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Article

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

Department of Agronomy, Biotechnical Faculty, University of Ljubljana Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
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Author to whom correspondence should be addressed.
Biology 2021, 10(6), 535; https://doi.org/10.3390/biology10060535
Submission received: 24 May 2021 / Revised: 7 June 2021 / Accepted: 10 June 2021 / Published: 15 June 2021
(This article belongs to the Section Plant Science)

Abstract

:

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.

1. 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 use [3]. Walnut agro-residues include: (i) the hard woody shell that protects the seed; (ii) the green husk that is rich in phenolic compounds (phenolics) and that further protects the woody shell in early stages of development; and (iii) the twigs and branches that are usually mulched after the winter pruning, with the bark and buds potentially also containing high levels of phenolics. All of these walnut agro-residues are usually discarded as landfill, or burnt or left to decompose in the orchard [4]. 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 [3].
As many different natural agro-residues are inexpensive and available in large quantities, there have been increasing tendencies over the last few decades towards their reuse as natural ingredients instead of chemical treatments [5]. In more recent years, several new research ideas for using walnut agro-residues have been proposed, with most studies focusing on the woody shells as a sorbent for oil [6], for hazardous material removal [7], as an ingredient in the cosmetics industry and as a blasting medium, among others [3].
The situation is quite different for walnut husks, and in particular the branches that include the buds and bark. While some studies have reported on the content of bioactive compounds in walnut husks, which have demonstrated antiradical and antimicrobial effects [3], little or nothing is known to date about the bioactive compounds in the buds and bark. Potentially, if walnut husks are used as a valuable source of bioactive compounds (i.e., mostly phenolics), they might still be reused for the removal of heavy metals from contaminated wastewaters [3,8], as a biofuel [9], for cosmetics [10] and as a natural dye [10,11]. Similarly, walnut bark might be reused as a natural dye [12], to truly make the most of these agro-residues.
Bioactive compounds are extra nutritional constituents that naturally occur in plant and food products. Most of these are secondary metabolites, such as alkaloids, pigments, mycotoxins, plant growth factors and phenolics. In recent years, numerous studies have been carried out that have promoted the benefits of such bioactive compounds for human health, in terms of potential protection against some degenerative diseases, like cancers and diabetes, and against cardiovascular diseases, and as anti-allergens, anti-microbials, anti-inflammatories and antioxidants, among others [13]. Phenolics have also been used effectively as functional ingredients in foods, as they can prevent mould and bacterial growth, and lipid oxidation [14].
Due to these countless benefits of such bioactive compounds, and of the phenolics in particular, studies have intensified to identify vegetables, fruit, plants and agricultural and agro-industrial residues as sources of phenolics. For walnuts, naphthoquinones and flavonoids have been reported to be the major phenolics [15].
Naphthoquinones are secondary metabolites that have been identified in about 20 plant families, they are most commonly found in Bignoniaceae, Droseraceae, Plumbagi-nace, Boraginaceae and Juglandaceae families, and they comprise a wide variety of chemical structures based on the naphthalene skeleton. They participate in multiple oxidative processes, serve as important links in electron transport chains, and might also act as defensive compounds in interspecies chemical warfare (i.e., alelopathy). On the basis of these traits, many studies have been exploring the biological and toxicological activities of naphthoquinones, to potentially discover and develop new drugs (e.g., antibacterial, antifungal, antiviral, antiparasitic and antitumor) [16].
Over time, microorganisms tend to develop resistance to antimicrobial agents that are used as therapeutics, which has prompted the search for new effective antimicrobials. There are numerous studies that have documented activities of a variety of naphthoquinones against an array of microorganisms, including viruses, bacteria, fungi and parasites [16]. The most studied naphthoquinones are vitamin K (e.g., anti-inflammatory, decrease of gap-junctional intercellular communication), juglone (e.g., apoptotic, cell-cycle arrest, anti-inflammatory) and plumbagin (e.g., apoptotic, cell-cycle arrest, inhibition of cell invasion, migration and proliferation, anti-inflammatory and neuroprotection) [17].
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.

2. Materials and Methods

2.1. 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.

2.2. 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].

2.3. 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 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.

2.4. 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.

2.5. Chemicals

The following standards were used to identify and quantify the phenolics: apigenin 7-glucoside, kaempferol-3-glucoside, procyanidin B1, quercetin-3-glucoside, ferulic acid, p-coumaric acid (Fluka Chemie GmbH, Buchs, Switzerland); (+) catechin (Roth, Karlsruhe, Germany); 4-O-caffeoylquinic acid, neochlorogenic acid (3-caffeoylquinic acid), myricetin-3-galactoside, quercetin-3-galactoside, quercetin-3-rhamnoside, juglone (5-hydroxy-1,4-naphthoquinone), 1,4-naphthoquinone, caffeic acid, galic acid, ellagic acid, naringenin, (−)epicatechin (Sigma–Aldrich Chemie GmbH, Steinheim, Germany); and myricetin-3-rhamnoside, quercetin-3-arabinofuranoside, quercetin-3-arabinopyranoside (Apin Chemicals, Abingdon, UK).
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).

2.6. 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.

3. Results and Discussion

3.1. 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., MS2, MS3, MS4, MS5). 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.
Seven of the naphthoquinones were identified in all of the plant tissues of all of the cultivars: juglone, hydrojuglone, hydrojuglone β-D-glucopyranoside, hydrojuglone rutinoside, hydrojuglone derivative pentoside 2, hydrojuglone derivative rhamnoside and dihydroxytetralone hexoside. As well as these, the inner and outer husks contained a few hydrojuglone derivatives and mostly other naphthoquinones, while the buds and bark contained mainly hydrojuglone derivatives. To the best of our knowledge, 13 of the phenolics indicated here have not been reported for the walnut J. regia, or for any other Juglans species, or indeed, for any plant tissues: hydrojuglon, hydrojuglon rutinoside, hydrojuglone dihexoside, hydrojuglone derivative 1, hydrojuglone derivative 2, hydrojuglone derivative 3, hydrojuglone derivative 4, hydrojuglone derivative 5, hydrojuglone derivative pentoside 1, hydrojuglone derivative pentoside 2, hydrojuglone derivative pentoside 3, hydrojuglone derivative rhamnoside, hydrojuglone pentose galloyl derivative and hydrojuglone hexoside derivate.
Overall, for the inner and outer husks, 38 phenolics were identified, as 17 naphthoquinones, 11 hydroxycinnamic acids, 3 hydroxybenzoic acids, 3 flavanols, 2 flavones and 2 flavonols. For the buds, 57 phenolics were identified, as 13 naphthoquinones, 6 hydroxycinnamic acids, 6 hydroxybenzoic acids, 12 flavanols, 2 flavones, 1 flavanone and 17 flavonols. For the bark, 29 phenolics were identified, as 11 naphthoquinones, 3 hydroxybenzoic acids, 3 flavanols, 2 flavones and 10 flavonols.
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]–H2O–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 MS2 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 MS2 fragment of m/z 161 and MS3 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 MS3 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 MSn m/z 175 and MSn+1 m/z 131, 157, 103, 147 and 115, as seen for the fragmentation of hydrojuglone β-D-glucopyranoside; 5-hydroxy-2,3-dihydro-1,4-naphthalenedione through its fragmentation pattern of MS2 ions m/z 131 [M–H–CO2], 147 [M–H–CO], 157 [M–H–H2O] and 129 [M–H–CO–H2O]; regiolone through its fragmentation pattern of MS2 ions m/z 159 [M–H–H2O], 175 and 131 [M–H–H2O–CO]; 4,5,8-trihydroxynaphthalene-5-D-glucopyranoside through its fragmentation pattern of MS2 ions m/z 331 [M–H–C10H8O3] and 271 [M–H–C12H12O5], and MS4 ions m/z 211 [M–H–C14H16O7] and 169 [M–H–C16H18O8]; 1,4,8-trihydroxynaphthalene-1-D-glucopyranoside through its fragmentation pattern of MS2 ion m/z 327 [M–H–C10H8O3] and MS3 ions m/z 183 [M–H–C16H16O7] and 225 [M–H–C14H14O6]; bis-juglone through its fragmentation pattern of MS2 ions m/z 345 [M–H–H2O], 317 [M–H–H2O–CO] and 301 [M–H–CO2]; and p-hydroxymethoxybenzobijuglone through its fragmentation pattern of MS2 ions m/z 383 [M–H–CH4O] and 355 [M–H–CH4O–CO], as reported by Huo et al. [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, MS2 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 MSn ion m/z 193 and MSn+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 MSn 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 MS2 ions m/z 301 and 275, and MS3 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 MSn m/z 577 and MSn+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 5,7-dihydroxy-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.
The flavonols included the identification of three groups of compounds: (i) myricetin glycosides through their fragmentation pattern of MS2 ions m/z 316, 317 and MS3 ions m/z 179, 191; (ii) quercetin and quercetin glycosides through their clear fragmentation pattern of MS2 m/z 301 and MS3 m/z 179, 151; and (iii) kaempferol and kaempferol glycosides through their fragmentation pattern of MS2 m/z 284 and 285 and MS3 m/z 255 and 227, as reported by Santos et al. [31] and Vieira et al. [22]. Fragmentation patterns with the loss of hexosyl (-162), pentosyl (-132) and rhamnosyl (-146) residues were seen here, as reported by Vieira et al. [22]. Kaempferol-7-hexosides were identified through their fragmentation pattern of MS2 ion m/z 285 and MS3 ions 165, 119 and 93, as reported by Chen et al. [32]. Kaempferol-7-hexosides have been reported previously for Rhamnus davurica [32], but this is the first time for walnut.

