Biomolecules, Vol. 12, Pages 1797: Metabolic Profiling of Chestnut Shell (Castanea crenata) Cultivars Using UPLC-QTOF-MS and Their Antioxidant Capacity

3.1. The Metabolic Composition of Whole C. crenata ShellsTo evaluate the differences in chemical composition between C. crenata cultivars, non-target metabolite profiling of chestnut extracts was performed using UPLC–QTOF/MS. The metabolites were tentatively identified with the exact mass values of the precursor and the MS/MS spectral data of authentic standards, previously published data, or the MassBank and METLIN databases [24]. The total ion chromatograms (TIC) of the chestnut extracts are given in Figure S2. The characteristic ions corresponding to each compound are showed in Table 1. The mass accuracy of the [M+H]+ or [M−H]− ions was within ±8.89 ppm.The primary compounds identified from the C. crenata shell extracts were ellagitannins. Ellagitannins are hydrolyzable tannins containing polyphenolic compounds and a sugar core [25]. These compounds protect against oxidative stress-related diseases and prevent degenerative diseases, such as cardiovascular and inflammatory diseases and cancers [4,11,13]. Each compound was tentatively characterized through loss of a hexahydroxydiphenyl (HHDP), a sugar unit, and gallic acid moieties. The predominant ion at m/z 301 was formed from the ellagic acid anion produced by the rearrangement of the HHDP moiety [5]. Bis-HHDP-glucose showed [M−H]− ion at m/z 783 and fragment ions at m/z 765 [M-H-H2O]−, 481 [M-H-HHDP]−, and 275 [M-H-HHDP-glucose-CO2]−. Tri-, di-, and galloyl-HHDP-glucoside produced [M−H]− ions at m/z 937, 785, and 633. The most characteristic ions of galloyl-HHDP-glucosides are matched by the loss of a gallolyl unit at m/z 463 and 300, corresponding to [M-H-Gal-glucose]− [26].A valoneoyl group consists of an HHDP group and a galloyl moiety connected by a C–O–C bond [27]. In the MS/MS experiment, HHDP-valoneoyl-glucose exhibited fragment ions at m/z 907 and 783, which corresponded to the loss of CO2 ([M−COOH]−) and a gallolyl unit from the [M−H]− ion, respectively. NHTP (nonahydroxytriphenoyl-) glucose derivatives are based on a core of glucose esterified with HHDP or NHTP groups [28].NHTP-HHDP-glucose showed a [M−H]− ion at m/z 933 and fragment ions at m/z 915 [M-H-H2O]−, 631 [M-H-HHDP]−, and 425 [M-H-HHDP-glucose-CO2]− in the MS/MS experiment. Ellagitannins were also reported in C. sativa Mill. shells in a previous study, and they possessed a wide range of biological activities [5].In agreement with a previous report, various proanthocyanidins were detected in chestnuts [29]. The highly enriched proanthocyanidins showed significant anti-inflammatory activity. Proanthocyanidins are condensed tannins found in the nuts, bark, fruits, and seeds of various plants [30] and are anti-inflammatory and have beneficial effects on metabolic syndrome, atherosclerosis, and cancer [29]. Proanthocyanidins are formed by catechins and epicatechins [31]. These compounds produced the same fragment ions at m/z 289, 303, and 305 in the MS/MS experiment, suggesting that these compounds were composed of either catechin (C) or gallocatechin (GC) aglycones [32]. The loss of 170 Da or 188 Da (170 + 18) was caused by the release of a gallic acid and a water molecule.Flavonoids are one of the major classes of phenylpropanoids abundantly found in various foods and beverages [33]. Flavonoids were tentatively identified in the chestnut shells, including flavonol, flavone, flavanone, and their sugar conjugates. Kaempferol showed the [M+H]+ ion at m/z 287 in the positive ion mode, with a well-shaped peak observed for kaempferol derivatives. Kaempferol coumaroyl hexose was confirmed by the identification of the fragment ion at m/z 309 via the loss of a kaempferol moiety from [M+H]+. Luteolin displayed the same [M+H]+ ion as kaempferol at m/z 287, confirmed by comparing their retention times and MS/MS spectra with the relevant standards. The most abundant ions of quercetin derivatives at m/z 301 represented the loss of a sugar moiety. Rutin is characterized using the fragment ions at m/z 301 and 273 in negative mode, which result from the loss of the rhamnose-glucose unit and the serial loss of CO, respectively.Myricetin, isorhamnetin, and apigenin were detected at m/z 317, 315, and 269 from [M−H]−. The flavanones eriodictyol and naringenin were tentatively identified by the presence of m/z 289 and 273 ions in positive mode. The most characteristic ion of these compounds at m/z 151 (negative mode) or 153 (positive mode) were generated by retro-Diels–Alder fragmentation [34]. Myricetin-hexoside showed the loss of the glucose unit (m/z 479 → m/z 317) in negative mode. Naringin and naringenin glucosides were identified using the predominant ion at m/z 271 from the aglycone of naringenin in negative mode.Ellagic acid is a phenolic acid found in various fruits and vegetables, with antioxidant and antiviral properties [35,36]. Ellagic acid derivatives, such as ellagic acid hexose, -pentose, and -deoxyhexose, yielded the predominant fragment ion at m/z 301 in negative mode from an ellagic