3.2. Biologically Active Compounds in Relation to Cultivar and Tissue Type
The question about the concentration of phenolic compounds can be classified as the most frequently asked query in the analysis of plant-derived foods [2
]. This extremely diverse group of secondary metabolites, and one that is involved in numerous functions in plants, are also known for high biological activity. The relationship between (poly) phenol-rich foods and human health has been demonstrated in numerous epidemiological studies [11
]. Phenolic compounds are characterized by a high antioxidant activity, and in many cases, the quali-quantitative phenolic compounds pattern determines the antioxidant power of fruits or vegetables [25
]. In this study, 2 methods were used to assess the total phenolic compound concentrations: the commonly used method with Folin’s reagent and the method recently developed by Medina [34
] using FBBB. The search for a new, simple, and inexpensive method for the determination of the sum of phenolic compounds results from the fact that many non-phenolic compounds often found in fruits and vegetables form blue complexes with Folin’s reagent. However, the method utilizing Fast Blue BB diazonium salt is based on the coupling of phenolic compounds with the diazonium salt resulting in the formation of azo complexes and may be more specific [34
]. Therefore, we included this method in the analysis of total phenolic compounds concentration in apple fruit to evaluate the result differences between methods.
The results of the statistical analysis differed depending on the method used to determine phenolic compounds. The influence of the cultivar on the phenolic concentration was not proven in the case of the Folin method, and in the case of the FBBB method, the effect of the growing season on the concentration of these compounds was not confirmed (Table 1
). Therefore, the method used to measure total phenolic concentrations may give different final results regarding the influence of tested factor (s). The differences in the phenolic concentrations between apple peel and apple pulp ranged from 5.6- (FBBB) to 7.1-fold (Folin) on average (Table 2
). Furthermore, the concentration of phenolic compounds was on average about 3 times higher in the apple peel and 3.8 times higher in the pulp, respectively, in the FBBB method opposed to Folin (Table 2
). The FBBB method revealed not only a greater concentration of phenolic compounds in general, but also a greater presence of these compounds in the flesh, which narrows the differences between their amount in the epidermal zone and downstream of the epidermal layer. The author of the FBBB method [34
], comparing the concentration of phenolic compounds determined by the FBBB and Folin methods in barley and wheat grains, obtained a concentration of phenolic compounds twice as high using a newly developed method. When the research material was that of different types of tea, the differences in phenolic compounds concentration between these two methods were up to 6-fold. The method, however, requires further verification on larger plant samples, along with the evaluation of individual phenolic compounds by HPLC, so that it could be stated with greater certainty that it is more selective in relation to phenolic compounds and allows for a better estimation of their total concentration than the widely used Folin method. Similar conclusions were drawn by other researchers analysing the concentration of phenolic compounds in strawberry fruits [39
Irrespective of the method used, the flesh phenolic compounds concentration of all tested apple cultivars was similar (Table 2
). In turn, the tested apple peel differed considerably in terms of the phenolic concentrations, but the measurement method also influenced the results. Based on the Folin analysis, the ‘Chopin’ cultivar did not differ significantly in the concentrations of peel phenolic compounds in relation to the red-skinned ‘Gala S.’. According to the FBBB method, the ‘Gala S.’ cultivar distinctly differed from the other two tested cultivars in terms of the phenolic compounds concentration in the apple peel. In both methods green peel apples ‘Granny S.’ and ‘Chopin’ were characterized by similar total phenolic concentrations.