3.2. 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 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; Table 4, Table 5, Table 6 and Table 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.
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 Table 4, Table 5, Table 6 and Table 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 agro-residues 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.

4. 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.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/biology10060535/s1. Figure S1: MS1 spectra of hydrojuglone β-D-glucopyranoside (naphthoquinone) in a negative ion mode., Figure S2: MS2 spectra of hydrojuglone β-D-glucopyranoside (naphthoquinone) in a negative ion mode., Figure S3: MS1 spectra of gallic acid derivative 5 (hydroxybenzoic acid) in a negative ion mode., Figure S4: MS1 spectra of (epi)catechin derivative 5 (flavanol) in a negative ion mode., Figure S5: MS2 spectra of (epi)catechin derivative 5 (flavanol) in a negative ion mode., Figure S6: MS1 spectra of santin (flavone) in a negative ion mode., Figure S7: MS2 spectra of santin (flavone) in a negative ion mode., Figure S8: MS1 spectra of quercetin-rhamnoside (flavonol) in a negative ion mode., Figure S9: MS2 spectra of quercetin-rhamnoside (flavonol) in a negative ion mode., Figure S10: MS1 spectra of p-coumaric acid derivative 4 (hydroxycinnamic acid) in a negative ion mode., Figure S11: MS2 spectra of p-coumaric acid derivative 4 (hydroxycinnamic acid) in a negative ion mode.

Author Contributions

Conceptualization, A.M. and R.V.; Data curation, A.M.; Formal analysis, A.M.; Funding acquisition, M.H.; Investigation, A.M.; Methodology, A.M., J.J. and R.V.; Project administration, R.V.; Resources, A.M., M.H. and A.S.; Software, A.M. and J.J.; Supervision, R.V.; Validation, A.S. and R.V.; Visualization, A.M.; Writing—original draft, A.M.; Writing—review & editing, J.J., M.H., A.S. and R.V. All authors have read and agreed to the published version of the manuscript.

Funding

This study is part of programme P4-0013-0481, which is funded by the Slovenian Research Agency (ARRS).

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.

Abbreviations

MSmass spectrometer
UHPLCultra-high performance liquid chromatography
HPLChigh performance liquid chromatography

