Walnut (J. regia) Agro-Residues as a Rich Source of Phenolic Compounds

Simple Summary Agro-residues are usually discarded as landfill, or burnt or left to decompose in the orchard. The efficient use of these walnut agro-residues would be a strategy that simultaneously helps to preserve the environment and boosts the economic outcome for farmers and companies. While some studies have reported on the content of bioactive compounds in walnut husks, little or nothing is known to date about the bioactive compounds in the buds and bark. Potentially, if walnut parts are used as a valuable source of bioactive compound, they might still be reused for other purposes. The identification and quantification of new phenolics between the different parts of the plant was carried out. It provided valuable data on their phenolic contents, and demonstrated where the extraction of individual phenolics would be meaningful. These data also show origin-related phenolic contents across the cultivars, and thus these phenolic profiles might serve to define the origins of different walnut cultivars. The study will help to propose new directions for further studies essential for agro-food, cosmetics and pharmacy industries. Abstract The present study was designed to identify and quantify the major phenolic compounds (phenolics) in the inner and outer husks, buds and bark of the Persian walnut, Juglans regia L. A comparison across six different cultivars grown in Slovenia was also carried out: ‘Fernor’, ‘Fernette’, ‘Franquette’, ‘Sava’, ‘Krka’ and ‘Rubina’. A total of 83 compounds were identified, which included 25 naphthoquinones, 15 hydroxycinnamic acids, 8 hydroxybenzoic acids, 13 flavanols, 2 flavones, 1 flavanone and 19 flavonols. For the first time, 38 phenolics in the husks, 57 phenolics in the buds and 29 phenolics in the bark were presented in J. regia within this study. Naphthoquinones were the major phenolics determined, approximately 75% of all analysed phenolics in the inner husk, 85% in the outer husk, 50% in buds and 80% in bark. The highest content of phenolics was found in the walnut buds, followed by the bark, the inner husk and the outer husk. On the basis of these high phenolic contents, walnut husks, buds and bark represented valuable by-products of the walnut tree. These data also show origin-related phenolic contents across the cultivars, and thus these phenolic profiles might serve to define the origins of different walnut cultivars.


Introduction
Persian or English walnut (Juglans regia L.) is the second most cultivated tree nut worldwide. Walnuts are native to the region stretching from the Balkans eastward to the Himalayas and southwest China. Nowadays, it is widely cultivated across Europe [1]. Walnuts are the third most consumed nut in the world, after almonds (Prunus amygdalus Batsch) and hazelnuts (Corylus avellana L.) [2]. The walnut kernel represents 50% of the total fruit weight, and it is the only edible part; therefore, it is clear that a lot of walnut agro-residues are generated every year that might have the potential for further, alternative Therefore, the recovery of these secondary metabolites from walnut agro-residues might generate functional ingredients, and might also add more value to the walnut industry. To effectively recover and use the phenolics from walnut husks, buds and bark, the chemical profiles of each of these agro-residues need to be defined, especially in terms of their individual phenolics. Due to their non-specific mechanisms of action, naphthoquinones also show significant toxicity [16], which can be seen for juglone and its allelopathic effects [18]. However, adequate modifications to naphthoquinone structures might instead produce new and valuable drugs [16].
Therefore, the objective of this study was to define the phytochemical compositions of walnut husks, buds and bark, and expand the discussion on the use of bioactive molecules in these walnut agro-residues. The identification of new phenolics such as naphthoquinones, in particular, and their quantification between the different parts of the plant will provide valuable data on their phenolic contents, and it will demonstrate where the extraction of individual phenolics would be meaningful. Thus, the identification and quantification of the phenolics in different parts of walnut agro-residues might help to propose new directions for further studies essential for agro-food, cosmetics and pharmacy industries. This study follows and upgrades an earlier study by Medic et al. [19] on walnut (peeled kernel and pellicle) on the phenolic and dicarboxylic acid content of the same six cultivars.

