2.1. Changes of Total Phenolic Content, Condensed Tannins, and Total Flavonoid Content
The highest TPC appeared at the water stages (S1–S3) of fruit ripening of all five cultivars (Figure 1
). Then, TPC declined quickly and reached the lowest point at the mike stage (S4–S5) or dough stages (S7–S8), with a slightly bounce at the kernel stages (except Jinhua). CT and TFC shared similar change patterns: the highest value appeared at milk stages (S4–S5) and the lowest value showed at dough stages (S6–S8). The highest TPC value was in Wichita (181.28 mg EAE g−1
) at S1, while the lowest was in Shaoxing at S8 (0.23 mg EAE g−1
). Both the maximum CT and TFC values appeared at S4 of Wichita (379.85 and 79.17 mg CE g−1
), while the minimum values showed at S7 of Wichita (0.3 and 0.1 mg CE g−1
). The TPC, CT, and TFC of mature kernels were 11.36–29.50 mg EAE g−1
, 29.5–84.3, and 6.1–14.8 mg CE g−1
, respectively. TPC, CT, and TFC increased in over-ripe samples, except for Jinhua, which did not have over-ripe samples, and Shaoxing, in which they all decreased. The TPC, CT, and TFC values were all significantly affected by cultivars.
Previous reports mainly focused on mature pecan kernels. Ellagic acid was chosen as the standard for TPC in this experiment because it is more representative of the type of phenolic compounds that exist in pecan kernels [19
]. Results of TPC of mature pecan kernels in previous reports were mostly around 18.2–26.2 mg EAE g−1
(using ellagic acid as standard) [19
] or 10–20 mg GAE g−1
(using gallic acid as a standard) [3
]. Though different standards made it hard to compare, we still see that our TPC values of mature kernels were in a similar range to most reports. Slight differences may be caused by the variation in geographic location, climate difference, or cultivation techniques. When comparing our CT values of mature kernels with previous reports, the result of cultivar Stuart was lower than previous reports, while results of cultivars Pawnee, Wichita, Jinhua, and Shaoxing were similar to previous reports [8
]. When comparing TFC values, our results were more similar to the reports of de la Rosa et al. [20
] and Yang et al. [22
]. Changes in TPC and TFC were widely investigated in fruits such as grapes and strawberries [27
], which usually decreased along with ripening, but rarely in nuts. The values of CT were higher than TPC values in pecan kernels, which is consistent with previous research on mature pecans [6
]. These results may attribute to the overestimation caused by the higher extinction coefficient of polymeric tannins to free catechin [29
]. These results also suggested that pecan kernels contain highly polymeric tannins.
2.3. Identification of Phenolics in Pecan Kernels Using UPLC-Q/TOF-MS
UPLC-Q/TOF-MS was used to identify the phenolics in pecan kernels. Cultivar Pawnee was chosen due to its relatively high antioxidant capacity among the five cultivars. Pecan kernels of Pawnee of 10 developmental stages were pooled and analyzed using UPLC-Q/TOF-MS. Both the positive and the negative mode were used to identify phenolic compounds, while results showed that the negative mode was more suitable for phenolics. So, subsequent analyses were carried out using negative mode.
A total of 40 phenolics were tentatively identified in pecan kernels, of which two were first reported in the genus Carya
, six were first reported in species Carya illinoinensis
and one was first reported in pecan kernels. The retention time (RT), molecular ion, fragment ions, molecular formula, and mass measurement errors (∆m) are shown in Table 1
More than half of the phenolics identified in pecan kernel were ellagic acid derivatives. The free ellagic acid (peak 20) was detected at RT of 6.36 min with a [M − H]−
300.9961 and confirmed by comparing with commercial standard. Ellagic acid hexoside (Peak 19) and ellagic acid pentose (peak 21) can easily be found by the characteristic fragment ion at m
301 along with the neutral losses of 162 amu (the loss of hexoside) and 132 amu (the loss of pentose), respectively. Peak 29 was tentatively identified as ellagic acid rhamnoside because of the same fragment ion at m
301 and a neutral loss of 146 amu [36
]. Peak 25 was assigned as ellagic acid rutinoside for a neutral loss of 308 amu, which corresponded to the loss of a rhamnose-glucose structure. To the best of our knowledge, both ellagic acid rhamnoside and ellagic acid rutinoside were identified in the Carya
genus for the first time.