References

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Figure 1. Relative contents of the phenolics groups for the different walnut tissues, as proportions of total phenolic compounds identified (A) and as g/100 g walnut tissue defined by the most relevant standards (B).
Figure 1. Relative contents of the phenolics groups for the different walnut tissues, as proportions of total phenolic compounds identified (A) and as g/100 g walnut tissue defined by the most relevant standards (B).
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Table 1. Tentative identification of the 38 phenolics from the walnut inner and outer husks, and the standard equivalents used.
Table 1. Tentative identification of the 38 phenolics from the walnut inner and outer husks, and the standard equivalents used.
PhenolicRt [M − H] Fragmentation Pattern (m/z)Equivalents Expressed
(min)(m/z)MS2MS3MS4MS5
p-Coumaric acid derivative 27.06343163, 325, 119 p-Coumaric acid
Ferulic acid derivative 18.20221149, 117 Ferulic acid
Neochlorogenic acid (3-caffeoylquinic acid) 9.90353191, 179, 135 Neochlorogenic acid
Ferulic acid derivative 211.43489193149, 134, 178, 117 Ferulic acid
(+)Catechin12.34289245, 205, 179, 125 (+)Catechin
3-p-Coumaroylquinic acid12.34337163, 191, 173 4-O-Caffeoylquinic acid
Dihydroxytetralone hexoside12.69339159, 177115, 75 Juglone
Caffeic acid derivative 213.95251207, 175179, 191 Caffeic acid
5-Hydroxy-2,3-dihydro-1,4-naphthalenedione13.97175131, 147, 157, 115, 103, 129 Juglone
p-Coumaric acid derivative 314.51325235, 265, 163163, 191, 119 p-Coumaric acid
Regiolone14.79177159, 175, 131115, 75 Juglone
159, 175, 131131, 147, 157, 103, 129, 119
(−)Epicatechin14.79289245, 205, 179, 125 (−)Epicatechin
Gallic acid derivative 415.87261243, 201, 187199, 225, 169, 125 Gallic acid
Hydrojuglone β-D-glucopyranoside 16.49337175131, 147, 157, 103 Juglone
Trihydroxytetralone galloyl hexoside17.77507331, 271271 Juglone
Hydrojuglone derivative rhamnoside18.02449303, 285285241, 175, 257213, 199, 197Juglone
241, 175, 257157, 147, 129, 119
Quercetin-3-galactoside18.23463301179, 151 Quercetin-3-galactoside
p-Coumaric acid derivative 419.64475265, 163205, 163, 119 p-Coumaric acid
Gallic acid derivative 519.80421313, 169169, 125 Gallic acid
(epi)Catechin derivative 524.04469289245, 205, 179, 125 (+)Catechin
4,5,8-Trihydroxynaphthalene-5-D-glucopyranoside20.55507331, 271271211, 169 Juglone
Ferulic acid derivative 321.03521473, 503, 337337193, 175 Ferulic acid
Hydrojuglone derivative pentoside 221.42435303, 285285 Juglone
Caffeic acid derivative 321.78519489161, 313, 179 Caffeic acid
Gallic acid derivative 322.10489271, 313211, 169, 125 Gallic acid
Quercetin-3-rhamnoside22.60447301179, 151 Quercetin-3-rhamnoside
1,4,8-Trihydroxynaphthalene-1-D-glucopyranoside24.11503327183, 225, 139 Juglone
Hydrojuglone hexoside derivative24.86497335175131, 157, 103, 147 Juglone
Hydrojuglone derivative 526.29517175, 341131, 157, 103, 147 Juglone
Caffeic acid derivative 426.62499341, 323, 281, 175251, 281, 179179 Caffeic acid
Hydrojuglone28.57175131, 103, 157, 175 Juglone
1,4-Naphthoquinone28.57173111, 155, 129, 145 1,4-Naphthoquinone
Hydrojuglone rutinoside29.56483175, 325131, 157, 103, 147 Juglone
Juglone 30.05189161117, 133 Juglone
bis-Juglone31.42363345, 317, 319, 301301 Juglone
p-Hydroxymetoxybenzobijuglone31.70415383, 355 Juglone
Santin32.37343328313, 285 Apigenin-7-glucoside
5,7-Dihydroxy-3,4-dimetoxyflavone32.60313298283, 255 Apigenin-7-glucoside
Rt, retention time; [M − H], pseudo-molecular ion identified in negative ion mode; bold numbers, fragments further fragmented; first fragment number, fragment that was further fragmented if no bold numbers are given.
Table 2. Tentative identification of the 57 phenolics from the walnut buds, and the standard equivalents used.
Table 2. Tentative identification of the 57 phenolics from the walnut buds, and the standard equivalents used.
PhenolicRt[M − H]Fragmentation Pattern (m/z)Equivalents Expressed
(min)(m/z)MS2MS3MS4MS5
Gallic acid derivative 18.01345169, 125, 175125 Gallic acid
Procyanidin dimer derivative 18.84593425, 467, 407, 289 Procyanidin B1
Neochlorogenic acid (3-caffeoylquinic acid)9.75353191, 179, 135 Neochlorogenic acid
(epi)Catechin derivative 19.90357289, 311245, 205, 179, 125 (+)Catechin
bis-HHDP-glucose10.40783301, 275257, 229, 185 Gallic acid
Procyanidin dimer 110.58577425, 407, 451, 289 Procyanidin B1
Procyanidin dimer 211.62577425, 407, 451, 289 Procyanidin B1
(+)Catechin12.45289245, 205, 179, 125 (+)Catechin
3-p-Coumaroylquinic acid12.55337163, 191, 173 4-O-Caffeoylquinic acid
Dihydroxytetralone hexoside12.76339177, 159 Juglone
(epi)Catechin derivative 213.33325289, 163, 179245, 205, 179, 125 (+)Catechin
Hydrojuglone dihexoside13.89499175131, 157, 103, 129, 147 Juglone