In order to elucidate further differences with respect to phenolic compounds and tissue type, selected individual phenolic compounds were measured using the HPLC technique. The dominant compounds, such as (+)-catechin, (−)-epicatechin, rutin, phloridzin, and chlorogenic acid, were quantified (Table 3
). The concentration of individual phenolic compounds did not fully reflect the differences that were obtained with the total concentration of phenolic compounds (Folin vs. FBBB method). Tested cultivars average in two seasons did not differ significantly in the concentration of rutin in the peel and phloridzin, both in the peel and pulp. Among the determined phenolic compounds, rutin was present in the highest concentration, but rutin was detected only in the apple peel. The lowest concentration was recorded for phloridzin irrespective of tested cultivars. Savatovic et al. [40
], when determining phenolic compounds similarly to the presented study, recorded the highest concentration of rutin and many times, as much as 26 times lower the concentration of phloridzin. In the other studies [7
], (−)-epicatechin, (+)-catechin, and chlorogenic acid were determined approximately 4 times less than the concentration of rutin. In turn, based on the dry weight of the apple peel, the highest concentration was recorded for rutin while the lowest of chlorogenic acid. Taking into account the whole fruit (AP and AF), chlorogenic acid was at the highest concentration and phloridzin at the lowest. However, it should be pointed out that great differences between cultivars were documented [7
There were no significant differences between examined cultivars in the chlorogenic acid and (+)-catechin concentrations in apple pulp, but the peel differed significantly. Regarding examined cultivars, ‘Gala S.’ peel exhibited a significantly higher concentration of chlorogenic acid and (−)-epicatechin compared to ‘Chopin’ and ‘Granny S.’, but ‘Chopin’ was the second highest. For (+)-catechin, peel of ‘Chopin’ and ‘Gala S.’ form the same homological group (Table 3
). In contrast to other phenolic compounds, the case of (−)-epicatechins is interesting, the concentration of which is significantly different in the tested cultivars, both in the apple peel and in the flesh. Apple flesh of ‘Chopin’ expressed significantly higher (−)-epicatechin concentrations compared to ‘Gala S.’; the opposite situation was for the peel of these cultivars. According to Petkovsek et al., scab-resistant apple cultivars had a significantly higher concentration of some individual and total phenolic concentrations in comparison with the scab susceptible, especially the pulp [16
]. In this study ‘Chopin’ expressed a higher concentration of flavanols, particularly compared to ‘Granny S.’ In turn, compared to ‘Gala S.’, ‘Chopin’ was richer in these compounds in the flesh. Of course, these compounds do not represent all the phenolic compounds identified in apples [41
], and the issue should be further explored. An important group of apple phenolic compounds are oligomeric flavanols, located primarily in the peel of apples.
In addition, the ‘Chopin’ cultivar was definitely distinguished by the concentration of ascorbate in both the peel and the flesh (Table 3
). Apple peel of ‘Chopin’ was 1.6 and 3-fold higher while its flesh was 2.6- and 3.6-fold higher in ascorbate concentration compared to ‘Granny S.’ and ‘Gala S.’ peels, respectively. The concentration of ascorbic acid in commercial apple cultivars is low compared to other fruits, amounting on average to 10 mg per 100 g fresh fruit weight [43
]. Since the number of apple cultivars is huge, the variation in ascorbic acid concentration between them can range even between 1.7- and 3.3- in whole apples and may differ nearly 5 times in apple peels [5
]. The share of the peel as a source of ascorbate in relation to the whole apple fruit may amount to even 30%, although its share in the fresh weight of the fruit does not generally exceed 10% [2
]. Many external factors influence the metabolism of antioxidants, to which the response of individual cultivars may depend on the genetically defined total antioxidant potential [10
Due to a large number of bioactive compounds in the plant tissue, several assays have been developed to help assess total antioxidant activity/capacity [14
]. They are useful in the overall assessment of the health benefit potential of different types of food. Sometimes, more than one method is used when evaluating the antioxidant properties of different food types. This is due to the presence of two groups of antioxidants, hydrophilic, and lipophilic, but within each group, a great number compounds with individual physico-chemical characteristics exist. While the ABTS test can measure both hydrophilic and lipophilic antioxidants, the FRAP method only measures hydrophilic ones while the DPPH test is applicable to the hydrophobic counterparts [14
]. However, each of these tests has its limitations with respect to individual compounds; therefore, it is suggested to involve more than one method to assess total antioxidant capacity [46
]. By evaluating hydrophilic and hydrophobic antioxidants in separate tests, differences in the range of a given group of compounds in the tested samples can be detected. Moreover, because the authors of the studies use different tests, this gives a greater opportunity to compare different study results. In this study, it was decided to measure total antioxidant capacity using three methods—ABTS, DPPH, and FRAP-assays (Table 4