Ellagic acid is formed from the condensation reaction of two gallic acids. Galloyl-linked ellagic acid derivatives can also be found in pecan kernels. Peak 33 with [M − H]− ion at m/z 585 produced ions at m/z 433 (M − H − 152, loss of galloyl) and m/z 301 (M − H − 284, loss of galloyl and pentose) was assigned as ellagic acid galloyl pentose. Peak 23 with [M − H]− ion at m/z 615 produced ions at m/z 463 (M − H − 152, loss of galloyl) and m/z 301, which corresponded to the deprotonated ellagic acid, was assigned as digalloyl ellagic acid.
Similar methyl ellagic acid derivatives can be identified with the characteristic fragment ion at m/z 315, including methyl ellagic acid (peak 30), methyl ellagic acid hexoside (peak 24), methyl ellagic acid pentose (peak 31), and methyl ellagic acid galloyl pentose (peak 36, 37 and 38). Dimethyl ellagic acid derivatives added an extra 15 amu (another methyl group) to form the main ion at m/z 328. Several peaks that contained this kind of structure were assigned, including dimethyl ellagic acid (peak 39 and 40) and dimethyl ellagic acid hexoside (peak 27).
Another ellagic-acid-related derivative family was the ellagitannins (ETs), which contain the hexahydroxydiphenoyl (HHDP) group (Figure 3
). HHDP-glucose was found at RT 0.94 min with [M − H]−
ion at m
481 (peak 3), fragment ion at m
301 and a neutral loss of 180 amu.
Bis-HHDP-glucose (pedunculagin/casuariin isomer), which contains two HHDP groups, was found with [M − H]−
ions at m
783 (peak 6 and 9). MS/MS spectra yielded fragment ions at m
481 (M − H − 302, loss of HHDP) and 301 (M − H − 482, loss of HHDP-glucose), confirmed the assignment. Peak 10 was found to yield similar ions at m
783, 481, 301 as bis-HHDP-glucose (peak 6 and 9) along with extra ions at m
907 (M − H − 44, loss of carboxyl) and 951 [M − H]−
. From this fragmentation pattern, we can see that another gallic acid group was linked in this compound. So, this ET was assigned the name HHDP-valoneoyl-glucose (praecoxin A/platycariin isomer). A peak showing [M − H]−
signal at m
649 (peak 28) with fragment ions at m
605 (M − H − 44, loss of carboxyl), 481 (M − H − 168, loss of gallic acid) and 301 (M − H − 348, loss of gallic acid and glucose) was assigned as valoneoyl-glucose. To our knowledge, bis-HHDP-glucose (pedunculagin/casuariin isomer), HHDP-valoneoyl-glucose (praecoxin A/platycariin isomer), and valoneoyl-glucose were identified for the first time in pecans. They had been reported previously in walnut kernels [30
Galloyl-HHDP-glucose (strictinin/isostrictinin) was identified at RT 4.59 min with [M − H]− ion at m/z 633 (peak 16) and fragment ion at m/z 301 (M − H − 332, loss of galloyl-glucose). Another peak with [M − H]− ion at m/z 785 (peak 17) was detected. Fragment ions were at m/z 483 (M − H − 302, loss of HHDP) and 301 (M − H − 484, loss of digalloyl group and glucose), which corresponded to deprotonated digalloyl glucose and deprotonated HHDP, respectively. This fragmentation pattern complied well with digalloyl-HHDP-glucose (tellimagrandin I).