Hydrojuglone derivative 114.44355175, 169, 265, 193131, 147, 157, 103, 129 Juglone
(−)Epicatechin14.74289245, 205, 179, 125 (−)Epicatechin
(epi)Catechin derivative 314.74325163, 179, 289, 119245, 205, 179, 125 (+)Catechin
p-Coumaric acid derivative15.33281163, 135, 119119 p-Coumaric acid
Hydrojuglone β-D-glucopyranoside 16.54337175131, 103, 157, 145 Juglone
Procyanidin dimer derivative 217.15729577425, 407, 451, 289 Procyanidin B1
Ellagic acid derivative17.42467391, 301301257, 229, 185 Ellagic acid
Hydrojuglone derivative 217.98465301, 339215, 257, 283, 175, 151187, 171, 143 Juglone
215, 257, 283, 175, 151147, 131, 157, 129
Myricetin-3-galactoside18.13479316271, 287, 179 Myricetin-3-galactoside
Hydrojuglone derivative pentoside 118.51435285241, 199, 175, 151, 135213, 199, 197 Juglone
Gallic acid derivative 219.28491271, 331211, 169, 125 Gallic acid
Quercetin galoyll hexoside19.71615463301179, 151 Quercetin-3-glucoside
Myricetin pentoside19.99449317, 316179, 151, 191 Myricetin-3-galactoside
Galloyl-3-(epi)catechin20.36441289245, 205, 179, 125 (+)Catechin
Quercetin-3-galactoside20.36463301179, 151 Quercetin-3-galactoside
Myricetin-3-rhamnoside20.61463316271, 287, 179, 151 Myricetin-3-rhamnoside
Quercetin-3-glucoside20.80463301179, 151 Quercetin-3-glucoside
Hydrojuglone derivative rhamnoside21.17449303, 285 Juglone
Hydrojuglone derivative pentoside 221.48435303, 285285, 177241, 175, 161213, 199, 197Juglone
Gallic acid methyl ester21.86183168, 12412495 Gallic acid
Quercetin-3-arabinopyranoside21.99433301179, 151 Quercetin-3-rabinopyranoside
Gallic acid derivative 322.17489271211, 169168, 124 Gallic acid
Quercetin-3-arabinofuranoside22.41433301179, 151 Quercetin-3-arabinofuranoside
Quercetin-3-rhamnoside22.55447301179, 151 Quercetin-3-rhamnoside
Kaempferol pentoside 123.22417284255, 227, 151 Kaempferol-3-glucoside
Kaempferol pentoside 223.48417284255, 227, 151 Kaempferol-3-glucoside
Caffeic acid hexoside derivative23.84501341251, 281, 179, 323179, 135 Caffeic acid
Kaempferol pentoside 324.06417285257, 267, 229 Kaempferol-3-glucoside
Kaempferol-3-rhamnoside24.32431285257, 268, 229 Kaempferol-3-glucoside
(epi)Catechin derivative 424.56463289245, 205, 179203, 187, 161 (+)Catechin
Hhydrojuglone pentose galloyl derivative24.85587455303, 285285, 259, 177, 241, 175 Juglone
Quercetin hexoside derivative 125.13669463301179, 151 Quercetin-3-glucoside
Procyanidin dimer derivative 325.71903729603, 577 Procyanidin B1
Quercetin hexoside derivative 226.15639463301179, 151 Quercetin-3-glucoside
Caffeic acid derivative 126.62499341, 323, 281, 175251, 221, 179179, 135 Caffeic acid
Diferuoyl hexoside27.73531337193, 178134, 149 Ferulic acid
Hydrojuglone28.60175131, 103, 157, 175 Juglone
Quercetin29.34301179, 151 Quercetin-3-glucoside
Hydrojuglone rutinoside29.63483175131, 157, 103 Juglone
Hydrojuglone derivative 329.73513175, 337131, 157, 103 Juglone
Juglone 30.14189161117, 133 Juglone
Naringenin30.33271151, 177 Naringenin
Kaempferol30.64285151 Kaempferol-3-glucoside
Santin31.97343328313, 285 Apigenin-7-glucoside
5,7-Dihydroxy-3,4-dimetoxyflavone32.34313298283, 255 Apigenin-7-glucoside
Rt, retention time; [M − H], pseudo-molecular ion identified in negative ion mode; bold numbers, fragments further fragmented; first fragment number, fragments that were further fragmented if no bold numbers are given.
Table 3. Tentative identification of the 29 phenolics from the walnut bark, and the standard equivalents used.
Table 3. Tentative identification of the 29 phenolics from the walnut bark, and the standard equivalents used.
PhenolicRt[M − H]Fragmentation Pattern (m/z)Equivalents Expressed
(min)(m/z)MS2MS3MS4
Procyanidin dimer 211.67577425, 407, 451, 289 Procyanidin B1
(+)Catechin12.43289245, 205, 179, 125 (+)Catechin
Dihydroxytetralone hexoside12.82339177, 159 Juglone
Hydrojuglone β-D-glucopyranoside 16.58337175131, 147, 157, 103 Juglone
Procyanidin dimer derivative 217.16729577425, 407, 451, 289 Procyanidin B1
Ellagic acid derivative17.47467391, 301301257, 229, 185Ellagic acid
Hydrojuglone derivative 417.83451319, 325, 301, 193, 151193, 301, 179, 125165, 175, 121, 131Juglone
319, 325, 301, 193, 151192, 235
319, 325, 301, 193, 151215, 257, 283, 175, 151147, 131, 157, 129
319, 325, 301, 193, 151215, 257, 283, 175, 151187, 171, 143
Hydrojuglone derivative 218.02465301, 339, 319, 151, 193215, 257, 283, 175, 151187, 171, 143Juglone
Hydrojuglone derivative pentoside 118.21435285, 301241, 175, 199, 257, 151 Juglone
285, 301229, 179, 151, 257, 137
Hydrojuglone derivative pentoside 318.55435285, 301 Juglone
Myricetin pentoside19.20449317179, 151 Myricetin-3-galactoside
Gallic acid derivative 219.32491271211, 169, 125168, 124Gallic acid
Quercetin galloyl hexoside19.74615463301179, 151Quercetin-3-glucoside
Myricetin-3-rhamnoside20.33463316271, 287, 179, 164243, 227, 215, 183Myricetin-3-rhamnoside