acid moiety. Methylated ellagic acids were generated as methyl, dimethyl, and trimethylellagic acid at m/z 315, 329, and 243, respectively, by the addition of methyl groups to ellagic acid.Gallic acid and derivatives showed high antioxidant activity and may play protective roles, including anticancer, antiviral, antifungal, and antibacterial activities [37]. Gallic acid displayed [M−H]− ions at m/z 169 and an abundant fragment ion at m/z 125, due to the loss of carboxylic acid. Digalloyl-, trigalloyl-, and tetragalloyl glucose were characterized by a product ion at m/z 465, which could be attributed to the loss of H2O and gallic acid moieties from the [M−H]− ion. 3.2. Differences in Metabolite Levels Associated with Whole C. crenata ShellsTo assess the differences in the chemical compositions of the C. crenata cultivars, principal component analysis, specifically partial least squares discriminant analysis (PLS-DA), was performed on the mass spectra. The PLS-DA score plots derived from the positive (Figure S3A) and negative (Figure S3B) modes showed a significant separation in C. crenata cultivars (positive, R2 = 0.547, Q2 = 0.371; negative, R2 = 0.770, Q2 = 0.711). The five C. crenata cultivars in this study were clustered in both polarity modes. Okkwang, Porotan, and Ishizuuchi were separated from the other cultivars, Daebo and Riheiguri, which formed a separate group. These results showed the close relationship due to their origins. Daebo originated by crossbreeding with “Sangmyeon,” a Korean native cultivar, and Riheiguri (Table S1).In order to visualize the relationship of the differential metabolites identified in the C. crenata cultivars, a heatmap visualization was employed (Figure 1). In Figure 1, the chestnut shell samples were divided into three branches according to their cultivars: Daebo and Riheiguri formed one group, Okkwang formed another group, and Porotan and Ishizuuchi formed a final group. The differential compounds were separated into four groups, which indicated that the identified metabolites could be used to identify the cultivars of chestnuts from their shells. The identified metabolites included in each group are shown in Table S3. To obtain an insight into the behavior of these metabolites in chestnut shells from different cultivars, the relative contents of each group of metabolites are displayed as boxplots in Figure 2. The Group 1 compounds are most abundant in Porotan and Ishizuuchi, which contain high levels of flavonols and methylellagic acids. The Group 2 compounds, which were higher in Okkwang, are mainly ellagitannins and ellagic acid glucosides. Most ellagic acid is present as hydrolyzable ellagitannin in the vacuoles of plant cells, and ellagitannins produce ellagic acid during hydrolysis of the HHDP group [38]. In this study, the level of ellagic acid was positively correlated with ellagitannins. The compounds in Group 3, which are mainly flavonoids and proanthocyanidins, had a high degree of variation within the samples in each group. Amino, organic, and phenolic acids were the dominant compounds in Group 4 and were abundant in Daebo and Riheiguri.To determine the relationship between the metabolite contents and the antioxidant effects, the total phenolic content and the antioxidant capacity of the C. crenata shell extracts were measured. The results of the total phenolic content, DPPH radical scavenging assay, and FRAP assay are shown in Table 2. The antioxidant effects of the C. crenata shell extracts determined using the DPPH and FRAP assays were correlated with the total phenolic content. The Okkwang, Porotan, and Riheiguri extracts presented higher antioxidant activities than the Daebo and Riheiguri extracts, similarly to the results obtained from the metabolite profiling of C. crenata. In previous reports, differences in the chemical composition and antioxidant capacities among chestnut (C. sativa Mill. and C. mollissima) cultivars were observed [5,39,40,41]. 3.3. Metabolite Quantification in Inner and Whole Shells of C. crenataChestnut inner shells are a rich source of total phenols and hydrolyzable tannins and flavonoids [10,42], but they are separated from the kernel with the outer shell during industrial peeling processes and are considered waste materials. To separate only the inner shell of the chestnuts is a difficult, time-consuming, and labor-intensive process. To re-evaluate the effective use of whole shells, the metabolic composition and antioxidant capacities of the inner and whole shells of chestnuts were compared.To investigate the contribution of the different parts of the shells to the phytochemical composition and biological activity, chestnut extracts from the inner parts and whole shells were prepared separately, as described in Section 2.2. The metabolic profiling data showed that bioactive compounds, such as phenolic acid derivatives, flavonoids, tannins, and proanthocyanidins, had the highest intensities in the Okkwang, Porotan, and Ishizuuchi cultivars (Figure 1). Thus, these three cultivars were selected to determine the differences between the inner and whole shells of C. crenata.