Plant Materials
Samples of walnut husks, buds and bark were obtained for six walnut cultivars three French cultivars: 'Fernor', 'Fernette', 'Franquette' and three Slovenian cultivars: 'Sava', 'Krka' and 'Rubina'. All of these samples were collected on 23 September 2019, at the Experimental Field for Nut Crops in Maribor (Slovenia; 46 • 34 01 N; 15 • 37 51 E; 275 m a.s.l.). They were obtained from 24-year-old trees at a planting density of 10 m × 10 m, with all under the same agronomical management and soil and climate conditions. The samples were collected from four trees for each cultivar, for a total of four repetitions per analysis. The samples were collected from the middle third of the branches on the east side of the trees, put in plastic bags and frozen immediately. The inner and outer husks were separated using a peeler, where~1 mm of the husk was peeled away as outer husk, with the remaining peeled husk as the inner husk. The terms inner and outer husk are used here because the exocarp and mesocarp of the walnut husk cannot be separated completely. Therefore the inner husk represents only the husk mesocarp, while the outer husk represents the exocarp and part of the mesocarp. The samples were then transported to the laboratory of the Department of Agronomy in the Biotechnical Faculty, of the University of Ljubljana (Slovenia), where they were lyophilised, ground into a powder with liquid nitrogen, and stored at −20 • C prior to further analysis.

Extraction of the Individual Phenolic Compounds
Briefly, 0.25 g of inner and outer husk and bark, or 0.1 g of buds, were extracted using 100% methanol (Sigma-Aldrich, Steinheim, Germany) at a 1:20 (w/v) tissue:methanol ratio. The protocol followed that described by Medic et al. [19].

HPLC-Mass Spectrometry Analysis of Individual Phenolic Compounds
The phenolics were analysed on an UHPLC system (Surveyor Dionex UltiMate 3000 series; Thermo Finnigan, San Jose, CA, USA) with a diode array detector at 280 nm for hydroxycinnamic acids, hydroxybenzoic acids, flavanols, flavanones and naphthoquinones, and at 350 nm for flavones and flavonols. The spectra were recorded between 200 nm and 600 nm. A C18 column (Gemini 150 × 4.60 mm; 3 µm; Phenomenex, Torrance, CA, USA) was used to separate the phenolics, at 25 • C, as previously described by Medic et al. [19].
The identification of the phenolics was done by tandem mass spectrometry (LCQ Deca XP Max; Thermo Scientific, Waltham, MA, USA) with heated electrospray ionisation Biology 2021, 10, 535 4 of 24 operating in negative ion mode, using the parameters as described by Medic et al. [19]. The mass spectrometry (MS) scanning for analysis was from m/z 50 to 2000, with data acquisition using the Xcalibur 2.2 software (Thermo Fischer Scientific Institute, Waltham, MA, USA). The phenolics were fragmented, with external standards used for the identification and quantification of known compounds. Literature data and MS fragmentation were used for identification of the unknown compounds, which were quantified using similar standards. The levels of the individual phenolics are expressed as mg/100 g dry weight, with their quantification according to the most relevant standard.

Analysis of Total Phenolics Content
For the full comparisons across the different walnut cultivars, the total phenolics content is represented first as the sum of all of the individual identified phenolics, each of which is expressed in mg/100 g dry weight according to the most relevant standard. A second determination of the total phenolics content was also carried out for the different walnut samples from the different walnut cultivars, with the extractions according to the same protocol as for the individual phenolics. These values for the total phenolics content of the extracts were determined using the Folin-Ciocalteau phenol reagent, as described by Singleton et al. [20], and then processed as described by Medic et al. [19] and Zamljen et al. [21]. These values are expressed in mg gallic acid equivalents/100 g dry weight.
The water used for all sample preparation, solutions and analyses was bi-distilled and purified using a Milli-Q water purification system (Millipore, Bedford, MA, USA). The acetonitrile and formic acid for the mobile phases were HPLC-MS grade (Fluka Chemie GmbH, Buchs, Switzerland).

Statistical Analysis
The data were collated using Microsoft Excel 2016, and analysed using R commander. Samples of the inner and outer husks, buds and bark were assayed as four repetitions. The data are expressed as means ± standard error (SE). For determination of significant differences between the data, one-way analysis of variance (ANOVA) was used, with Tukey's tests. Statistical means at 95% confidence level were calculated to determine the significance of the differences.