Quercetin-3-galactoside20.64463301179, 151 Quercetin-3-galactoside
Quercetin-3-glucoside20.84463301179, 151 Quercetin-3-glucoside
Hydrojuglone derivative rhamnoside21.22449303, 285181, 153, 285 Juglone
303, 285241, 175, 257, 199, 151
Hydrojuglone derivative pentoside 221.47435285241, 175, 257 Juglone
Quercetin-3-arabinopyranoside22.04433301179, 151 Quercetin-3-arabinopyranoside
Gallic acid derivative 322.19489271, 313211, 169, 125168, 124Gallic acid
271, 313169, 125125
Quercetin-3-arabinofuranoside22.39433301179, 151 Quercetin-3-arabinofuranoside
Quercetin-3-rhamnoside22.67447301179, 151, 273, 257, 229 Quercetin-3-rhamnoside
Kaempferol-7-hexoside 123.06447285165, 119, 93 Kaempferol-3-glucoside
Kaempferol-7-hexoside 228.34447285165, 119, 93 Kaempferol-3-glucoside
Hydrojuglone28.56175131, 103, 157, 175 Juglone
Hydrojuglone rutinoside29.64483175131, 103, 157 Juglone
Juglone 30.14189161117, 133 Juglone
Santin31.98343328313, 285 Apigenin-7-glucoside
5,7-Dihydroxy-3,4-dimetoxyflavone32.34313298283, 255 Apigenin-7-glucoside
Rt, retention time; [M − H], pseudo-molecular ion identified in negative ion mode; bold numbers, fragments further fragmented; first fragment number, fragments that were further fragmented if no bold numbers are given.
Table 4. Individual phenolics for the walnut inner husks across the six selected cultivars.
Table 4. Individual phenolics for the walnut inner husks across the six selected cultivars.
PhenolicInner Husk Phenolic Content per Cultivar (mg/100 g Dry Weight)
‘Fernor’‘Fernette’‘Franquette’‘Sava’‘Krka’‘Rubina’
Naphthoquinones
1,4-Naphthoquinone1148.5 ± 109.0 b1067.5 ± 95.2 b435.3 ± 73.6 a1521.8 ± 47.3 b2231.8 ± 233.2 c1547.1 ± 105.5 b
Juglone 593.2 ± 45.9 a776.3 ± 39.8 ab533.6 ± 34.3 a598.9 ± 61.8 a852.1 ± 93.4 b852.0 ± 38.8 b
Hydrojuglone143.2 ± 13.6 b133.1 ± 11.9 b54.3 ± 9.2 a189.8 ± 5.9 b278.3 ± 29.1 c192.9 ± 13.2 b
Hydrojuglone β-D-glucopyranoside502.9 ± 9.9 b250.9 ± 17.4 a266.3 ± 21.4 a761.9 ± 50.1 c624.9 ± 31.8 bc734.7 ± 69.4 c
Hydrojuglone rutinoside125.2 ± 2.3 ab93.4 ± 10.6 a90.7 ± 14.7 a179.2 ± 14.7 bc234.1 ± 17.3 c200.8 ± 15.5 c
Hydrojuglone derivative 5153.9 ± 7.0 b75.3 ± 5.1 a58.3 ± 10.3 a294.1 ± 31.3 c286.2 ± 12.4 c187.2 ± 22.2 b
Hydrojuglone derivative pentoside 2154.8 ± 16.6 bc95.9 ± 9.6 ab65.4 ± 11.8 a246.7 ± 23.2 d236.0 ± 21.7 cd274.2 ± 22.2 d
Hydrojuglone derivative rhamnoside324.2 ± 8.4 bc155.7 ± 11.6 a208.8 ± 25.2 ab572.1 ± 45.0 e486.3 ± 45.0 de403.8 ± 45.1 cd
Hydrojuglone hexoside derivative137.9 ± 13.6 ab97.6 ± 11.8 a85.2 ± 20.2 a239.9 ± 16.4 c194.5 ± 26.3 bc153.6 ± 19.7 ab
bis-Juglone110.7 ± 7.0 a125.8 ± 11.0 a133.8 ± 17.8 ab247.9 ± 16.0 c194.4 ± 19.1 bc175.6 ± 13.2 ab
p-Hydroxymetoxybenzobijuglone680.4 ± 92.1 a494.3 ± 85.9 a440.1 ± 46.6 a490.5 ± 60.1 a641.6 ± 8.8 a528.1 ± 46.9 a
Regiolone672.3 ± 18.6 ab511.3 ± 39.8 a775.1 ± 73.8 bc808.6 ± 44.6 bc936.3 ± 42.8 c920.9 ± 38.8 c
4,5,8-Trihydroxynaphthalene-5-D-glucopyranoside709.0 ± 44.7 bc479.5 ± 32.6 ab412.6 ± 15.3 a1276.7 ± 45.1 d1241.5 ± 58.5 d913.6 ± 88.1 c
1,4,8-Trihydroxynaphthalene-1-D-glucopyranoside335.7 ± 42.6 bc151.1 ± 11.3 a156.3 ± 21.3 a478.0 ± 50.7 c339.1 ± 23.2 bc262.1 ± 23.6 ab
Dihydroxytetralone hexoside88.2 ± 3.6 b21.4 ± 9.3 a68.9 ± 14.7 ab106.8 ± 16.7 b113.4 ± 11.9 b104.2 ± 12.2 b
Trihydroxytetralone galloyl hexoside169.6 ± 10.2 b61.2 ± 10.4 a78.4 ± 20.7 a240.8 ± 27.2 b182.7 ± 12.7 b174.0 ± 23.1 b
Hydroxycinnamic acids
Neochlorogenic acid (3-caffeoylquinic acid)28.5 ± 0.8 ab17.0 ± 1.2 a21.6 ± 1.0 ab47.6 ± 5.3 d43.0 ± 2.6 cd31.9 ± 3.5 bc
3-p-Coumaroylquinic acid494.7 ± 4.3 a496.9 ± 26.7 a443.4 ± 18.4 a712.2 ± 26.1 b628.7 ± 32.1 b474.2 ± 17.8 a
p-Coumaric acid derivative 211.6 ± 0.5 ab8.8 ± 0.8 a10.8 ± 0.8 ab21.6 ± 1.5 d17.7 ± 0.6 cd14.4 ± 1.4 bc
p-Coumaric acid derivative 316.7 ± 0.6 ab12.5 ± 1.1 a17.8 ± 1.3 ac20.3 ± 2.5 bc22.0 ± 1.0 bc23.8 ± 1.5 c
p-Coumaric acid derivative 411.1 ± 0.3 b6.5 ± 0.5 a4.8 ± 0.6 a13.3 ± 0.9 b11.8 ± 0.6 b12.2 ± 1.2 b
Caffeic acid derivative 29.5 ± 0.4 ab5.0 ± 1.4 a 9.3 ± 1.6 ab14.1 ± 1.6 b14.9 ± 0.9 b13.1 ± 1.3 b
Caffeic acid derivative 352.9 ± 3.0 b29.7 ± 2.3 a25.1 ± 2.9 a85.2 ± 4.2 c61.4 ± 4.8 b64.3 ± 5.6 b
Caffeic acid derivative 44.8 ± 0.5 ab3.2 ± 0.8 a2.8 ± 0.9 a5.4 ± 0.9 ab7.2 ± 1.3 b4.9 ± 0.8 ab
Ferulic acid derivative 129.8 ± 3.1 bc10.5 ± 1.1 a18.3 ± 2.0 ab34.8 ± 2.7 cd40.3 ± 2.2 cd43.7 ± 4.3 d
Ferulic acid derivative 219.8 ± 1.3 ab12.4 ± 0.6 a18.2 ± 1.3 a34.5 ± 3.0 c27.0 ± 1.2 b17.8 ± 2.0 a
Ferulic acid derivative 315.2 ± 1.5 a 11.2 ± 0.4 a10.5 ± 1.5 a28.9 ± 1.8 b26.9 ± 1.1 b28.6 ± 2.2 b
Hydroxybenzoic acids
Gallic acid derivative 3335.7 ± 9.9 b148.7 ± 10.4 a240.6 ± 14.6 ab897.8 ± 41.4 d766.9 ± 27.5 c817.6 ± 31.2 cd
Gallic acid derivative 453.8 ± 3.1 b21.3 ± 2.2 a30.7 ± 2.5 a41.4 ± 7.4 ab42.9 ± 5.8 ab42.6 ± 6.4 ab
Gallic acid derivative 526.4 ± 1.9 bc21.0 ± 2.0 ab12.7 ± 2.4 a38.8 ± 5.0 cd44.8 ± 2.9 d31.9 ± 1.6 bc