Another dominant class of compounds in pecan kernels was catechin and its derivatives. (+)-Catechin (m
289, peak 15), (−)-epicatechin (m
289, peak 18), (epi)catechin gallate (m
441, peak 26), epigallocatechin gallate (m
457, peak 34), and catechin hexoside (m
451, peaks 7 and 8) were identified by comparing MS/MS fragment ions with reference reports or commercial standards. Procyanidin dimer B-type [(epi)catechin→B→(epi)catechin] was identified with [M − H]−
ions at m
577 (peak 11) and fragment ions at m
425 (M − H − 152, loss of galloyl), which complied with the reports in pecan and peanut skins [9
]. B-type means that two (epi)catechin structures are linked together through C4→C8 or C4→C6, in which C4→C8 is more common due to low space resistant. Peak 13 yielding [M − H]−
ion at m
865 and fragment ions at m
577 (M − H − 288, loss of catechin) and 289 (M − H − 576, loss of procyanidin dimer) was assigned the name procyanidin trimer (C1) [(epi)catechin→B→(epi)catechin→B→(epi)catechin] [9
Gallic acid derivatives can also be found in pecan kernels. The free gallic acid (peak 4) can be identified at RT of 1.15 min and confirmed by comparing with a commercial standard. Several other gallic acid derivatives were also identified, including gallic acid hexoside (m
331, peak 2), methyl gallate (m
183, peak 12), and digalloyl-glucose (m
483, peak 14). Methyl gallate was previously found in pecan leaves [31
], while this is the first time it has been reported in pecan kernels.
Two dicarboxylic acid derivatives were detected in pecans for the first time. Glansreginin A and B were identified by the characteristic [M − H]−
ions at m
592 (peak 32) and 565 (peak 22) and fragment ions at m
403/343/241, which were identical to previous reports [33
]. These two compounds were first isolated and elucidated in walnut kernels [38
]; they have also been found in hazelnut kernels [39
2.4. Quantification of Phenolics in Pecan Kernels Using HPLC
Although UPLC-Q/TOF-MS is very sensitive and suitable for identification of compounds, due to its relatively low peak resolution within a short procedure, HPLC was used to quantify the dynamic changes of several representative phenolics.
The contents of gallic acid, (+)-catechin, (−)-epicatechin, EGCG, and ellagic acid were analyzed in five cultivars of 10 developmental stages (Figure 4
). The contents of five individual phenolics were all highest at water stages, lowest at dough stages, and varied at the kernel stages, similar to the change patterns of TPC, CT, and TFC. Similar results can be found in research on Anacardium occidentale
L., while phenolic contents (like gallic acid and EGCG) decreased significantly from unripe to medium-ripe and ripe [40
]. This fall may be attributable to their conversion to other downstream forms such as esterified or glycoside forms, along with the nut development. It may also attribute to the high proportion of testa at immature stages. In immature pecan kernels, the proportion of testa was high, and decreased gradually with the nut development. Senter et al. reported firstly that pecan testa might contain powerful antioxidant phenolics and act as a natural screen to prevent infection by viruses, bacteria, and molds [41
]. They speculated that phenolics existed mainly in the outer testa or pellicle of the pecan kernels [42
]. Research showed that contents of (+)-catechin and (−)-epicatechin in the testa of cashew nut were 20 times and five times higher than in green tea and dark chocolate, respectively [43
]. Further experiments are needed to obtain more accurate data on phenolics in different organs of pecan kernels.
In immature pecan kernels, the most abundant phenolic was (+)-catechin, followed by (−)-epicatechin, while EGCG had the lowest content. The content of (+)-catechin was much higher, e.g., over 10 times (17.7100 mg g−1
at S1) higher than ellagic acid (1.4982 mg g−1
at S1) in Shaoxing, than other phenolics during immature stages. This was also probably due to the fact that they were used as building blocks to form proanthocyanidins. Two or three catechin units linked together formed proanthocyanidins (dimers and trimers), while three or more catechin units linked together formed oligomeric proanthocyanidins [45
]. Both proanthocyanidins and oligomeric proanthocyanidins are abundant in pecan kernels. Two proanthocyanidins were identified in this experiment. So, catechin was very likely to be stored at a high concentration at the beginning of kernel formation for future use.
In mature kernels (Pawnee, Wichita, and Jinhua at S8; Stuart and Shaoxing at S9), the contents of ellagic acid were highest in Pawnee, Stuart, Jinhua, and Shaoxing, but in Wichita the content of (+)-catechin was the highest. This result was consistent with previous reports on pecans, in which most of them reported the most abundant free phenolic was ellagic acid [9
], while some reported it to be (+)-catechin [46
]. Phenolics are secondary metabolites, so environmental factors such as temperature and sun irradiation could affect their concentration [47
]. For example, a walnut husk cultivated at higher altitude contained higher total phenolic content than one cultivated at a lower altitude [48
]. The temperature and sunshine duration during pecan kernel mature were recorded; however, the effects of these environmental factors on phenolic contents await further study.