406 μmol TE/100 g FW in iceberg lettuce to 2476 μmol TE/100 g FW in red cabbage, by HE-ORAC, and 9 to 162 μmol TE/100 g FW in beet and roman lettuce, respectively, by LE-ORAC. For TPC, the values ranged from 11 mg GAE/100 g FW in red lettuce to 231 mg GAE/100 g FW in red cabbage. Our results were different and there was no correlation. However, parameters such as the location of the samples, the time of collection, the maturity, processing, and storage could all account for these differences.
Deng, Lin, Xu, Gao, Xie, and Li [25
] analyzed the antioxidant capacity of 56 vegetables by FRAP, TEAC and TPC assays. Tetrahydrofuran for the lipophilic fraction and a methanol–acetic acid–water mixture (50:3.7:46.3, v
) for the hydrophilic fraction were used as solvents. Twenty five of the samples were leaves and four were similar to those analyzed in the present study (Chinese cabbage, green cabbage, green lettuce, and spinach). The values obtained by these authors were higher than ours. The geographic origin (China), the variety of vegetable or differences in the extraction procedure may account for these differences. However, as in our study, the correlations between TEAC- FRAP, TPC-FRAP, and TPC-TEAC were very high and statistically significant.
As is the case with common vegetables, the TAC and TPC of MO have been widely studied, but the results obtained vary widely according to the analysis method employed, the extraction procedure and the origin and time of collection of the moringa leaves. Thus, in their analysis of moringa leaves from India, Nicaragua, and Niger, Siddhuraju and Becker [3
] measured higher TPC values when 80% methanol was used versus 70% ethanol. The TPC for the plants from Nicaragua was 1.5 times higher than that for the plants from India.
Iqbal and Bhanger [4
] analyzed moringa leaves from different regions of Pakistan, obtained at various times, using methanol 80% as the solvent. The TAC values for the plants from the Chakwal region were about 24 times lower than those for the plants collected elsewhere. Pakade et al. [5
] in a study of moringa leaves conducted in South Africa measured TPC values 1.6 times higher in the Limpopo region than in Atteridgeville, using an 80:20 acetone–water mixture as the solvent. These TPC values were about 2.5 times and 2 times higher than those for cabbage and spinach, respectively.
Rodriguez-Perez, Quirantes-Piné, Fernández-Gutiérrez, and Segura-Carretero [26
] studied the effects on the TPC content of moringa leaves collected in Madagascar, using either acetone, methanol, or an ethanol–water mixture in various proportions. The best procedures were found to be ethanol and ethanol–water 50%. Rodriguez-Perez et al. [27
] used pressurized liquid and microwave-assisted extraction methods and reported variations of up to nine times in the TEAC and TPC contents between the lowest and highest values obtained.
Principal component analysis was performed to discriminate the samples. As shown in Figure 1
, PC1 explains 91.5% of total variation and was related to antioxidant activity and TPC; in brief, PC1 discriminates the samples with high TPC content (>197 mg GAE/100 g FW) from the others. PC2 explains 5.3% of the variation and appears to be correlated to TEAC. With respect to the sum of hydrophilic and lipophilic extracts, as shown in Figure 2
, PC1 explains 97.4% of the variance and, in particular, discriminates the samples with high TPC content (>74 mg GAE/100 g FW).
4.3. TAC and TPC Values for Fourth-Range Vegetable Packs, with or without, Moringa Leaves
TAC and TPC values were determined for three IV-gamma salads selected because in each case the percentage content of each ingredient is stated on the label: Vital salad 150 g (escarole 45%, red cabbage 25%, spinach sprouts 10%, lollo rosso lettuce 10%, and rocket 10%), Capriccio salad 100 g (green lettuce sprouts 40%, red lettuce sprouts 40%, and rocket 20%) and Gourmet salad mix 350 g (escarole 60%, red chicory leaves 20%, and lamb’s lettuce 20%).
The antioxidant capacity of these salads was estimated for the total extract (hydrophilic and lipophilic) by the TEAC and TPC methods, which presented the best correlation with the Tabart Index (r2 = 0.98084 TEAC-Tabart and r2 = 0.98092 TPC-Tabart).
The TEAC values were 191 µmol TE/100 g FW for the Vital salad, 60 µmol TE/100 g FW for Capriccio, and 262 µmol TE/100 g FW for the Gourmet salad mix. The TPC values were 33.5, 16.8, and 47.8 mg GAE/100g FW for Vital salad, Capriccio salad, and Gourmet salad mix, respectively.
When half of the ingredients presenting the lowest TEAC and TPC values were replaced by the same amount of moringa leaves, the antioxidant content increased sharply, by 145% to 686% for TEAC and by 128% to 340% for TPC (Figure 3
a,b). When the ingredient with the lowest antioxidant content was replaced by MO, the content rose by 1273% for TEAC and by 580% for TPC, in both cases for the Capriccio salad (Figure 3
The use of the leaves of MO as a food fortificant is common practice in African countries such as Ghana, Nigeria, and Ethiopia [33
]. They are also used as a source of antioxidants when added to other foods, like mayonnaise and bulk sunflower oil [34
]. Moreover, moringa leaves have good functional properties for use in ready-to-eat food products or snacks, such as ribbon-shaped toasted products [35
]. Due to its high oil absorption capacity, raw moringa leaf flour can be used in bakery food formulations, while alkali-pre-treated moringa leaf flour could be more suitable for making low-fat snack products [36