Flavanols
(+)Catechin151.7 ± 1.0 a151.5 ± 8.1 a136.1 ± 5.7 a218.1 ± 8.1 b192.1 ± 10.0 b145.4 ± 5.4 a
(−)Epicatechin398.9 ± 9.9 ab305.1 ± 23.8 a459.9 ± 44.9 bc480.6 ± 27.0 bc557.7 ± 25.7 c546.6 ± 23.7 c
Flavones
Santin61.3 ± 6.1 ab51.8 ± 1.3 a56.1 ± 4.7 a92.5 ± 11.4 c89.5 ± 3.5 bc51.2 ± 6.7 a
5,7-Dihydroxy-3,4-dimetoxyflavone43.1 ± 1.7 a30.4 ± 5.2 a23.1 ± 5.4 a166.8 ± 5.7 d124.4 ± 11.7 c74.3 ± 6.0 b
Flavonols
Quercetin-3-galactoside72.3 ± 3.1 abc49.2 ± 6.5 ab40.6 ± 3.5 a115.0 ± 15.3 d91.0 ± 10.1 cd85.1 ± 7.6 bd
Quercetin-3-rhamnoside18.3 ± 0.7 bc7.7 ± 2.1 ab6.3 ± 3.0 a22.1 ± 3.0 c24.5 ± 1.5 c24.4 ± 4.2 c
Total naphthoquinones6049.6 ± 224.4 b4590.5 ± 193.6 a3863.0 ± 294.6 a8253.6 ± 192.3 cd9073.1 ± 301.2 d7624.8 ± 361.0 c
Total hydroxycinnamic acids694.6 ± 5.0 ab613.6 ± 27.4 ab582.7 ± 22.6 a1017.7 ± 35.0 c900.2 ± 40.6 c728.9 ± 39.4 b
Total hydroxybenzoic acids415.8 ± 12.1 b190.9 ± 9.7 a284.0 ± 17.2 a977.9 ± 42.3 c854.6 ± 32.5 c892.1 ± 37.8 c
Total flavanols550.6 ± 9.4 ab456.6 ± 22.7 a596.1 ± 45.9 bc698.7 ± 31.3 cd749.8 ± 18.6 d692.0 ± 19.9 cd
Total flavones104.4 ± 7.7 a82.2 ± 5.7 a79.2 ± 9.6 a259.3 ± 16.4 b213.9 ± 13.8 b125.5 ± 2.0 a
Total flavanonesndndndndNdnd
Total flavonols90.6 ± 3.8 bc56.9 ± 6.5 ab46.9 ± 6.4 a137.1 ± 15.2 d115.5 ± 9.3 cd109.5 ± 11.5 cd
Total phenolics content (summation; relevant standards) x7905.5 ± 236.8 b5990.8 ± 204.2 a5451.8 ± 369.6 a11344.4 ± 298.0 cd11907.1 ± 228.9 d10172.8 ± 426.0 c
Total phenolics content (total extracts; mg gallic acid equivalents/100 g dry weight) y1447.2 ± 73.7 ab1327.2 ± 92.9 a1575.1 ± 92.0 ab1842.6 ± 175.9 b1804.5 ± 26.5 b1788.1 ± 85.5 b
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.
Table 5. Individual phenolics for the walnut outer husks across the six selected cultivars.
Table 5. Individual phenolics for the walnut outer husks across the six selected cultivars.
PhenolicOuter Husk Phenolic Content per Cultivar (mg/100 g Dry Weight)
‘Fernor’‘Fernette’‘Franquette’‘Sava’‘Krka’‘Rubina’
Naphthoquinones
1,4-Naphthoquinone128.8 ± 20.4 a260.6 ± 47.9 a296.5 ± 34.3 a800.8 ± 55.3 b1079.9 ± 44.7 c1026.5 ± 117.7 bc
Juglone 431.7 ± 25.1 a528.5 ± 37.1 ab519.8 ± 8.7 ab609.2 ± 18.7 b608.3 ± 9.6 b838.8 ± 19.5 c
Hydrojuglone25.7 ± 4.1 a52.0 ± 9.6 a59.2 ± 6.8 a159.8 ± 11.0 b215.5 ± 8.9 c204.8 ± 23.5 bc
Hydrojuglone β-D-glucopyranoside234.1 ± 9.8 a218.5 ± 21.7 a291.3 ± 24.6 ab361.7 ± 35.2 b278.7 ± 15.9 ab370.0 ± 19.5 b
Hydrojuglone rutinoside82.8 ± 7.3 ab80.3 ± 8.9 a70.2 ± 2.9 a97.6 ± 11.0 ab116.7 ± 4.3 bc132.2 ± 8.5 c
Hydrojuglone derivative 570.5 ± 14.6 a49.0 ± 3.3 a54.8 ± 4.7 a165.2 ± 18.8 b178.1 ± 16.6 b173.2 ± 6.2 b
Hydrojuglone derivative pentoside 2545.7 ± 40.8 c493.5 ± 54.4 bc327.9 ± 27.3 a480.2 ± 20.1 bc371.9 ± 11.6 ab328.8 ± 12.4 a
Hydrojuglone derivative rhamnoside172.5 ± 17.8 bc109.3 ± 11.5 a164.4 ± 10.3 ab202.2 ± 16.7 bd224.5 ± 13.1 cd244.7 ± 3.8 d
Hydrojuglone hexoside derivative107.8 ± 6.3 a101.8 ± 13.6 a134.1 ± 10.5 a79.7 ± 15.1 a75.9 ± 16.3 a124.2 ± 16.7 a
bis-Juglone102.1 ± 9.8 a150.6 ± 18.4 ab132.6 ± 9.4 ab177.5 ± 14.5 bc150.4 ± 9.1 ab215.3 ± 19.6 c
p-Hydroxymetoxybenzobijuglone128.7 ± 16.3 a133.0 ± 19.8 a168.3 ± 14.7 ab201.7 ± 15.4 ab193.9 ± 20.4 ab237.6 ± 20.5 b
Regiolone162.3 ± 11.8 a131.7 ± 11.7 a153.8 ± 9.1 a128.1 ± 9.2 a245.8 ± 12.3 b326.0 ± 22.0 c
5-Hydroxy-2,3-dihydro-1,4-naphthalenedione79.1 ± 11.6 ab120.1 ± 6.5 ab126.0 ± 19.6 b70.5 ± 9.5 a91.5 ± 9.8 ab103.9 ± 4.2 ab
4,5,8-Trihydroxynaphthalene-5-D-glucopyranoside459.8 ± 15.0 ab337.8 ± 30.3 a418.8 ± 28.0 ab672.3 ± 25.4 c529.1 ± 26.3 b392.3 ± 35.4 a
1,4,8-Trihydroxynaphthalene-1-D-glucopyranoside87.4 ± 8.4 a100.6 ± 14.3 ab119.0 ± 15.2 ab104.3 ± 19.1 ab161.6 ± 14.7 b148.5 ± 8.3 ab
Dihydroxytetralone hexoside58.9 ± 9.0 a63.7 ± 7.4 a79.5 ± 10.2 a70.5 ± 4.4 a57.9 ± 3.6 a115.3 ± 7.2 b
Trihydroxytetralone galloyl hexoside68.6 ± 8.7 a95.3 ± 15.5 a86.5 ± 6.0 a73.4 ± 8.6 a90.2 ± 9.7 a106.7 ± 12.7 a
Hydroxycinnamic acids
Neochlorogenic acid (3-caffeoylquinic acid)12.2 ± 0.7 a17.5 ± 1.7 a16.4 ± 2.1 a14.3 ± 1.1 a16.1 ± 1.4 a18.0 ± 1.3 a
3-p-Coumaroylquinic acid66.5 ± 3.6 ab101.7 ± 5.5 c104.9 ± 4.0 c70.1 ± 3.0 ab60.4 ± 2.3 a82.1 ± 2.9 b
p-Coumaric acid derivative 24.5 ± 0.5 ab4.0 ± 0.3 ab3.8 ± 0.4 a6.7 ± 1.0 ac7.8 ± 0.8 c6.9 ± 0.6 bc
p-Coumaric acid derivative 39.1 ± 0.6 b6.2 ± 0.3 ab7.2 ± 0.7 ab6.0 ± 0.2 a14.2 ± 1.0 c15.3 ± 0.8 c
p-Coumaric acid derivative 44.9 ± 0.6 a5.0 ± 0.3 a5.3 ± 0.5 a5.4 ± 0.4 a6.1 ± 0.3 a6.5 ± 0.4 a
Caffeic acid derivative 42.5 ± 0.9 a3.2 ± 0.2 ab3.1 ± 0.4 ab7.7 ± 0.8 c6.9 ± 1.4 bc9.7 ± 1.0 c
Ferulic acid derivative 215.3 ± 1.0 b20.2 ± 1.3 c21.8 ± 1.6 c15.1 ± 0.5 b9.9 ± 0.7 a15.1 ± 0.6 b
Ferulic acid derivative 36.3 ± 0.2 a8.1 ± 0.8 ab11.8 ± 1.0 bc8.7 ± 1.1 ab13.6 ± 1.0 c14.8 ± 1.2 c
Hydroxybenzoic acids