Identification of Individual Phenolics in Walnut Inner and Outer Husks, Buds and Bark
Based on the existing literature and the use of standard compounds, a total of 83 phenolics were tentatively identified for the inner and outer husks, buds and bark of these walnuts. Of these 83 phenolics, 14 were identified using standards, with fragmentation of both the standards and the addition of external standards used to confirm their identities. The remaining 69 phenolics were tentatively identified according to their pseudomolecular ions ([M − H] − ) and specific fragmentation patterns (i.e., MS 2 , MS 3 , MS 4 , MS 5 ). The selected MS spectra of the compounds can also be found in the Supplementary Material.
Most of the phenolics were identified for the buds, followed by the inner husk, the outer husk and the bark. The majority of naphthoquinones and hydroxycinnamic acids were in the inner and outer husks, and the majority of hydroxybenzoic acids and flavanols were in the buds. The only flavanone identified was in the buds.
Wherever possible, comparisons with authentic standards were performed. The data for all of these phenolics identified for these walnuts are summarised in Table 1 for the inner and outer husks, in Table 2 for the buds and in Table 3 for the bark.
These include mass spectrometry analysis (m/z, MS/MS fragmentation) and the standards according to which they were quantified.
In relation to these walnut naphthoquinones, dihydroxytetralone hexoside was identified by fragmentation ion m/z 159 ([M − H] − -H 2 O-180), and trihydroxytetralone galloyl hexoside by fragmentation ions m/z 331 and 271, as reported previously for walnut leaves [22]. Also, 1,4-Naphthoquinone was identified with the help of the standard at m/z 173, which yielded MS 2 fragments at m/z 111, 155, 129 and 145, which were previously mistakenly reported as juglone in Juglans mandshurica [23]. Juglone was identified with the help of the standard at m/z 189, which yielded an MS 2 fragment of m/z 161 and MS 3 fragments of m/z 117 and 133. Hydrojuglone β-D-glucopyranoside was identified from its fragmentation that yielded an ion at m/z 175, defining the loss of a hexosyl moiety (-162) [24]. The MS 3 m/z fragment of hydrojuglone β-D-glucopyranoside corresponded to the predicted LC-MS spectrum in a negative scan from the Human Metabolome Database, which yielded fragment ions of m/z 131, 157, 103 and 115.
Other phenolics identified through their fragmentation patterns included: hydrojuglone and its derivatives through the distinct fragment ions MS n m/z 175 and MS n+1 m/z 131, 157, 103, 147 and 115, as seen for the fragmentation of hydrojuglone β-D-glucopyranoside;  [23] in Juglans mandshurica. These compounds were previously reported in J. mandshurica, but are reported here for the first time in the walnut J. regia.     The 15 hydroxycinnamic acids identified through their fragmentation patterns included: neochlorogenic acid (3-caffeoylquinic acid) through its fragmentation, in addition to an external standard; 3-p-cumaroylquinic acid through its fragmentation pattern of MS m/z 337, MS 2 m/z 163, 191 and 173, as reported by Liu et al. [25] and Vieira et al. [22]; p-coumaric acid derivatives through the p-coumaric acid fragmentation pattern after being broken down, through the fragmentation patterns of ions m/z 163 and 119, as reported by Liu et al. [25] and Vieira et al. [22]; ferulic acid derivatives through their fragmentation patterns of MS n ion m/z 193 and MS n+1 ions m/z 149 and 117, as reported by Vieira et al. [22] and Šuković et al. [26]; and caffeic acid derivatives through their fragmentation pattern of MS n ion m/z 179 (caffeic acid-H), as reported by Vieira et al. [22].
Seven phenolics were identified for hydroxybenzoic acids: gallic acid derivatives, through the gallic acid fragmentation pattern after being broken down, through the fragmentation pattern of ions m/z 169 and 125, as reported by Li and Seeram [27] and Šuković et al. [26]; bis-(hexahydroxydiphenoyl)-glucose through its fragmentation pattern of MS 2 ions m/z 301 and 275, and MS 3 ions m/z 257, 229 and 185, as reported by Medic et al. [19] and Regueiro et al. [28]; and ellagic acid derivates through the typical fragmentation ions of ellagic acid at m/z 257, 229 and 185, as reported by Singh et al. [29].
There were 13 flavanols identified through their fragmentation patterns: (+)catechin and (−)epicatechin through their fragmentation patterns, in addition to an external standard, which produced fragment ions m/z 245, 205 and 179 for both (+)catechin and (−)epicatechin, thus suggesting that standards are needed when determining either of those compounds; epicatechin and catechin derivatives through the (+)catechin and (−)epicatechin fragmentation patterns after being broken down, through the ions m/z 245, 205 and 179, as seen in standard fragmentation patterns; and procyanidin dimers and procyanidin dimer derivatives through their characteristic fragmentation of MS n m/z 577 and MS n+1 m/z 425, 407 and 289 [14,30].
The two flavones identified were santin and 5,7-dihydroxy-3,4-dimetoxyflavone, through their fragmentation patterns according to Yan et al. [30]. Both santin and 5,7dihydroxy-3,4-dimetoxyflavone have been reported for walnut flowers [30], and now for the first time here for walnut inner and outer husks, buds and bark.
The flavanones included the identification of one compound: naringenin, through its fragmentation in addition to an external standard, through the fragment ions m/z 151 and 177.