). Regardless of the assay test and the cultivar examined, the antioxidant capacity determined for apple flesh did not differ significantly. This confirms the results obtained in the assessment of individual compounds discussed above, where their concentrations in the apple flesh were generally similar. ‘Chopin’ peel was characterized by the highest total antioxidant capacity based on the DPPH test while ‘Gala S.’ based on the ABTS and FRAP assays, as compared to the other two cultivars. However, in the case of the FRAP test, both mentioned above cultivars constituted one homologous group. These tests confirm the previously presented results, where ’Chopin’ and ‘Gala S.’ showed alternately the highest ascorbate and/or phenolic compound concentrations. In turn, in the case of the ABTS test, the peel of ‘Gala S.’ did not differ significantly from the ‘Granny S.’ one. Although the same results were not obtained for individual tests, it can be concluded that the total antioxidant capacity of the tested green-skinned cultivars did not differ so much from the red-skinned one. The variability in the total antioxidant capacity, both with respect to tested tissue type and cultivar, suggests that further differences between the tested cultivars in the content of an individual, hydrophilic and/or lipophilic, antioxidants can be expected, and the issue can be further studied. The greatest differences between the peel and flesh were noted for the ‘Gala S.’. This is likely related to the colour of the peel, indicating a higher concentration of anthocyanins compared to other cultivars. Different groups of antioxidant compounds have different contributions in the total antioxidant capacity. The strongest positive correlation between the total antioxidant activity, and the various compounds, regardless of the test used, was found in the case of phenolic compounds [6
]. Previous studies have found that phenolic compounds are the main group of antioxidant compounds in apples due to their activity and concentration [23
]. In this study, the correlation coefficients (the Pearson correlation analysis) between the total antioxidant capacity and the concentration of phenolic compounds were in the range of 0.84 (DPPH) to 0.96 (FRAP) for FBBB method and 0.79 (ABTS) to 0.92 (FRAP) in case of Folin method. For ascorbate, the values were lower and within the range 0.55 (FRAP, ABTS) to 0.72 (DPPH).
Tissue type was the most prominent factor that affected total ascorbate, total phenolic compounds, and individual phenolic compounds, such as (+)-catechin, (−)-epicatechin, rutin, phloridzin, and chlorogenic acid, as well as total antioxidant capacity (Table 1
, Table 2
, Table 3
, Table 4
and Table 5
). For all compounds, the differences in concentrations between apple peel and flesh were statistically significant. Apple peel was the richest source of bioactive compounds in all varieties. This is the expected result since similar conclusions can be found in other related studies where a more large number of cultivars were tested [18
]. Table 5
summarizes the range of the differences regarding antioxidant concentration between the peel and the flesh of tested apple cultivars. Differences between the peel and flesh in the concentration of the compound strongly depended not only on the cultivar, but also on the type of compoun. The size of the differences between the apple peel and the flesh concentration can be important in terms of the whole fruit compound content. In general, apple fruit of ‘Chopin’ and ‘Granny S.’ cultivars expressed much lower skin-to-flesh antioxidant potential differences than ‘Gala S.’. Fruits of ‘Gala S.’ and ‘Chopin’ cultivars were characterized by similar average fresh weight: 180 and 183 g per fruit, respectively. Granny S. was characterized by a bigger apple, i.e., approximately 224 g per fruit.
The lowest differences between tissue types were found in chlorogenic acid and flavan-3-ols, followed by total phenolic compounds and ascorbate. Except for phloridzin, ‘Gala S.’ exhibited the highest differences in global and individual phenolic compound concentrations as well as total antioxidant capacity between the apple peel and flesh. The range of differences between the peel and the pulp in the antioxidant concentration varied widely from 2.47 ((+)-catechin, Granny S.) to 15.4 ((−)-epicatechin, Gala S.) fold variation (Table 5
). The differences in the total antioxidant capacity between the tested tissues were characterized by a narrower range, i.e., 2.27 (FRAP, ‘Granny S.’) to 6.2 (FRAP, ‘Gala S.’) fold variation. In studies on a larger material, where 19 cultivars of apples were analysed but the peel and the whole fruit were tested [18
], the differences were smaller. A different pattern of compound distribution may indicate a different role of individual antioxidants under stress conditions. Enzymatic and non-enzymatic antioxidants are involved in several processes keeping level of active oxygen species, which are generated during biotic and abiotic stress as well as normal metabolic processes, under control [10
]. It is expected that cultivars with a more efficient antioxidant apparatus (antioxidant concentration/regeneration or activity) will be more resistant to stress. Simultaneously, a higher concentration of biologically active compounds increases the health-promoting value of the fruit.