Gallic acid derivative 3142.2 ± 14.1 a85.9 ± 10.6 a132.2 ± 3.5 a244.6 ± 18.6 b331.3 ± 16.1 c366.9 ± 11.8 c
Gallic acid derivative 416.7 ± 2.7 ab17.6 ± 2.7 ab23.6 ± 4.2 ab13.0 ± 1.7 a16.5 ± 2.6 ab25.4 ± 1.7 b
Gallic acid derivative 512.9 ± 2.4 a13.6 ± 2.2 a14.1 ± 2.8 a15.6 ± 1.6 a25.7 ± 2.0 b17.5 ± 1.3 ab
Flavanols
(+)Catechin53.7 ± 2.9 ab82.2 ± 4.4 c84.8 ± 3.2 c56.7 ± 2.4 ab48.9 ± 1.8 a66.4 ± 2.4 b
(−)Epicatechin69.4 ± 5.0 a56.3 ± 5.0 a65.7 ± 3.9 a54.8 ± 4.0 a105.0 ± 5.2 b139.3 ± 9.4 c
(epi)Catechin derivative 550.6 ± 8.9 a56.9 ± 8.2 ab68.8 ± 3.3 ac79.4 ± 4.7 bc88.0 ± 0.8 c80.9 ± 1.6 bc
Flavones
Santin14.1 ± 1.6 a13.3 ± 1.9 a15.0 ± 1.9 a35.6 ± 4.8 b49.4 ± 1.1 c46.2 ± 3.7 bc
5,7-Dihydroxy-3,4-dimetoxyflavone27.1 ± 0.4 bc12.5 ± 1.8 a16.2 ± 2.8 ab20.3 ± 2.0 ab40.5 ± 4.4 c39.8 ± 4.4 c
Flavonols
Quercetin-3-galactoside33.6 ± 1.8 ab39.8 ± 2.5 ab34.7 ± 2.1 ab42.3 ± 3.1 b35.8 ± 1.8 ab31.4 ± 0.6 a
Quercetin-3-rhamnoside12.7 ± 3.1 a22.7 ± 3.5 a20.6 ± 1.5 a16.8 ± 2.4 a17.4 ± 0.4 a16.3 ± 1.2 a
Total naphthoquinones2946.7 ± 116.4 a3026.2 ± 133.4 a3202.6 ± 142.9 a4454.6 ± 235.0 b4670.0 ± 74.7 b5088.8 ± 156.9 b
Total hydroxycinnamic acids121.2 ± 4.3 a166.0 ± 7.7 b174.3 ± 8.8 b134.0 ± 4.4 a135.0 ± 6.3 a168.4 ± 5.0 b
Total hydroxybenzoic acids171.8 ± 17.9 a117.1 ± 14.0 a169.9 ± 9.4 a273.2 ± 20.0 b373.5 ± 17.3 c409.8 ± 13.4 c
Total flavanols173.7 ± 11.3 a195.5 ± 15.3 a219.4 ± 8.7 ab190.9 ± 8.3 a241.9 ± 7.2 bc286.6 ± 8.9 c
Total flavones41.1 ± 1.8 ab25.7 ± 3.2 a31.2 ± 4.0 a55.9 ± 6.7 b89.9 ± 5.0 c86.0 ± 7.3 c
Total flavanonesndndndndNdnd
Total flavonols46.3 ± 4.1 a62.4 ± 5.2 a55.2 ± 3.4 a59.1 ± 4.8 a53.2 ± 2.0 a47.8 ± 1.7 a
Total phenolics content (summation; relevant standards) x3500.8 ± 137.3 a3592.8 ± 155.3 a3852.7 ± 173.1 a5167.7 ± 275.4 b5563.5 ± 81.0 bc6087.3 ± 179.6 c
Total phenolics content (total extracts; mg gallic acid equivalents/100 g dry weight) y1156.6 ± 51.4 a1532.7 ± 105.6 b1398.9 ± 80.2 ab1241.2 ± 56.6 ab1155.2 ± 22.9 a1398.5 ± 102.8 ab
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.
Table 6. Individual phenolics for the walnut buds across the six selected cultivars.
Table 6. Individual phenolics for the walnut buds across the six selected cultivars.
CompoundBud Phenolic Content per Cultivar (mg/100 g Dry Weight)
‘Fernor’‘Fernette’‘Franquette’‘Sava’‘Krka’‘Rubina’
Naphthoquinones
Juglone 573.8 ± 30.3 c407.5 ± 24.1 ab373.6 ± 9.9 a392.3 ± 5.9 a466.0 ± 18.2 ab508.8 ± 29.1 bc
Hydrojuglone149.4 ± 6.7 c100.4 ± 2.9 ab113.4 ± 10.7 bc70.7 ± 11.8 a88.0 ± 7.0 ab111.6 ± 9.0 bc
Hydrojuglone β-D-glucopyranoside2744.4 ± 58.1 c1636.6 ± 11.4 a3619.9 ± 100.3 d2326.5 ± 25.5 b2148.8 ± 37.8 b1688.2 ± 6.4 a
Hydrojuglone rutinoside314.7 ± 14.4 c191.1 ± 4.0 a283.0 ± 13.9 c189.4 ± 11.9 a261.1 ± 13.1 bc222.5 ± 6.7 ab
Hydrojuglone dihexoside509.0 ± 17.0 b404.3 ± 14.8 a683.1 ± 20.3 d606.5 ± 12.0 c454.7 ± 13.7 ab444.2 ± 14.0 ab
Hydrojuglone derivative 1642.3 ± 27.3 b348.1 ± 18.7 a951.0 ± 27.1 c432.1 ± 15.4a440.7 ± 8.5 a432.5 ± 16.1 a
Hydrojuglone derivative 2109.1 ± 6.2 a57.1 ± 3.6 a868.7 ± 14.7 e270.9 ± 7.2 b526.8 ± 17.8 c594.1 ± 12.6 d
Hydrojuglone derivative 3301.2 ± 11.5 c165.3 ± 8.6 a260.3 ± 17.5 bc161.0 ± 8.4 a180.7 ± 11.3 a216.1 ± 10.8 ab
Hydrojuglone derivative pentoside 11038.7 ± 18.2 d594.0 ± 18.9 a1995.1 ± 30.9 e973.0 ± 11.6 cd824.0 ± 17.6 b916.5 ± 27.1 bc
Hydrojuglone derivative pentoside 23855.8 ± 86.1 d2138.6 ± 29.4 ab3021.7 ± 34.9 c3166.0 ± 50.0 c2370.3 ± 46.4 b1940.6 ± 34.8 a
Hydrojuglone derivative rhamnoside1525.3 ± 31.7 c861.5 ± 18.3 a2429.3 ± 36.9 e1297.8 ± 33.8 b1933.2 ± 29.1 d1646.5 ± 14.1 c
Hydrojuglone pentose galloyl derivative433.9 ± 25.8 c234.5 ± 5.6 a525.5 ± 14.7 d268.0 ± 11.9 ab288.8 ± 24.0 ab341.7 ± 12.8 b
Dihydroxytetralone hexoside285.0 ± 7.8 c194.1 ± 13.7 a382.4 ± 13.1 d281.8 ± 9.0 bc219.5 ± 14.6 ab257.2 ± 20.0 ac
Hydroxycinnamic acids
Neochlorogenic acid (3-caffeoylquinic acid)74.2 ± 0.9 c 29.2 ± 2.6 a99.4 ± 6.8 d58.3 ± 3.1 bc62.0 ± 3.9 bc51.2 ± 4.3 b
3-p-Coumaoylquinic acid323.1 ± 5.2 c165.9 ± 4.7 a395.3 ± 10.4 d273.4 ± 4.6 b257.5 ± 2.2 b283.3 ± 4.3 b
p-Coumaric acid derivative 170.8 ± 0.9 c45.7 ± 0.8 a100.3 ± 1.5 d62.1 ± 0.9 b65.8 ± 0.6 b42.1 ± 0.4 a
Caffeic acid hexoside derivative20.5 ± 0.7 a18.5 ± 0.6 a48.5 ± 2.2 b22.2 ± 1.8 a18.9 ± 0.7 a23.0 ± 0.6 a
Caffeic acid derivative 19.0 ± 0.7 b6.5 ± 0.3 a12.0 ± 0.4 c4.9 ± 0.6 a6.8 ± 0.5 ab6.8 ± 0.4 ab
Diferuoyl hexoside8.4 ± 0.5 c5.6 ± 0.3 b8.0 ± 0.5 c2.8 ± 0.1 a7.1 ± 0.4 bc7.3 ± 0.3 bc
Hydroxybenzoic acids
Gallic acid derivative 155.2 ± 1.5 b37.4 ± 1.5 a60.7 ± 1.0 b41.9 ± 1.5 a42.3 ± 1.2 a42.2 ± 1.1 a
Gallic acid derivative 2705.9 ± 4.3 b369.8 ± 157.6 a1209.4 ± 26.3 c811.7 ± 22.3 b655.2 ± 6.3 ab685.1 ± 7.2 b
Gallic acid derivative 3541.6 ± 6.7 d305.1 ± 6.7 a424.2 ± 9.9 b485.5 ± 6.1 c520.6 ± 7.1 cd416.0 ± 9.5 b
Gallic acid methyl ester90.9 ± 1.6 b62.1 ± 5.7 a188.9 ± 9.5 c92.1 ± 4.5 b92.1 ± 4.2 b95.7 ± 2.3 b