A targeted analysis was performed on an LC-QTRAP/MS, to determine the quantities of differential metabolites in the inner and whole shells. Targeted analyses of C. crenata shell samples were performed for the phenolic acids, namely caffeic acid, ferulic acid, gallic acid, shikimic acid, chlorogenic acid, and coumaric acid; the flavonoids, namely catechin, quercetin, quercetin glucoside, rutin, apigenin, luteolin, and naringenin; and the amino acids, namely tryptophan. To quantify the metabolites, the phenylalanine-13C6 were used as internal standards, improving the quantification’s precision and accuracy.

The calibration standards were generated using a solution of combined standards for the appropriate range of each compound. The equations for the calibration curves and the linear regression coefficients for the targeted metabolites are given in Table S4. The calibration curves were constructed using a linear least squares regression analysis of the analyte and internal standard. The calibration curves of the compounds showed correlation coefficients (R2) greater than 0.99 within the given concentration ranges. Figure 3 shows the difference in the levels of significant metabolites in the different parts and cultivars of chestnut shells.

The inner shell extracts contained higher levels of flavonoids, including apigenin, luteolin, naringenin, and quercetin, than the whole shell extracts. In comparison, the whole shell extracts showed predominant concentrations of flavonoid glucosides, such as quercetin glucoside and rutin. The phenolic acids, including caffeic acid, chlorogenic acid, coumaric acid, ferulic acid, and shikimic acid, were more abundant in the whole shell extracts. However, the inner shell extracts contained more gallic acid. The distribution of catechin in the inner and whole shell extracts differed for each cultivar; in particular, tryptophan was barely detected in the inner shell extracts.

In a previous study of the potential anti-gastritis properties of chestnut (C. sativa Mill.), chestnut flour was devoid of any anti-inflammatory activity, while the inner and outer shells retained the ability to inhibit IL-8 secretion. In addition, it has reported that the inhibitory activity on IL-8 secretion was highly similar between the inner and outer shells [29]. We utilized a GES1 cell-based assay to evaluate the ability of the inner and whole shell extracts of C. crenata to reduce the cellular reactive oxygen species (ROS) production stimulated by H2O. The intracellular levels of ROS generated after stimulation of the GES1 cells with H2O2 were significantly reduced, by 100 μg/mL, for both the inner and whole shell extracts (Figure 4). The whole shell extracts more significantly reduced the intracellular levels of ROS than the inner shell extracts. In summary, the whole shells produced by mechanical peeling had as much antioxidant and biological activity as the inner shells.

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