Quantification of Total and Individual Phenolic Compounds for Walnut Inner and Outer Husks, Buds and Bark
The highest contents of phenolics were in the walnut buds, followed by the bark, the inner husk and the outer husk, as shown in Figure 1B.
The highest relative contents of hydroxycinnamic acids and flavones were seen for the inner husk, with the highest relative contents of hydroxybenzoic acids, flavanols, flavanones and flavonols for the walnut buds, as shown in Figure 1A. The higher absolute contents of phenolics in the walnut buds compared to the bark was mostly because of the higher content of flavanols, flavonols, hydroxycinnamic and hydroxybenzoic acids in the buds. The content of naphthoquinones was around 11 to 12 g/100 g plant material in both plant tissues. Therefore, walnut buds and bark represent an excellent source of naphthoquinones.
The highest contents of phenolics were in the walnut buds, followed by the bark, the inner husk and the outer husk, as shown in Figure 1B. The highest relative contents of hydroxycinnamic acids and flavones were seen for the inner husk, with the highest relative contents of hydroxybenzoic acids, flavanols, flavanones and flavonols for the walnut buds, as shown in Figure 1A. The higher absolute contents of phenolics in the walnut buds compared to the bark was mostly because of the higher content of flavanols, flavonols, hydroxycinnamic and hydroxybenzoic acids in the buds. The content of naphthoquinones was around 11 to 12 g/100 g plant material in both plant tissues. Therefore, walnut buds and bark represent an excellent source of naphthoquinones. The total naphthoquinones were the major phenolic group determined for the inner and outer husks, and for the buds and bark as well. These represented approximately just over 50% of all of the identified phenolics in the buds, 75% in the inner husk, 80% in the bark and 85 % in the outer husk, as shown in Figure 1A. As mentioned above, various naphthoquinones have shown activities against an array of microorganisms, including viruses, bacteria, fungi and parasites [16]. While the walnut buds were a better source of flavanols, hydroxybenzoic acids and flavonols, the inner and outer husks can also be considered as a source of naphthoquinones, with different naphthoquinones in the walnut buds and bark compared to the inner and outer husks, as seen in Table 1.
While the content of phenolics is usually higher in the peel of fruit compared to the flesh [19], here, interestingly, the content of phenolics for the outer husk was much lower than for the inner husk. When considering further the different tissues of walnut plants, the total phenolic contents (both as the summation and the total extracts; Tables 4-7) were higher than any previously reported for walnut shoots [15], leaves [18] or kernels [19], which further justifies the use of the husk, buds and bark as sources of the phenolics. Data are means ± standard error. x expressed as the sum of all of the individual identified phenolics (summation), in mg/100 g dry weight of the most relevant standard. y expressed as the separate analysis of the total phenolics for each extract (total extracts), in mg gallic acid equivalents/100 g dry weight. Means followed by different letters within a cultivar are significantly different (p ≤ 0.05; Tukey's tests); nd, not detected. Data are means ± standard error. x expressed as the sum of all of the individual identified phenolics (summation), in mg/100 g dry weight of the most relevant standard. y expressed as the separate analysis of the total phenolics for each extract (total extracts), in mg gallic acid equivalents/100 g dry weight. Means followed by different letters within a cultivar are significantly different (p ≤ 0.05; Tukey's tests); nd, not detected. Interestingly, the three Slovenian cultivars of 'Sava', 'Krka' and 'Rubina' had similar naphthoquinone contents in the walnut outer husk that were also higher than for the French cultivars 'Fernor', 'Fernette' and 'Franquette', which were also similar for their total naphthoquinone contents. The same was seen for the walnut inner husk, where the Slovenian varieties showed higher total naphthoquinone content than the French cultivars. This information that the Slovenian cultivars had higher total naphthoquinone contents than the French cultivars might also be useful in the future determination of the genetic origins of a cultivar, as cultivars that are bred in different climates might have specific naphthoquinone contents, as previously reported by Medic et al. [19]. The summarised total phenolic content of the buds identified in this study was compared with the total phenolic content of pellicle identified by Medic et al. [19], which was similar in terms of the order of phenolic compound content of the selected cultivars, with 'Franquette' containing the most phenols, followed by 'Fernor', 'Krka' and 'Sava' and 'Rubina' containing the second least phenols and the least 'Fernette'. Otherwise, no clear picture was seen linking phenolic content to different plant organs, suggesting that the total phenolics analysed may not be related between different parts of walnut, but rather a characteristic of the cultivar that dictates where the majority of phenolics are concentrated. Of note, this was observed only for the inner and outer husks, and not for the buds or bark, where the total naphthoquinone contents were not influenced by the origins of the cultivars.
Looking at individual naphthoquinones, in all these plant tissues, juglone was most abundant in the walnut inner husk, as can be seen in Tables 4-7. Among the walnut cultivars, 'Rubina' had the highest juglone content for the inner and outer husks and the bark, and the second highest juglone content for the buds, following 'Fernor'. This makes 'Rubina' an excellent choice for the purpose of juglone extraction. As juglone is used as a natural dye [12] and has anti-inflammatory effects [17], the efficient use of these agroresidues would represent a strategy that simultaneously helps to preserve the environment and potentially to boost the economic outcome for farmers and companies. This calls for further studies on the extraction of juglone from these, and other, plant tissues. The 83 phenolics identified across the different parts of the walnut tissues are shown in Table 8.

Conclusions
A total of 83 individual phenolics and the total phenolics content were identified and quantified for the inner and outer husks, buds and bark of six walnut cultivars. These 83 phenolics comprised 25 naphthoquinones, 15 hydroxycinnamic acids, 8 hydroxybenzoic acids, 13 flavanols, 2 flavones, 1 flavanone and 19 flavonols. Thirteen naphthoquinones have been reported for the walnut J. regia, or any other species for the first time, that may be unique to Juglandaceae family. To the best of our knowledge, this is the most complete study to describe the levels of the various phenolics for walnut husk, buds and bark. Furthermore, this is the first report to provide not only characterisation and quantification of the phenolics for walnut buds, but also a detailed characterisation and quantification of the separate husk layers (i.e., inner, outer). These data demonstrate the levels of the phenolics in these different walnut tissues, which are classified as agro-residuals. When considering the different walnut tissues, the total phenolic contents (both for the sum, and for the total extracts) were higher than previously reported for walnut shoots, leaves and kernels. This justifies the use of the husk, buds and bark as sources of phenolics. Furthermore, the Slovenian cultivars showed higher total naphthoquinone contents in the outer and inner walnut husks compared to the French cultivars. This information might be useful for the future determination of the genetic origin of a cultivar and also for the authentication of the walnuts belonging to each region, country, etc., as cultivars bred in different climates appear to show some specific variations in their naphthoquinone contents. Consequently, the present study provides useful information not only for agro-food industry (additives, pesticides) but also for the cosmetic and pharmaceutical industries.
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Part of the data presented in this study are available in Supplementary Material here. The remaining data presented in this study are available on request from the corresponding author. The remaining data are not publicly available due to privacy.

Conflicts of Interest:
The authors declare that they have no known competing financial interests or personal relationships that could appear to have influenced the work reported here.

MS
mass spectrometer UHPLC ultra-high performance liquid chromatography HPLC high performance liquid chromatography