bis-HHDP-glucose70.7 ± 3.7 bc46.1 ± 2.0 a80.8 ± 4.9 c72.9 ± 2.3 bc41.8 ± 1.3 a63.6 ± 0.7 b
Ellagic acid derivative 451.4 ± 3.9 d226.0 ± 13.5 a942.8 ± 17.0 e366.7 ± 7.1 c276.5 ± 3.6 b333.1 ± 10.4 c
Flavanols
Procyanidin dimer 1303.8 ± 18.4 b195.4 ± 21.8 a256.1 ± 11.5 ab289.7 ± 3.5 b275.9 ± 5.8 b267.1 ± 10.6 b
Procyanidin dimer 2494.3 ± 9.8 cd350.9 ± 25.4 a527.6 ± 16.2 d507.1 ± 17.2 cd378.9 ± 19.6 ab433.9 ± 7.9 bc
Procyanidin dimer derivative 1655.6 ± 24.0 c293.9 ± 10.1 a859.0 ± 40.5 d604.1 ± 11.7 c589.6 ± 2.7 c452,4 ± 10.8 b
Procyanidin dimer derivative 2431.7 ± 13.9 b261.6 ± 3.4 a823.9 ± 18.2 d445.2 ± 15.1 b413.3 ± 7.6 b630.5 ± 22.8 c
Procyanidin dimer derivative 3122.4 ± 7.3 c88.0 ± 5.6 ab193.8 ± 8.3 d72.4 ± 3.7 a116.1 ± 3.6 bc116.6 ± 7.9 bc
(+)Catechin838.6 ± 34.4 c469.0 ± 6.6 a1218.0 ± 25.9 d666.2 ± 22.9 b664.8 ± 16.2 b744.9 ± 26.5 bc
(−)Epicatechin266.6 ± 6.0 c178.3 ± 4.2 a347.4 ± 7.0 d198.5 ± 5.2 a238.5 ± 8.1 b192.8 ± 1.2 a
(epi)Catechin derivative 1210.9 ± 2.7 b160.5 ± 5.0 a273.1 ± 4.8 c205.8 ± 1.9 b205.5 ± 5.3 b209.8 ± 2.5 b
(epi)Catechin derivative 2267.3 ± 7.1 c185.1 ± 15.7 a384.7 ± 10.0 d269.3 ± 13.2 c256.4 ± 13.4 bc200.6 ± 19.6 ab
(epi)Catechin derivative 3435.9 ± 9.9 c291.5 ± 6.8 a568.0 ± 11.4 d324.5 ± 8.5 a390.0 ± 13.3 b315.3 ± 2.0 a
(epi)Catechin derivative 494.7 ± 4.1 a80.4 ± 4.5 a155.5 ± 4.4 b91.3 ± 3.2 a82.3 ± 3.9 a92.8 ± 2.9 a
Galloyl-3-(epi)catechin850.0 ± 9.3 d461.8 ± 15.3 a1396.0 ± 23.6 e644.6 ± 20.7 b724.0 ± 20.0 bc797.5 ± 6.3 cd
Flavones
Santin45.9 ± 2.3 c35.3 ± 1.7 ab26.6 ± 2.3 a32.2 ± 2.0 ab28.3 ± 1.6 ab36.8 ± 1.6 bc
5,7-Dihydroxy-3,4-dimetoxyflavone45.3 ± 2.3 c38.6 ± 2.6 bc28.5 ± 1.8 a29.2 ± 2.5 ab35.9 ± 1.7 ac58.4 ± 1.0 d
Flavanones
Naringenin83.5 ± 3.5 ab65.7 ± 4.7 a83.9 ± 4.7 ab73.9 ± 2.7 a70.3 ± 4.8 a98.4 ± 3.3 b
Flavonols
Myricetin galactoside259.0 ± 1.4 d140.2 ± 3.9 b214.0 ± 8.0 c141.4 ± 3.2 b131.5 ± 5.1 b107.7 ± 3.0 a
Myricetin pentoside105.4 ± 4.8 b54.4 ± 2.1 a242.3 ± 8.5 c92.4 ± 4.7 b95.6 ± 4.1 b104.8 ± 6.0 b
Myricetin-3-rhamnoside533.3 ± 12.2 c356.6 ± 2.1 a770.6 ± 14.9 d553.9 ± 9.9 c452.2 ± 12.6 b365.0 ± 7.7 a
Quercetin-3-galactoside227.5 ± 2.5 d123.6 ± 4.1 a373.6 ± 6.3 e172.5 ± 5.5 b193.8 ± 5.4 bc213.4 ± 1.7 cd
Quercetin-3-glucoside144.0 ± 4.3 bc113.9 ± 4.6 a262.0 ± 6.7 d147.9 ± 6.5 bc158.4 ± 4.1 c133.6 ± 3.4 ab
Quercetin-3-arabinopyranoside293.4 ± 3.6 b206.9 ± 3.1 a495.9 ± 9.8 c287.9 ± 6.5 b308.9 ± 2.5 b282.5 ± 5.3 b
Quercetin-3-arabinofuranoside250.8 ± 12.6 a207.2 ± 2.8 a613.0 ± 19.3 c244.3 ± 8.9 a335.3 ± 6.0 b320.2 ± 5.8 b
Quercetin-3-rhamnoside399.1 ± 7.6 bc332.5 ± 4.9 a745.4 ± 25.7 d371.0 ± 7.2 ab440.7 ± 8.5 c424.7 ± 7.6 bc
Quercetin galoyll hexoside130.8 ± 4.4 c90.5 ± 2.3 a177.5 ± 4.6 d143.7 ± 1.6 c98.4 ± 2.9 ab107.2 ± 2.5 b
Quercetin hexoside derivative 154.5 ± 2.8 c34.4 ± 1.6 ab68.6 ± 4.3 d27.2 ± 1.4 a50.6 ± 1.2 c41.8 ± 3.4 bc
Quercetin hexoside derivative 236.3 ± 1.0 bc30.4 ± 1.6 b40.2 ± 0.9 c19.6 ± 1.3 a30.7 ± 1.5 b31.5 ± 1.5 b
Quercetin29.0 ± 1.1 c21.4 ± 0.9 b29.6 ± 1.4 c15.6 ± 1.3 a21.3 ± 0.8 b26.8 ± 1.3 bc
Kaempferol pentoside 139.1 ± 1.0 b22.9 ± 1.9 a76.6 ± 4.9 c26.7 ± 0.9 a29.5 ± 2.1 ab40.2 ± 1.4 b
Kaempferol pentoside 268.8 ± 2.9 b43.1 ± 0.9 a114.6 ± 4.7 c61.0 ± 3.0 b56.5 ± 2.3 ab53.4 ± 4.4 ab
Kaempferol pentoside 336.2 ± 1.6 c17.9 ± 0.4 a66.5 ± 2.2 d28.4 ± 1.7 bc29.1 ± 2.0 bc27.8 ± 1.9 b
Kaempferol rhamnoside53.0 ± 1.4 bc37.5 ± 3.9 a83.4 ± 3.6 d45.0 ± 4.2 ab46.1 ± 2.5 ab60.9 ± 2.0 c
Kaempferol21.3 ± 1.2 a20.8 ± 1.6 a18.5 ± 1.7 a20.6 ± 1.7 a20.9 ± 1.5 a25.1 ± 1.4 a
Total naphthoquinones12482.7 ± 126.9 d7333.0 ± 102.7 a15507.1 ± 122.8 e10435.9 ± 42.1 c10202.6 ± 31.8 c9320.5 ± 91.8 b
Total hydroxycinnamic acids506.1 ± 4.9 c271.2 ± 7.9 a663.4 ± 17.7 d423.7 ± 10.1 b418.1 ± 2.0 b413.7 ± 3.2 b
Total hydroxybenzoic acids1915.8 ± 2.1 b1046.6 ± 156.6 a2906.8 ± 46.2 c1870.8 ± 21.0 b1628.5 ± 20.1 b1635.7 ± 10.9 b
Total flavanols4971.9 ± 61.0 c3016.5 ± 43.1 a7003.2 ± 129.6 d4318.6 ± 9.9 b4335.3 ± 59.0 b4454.1 ± 32.6 b
Total flavones91.3 ± 4.5 c73.9 ± 4.0 b55.1 ± 4.0 a61.4 ± 0.6 ab64.2 ± 1.2 ab95.3 ± 0.7 c
Total flavanones83.5 ± 3.5 ab65.7 ± 4.7 a83.9 ± 4.7 ab73.9 ± 2.7 a70.3 ± 4.8 a98.4 ± 3.3 b
Total flavonols2681.4 ± 7.6 d1854.1 ± 16.2 a4392.3 ± 42.3 e2399.0 ± 19.6 bc2499.3 ± 18.8 c2366.7 ± 13.8 b
Total phenolics content (summation; relevant standards) x22732.6 ± 189.8 d13661.1 ± 283.3 a30611.8 ± 130.3 e19583.4 ± 74.3 c19218.3 ± 21.1 c18384.4 ± 142.3 b
Total phenolics content (total extracts; mg gallic acid equivalents/100 g dry weight) y7236.4 ± 188.1 c4270.2 ± 144.4 a8232.8 ± 57.7 d5199.6 ± 166.3 b5017.7 ± 220.7 ab4853.8 ± 259.2 ab
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.
Table 7. Individual phenolics for the walnut bark across the six selected cultivars.
Table 7. Individual phenolics for the walnut bark across the six selected cultivars.
CompoundBark Phenolic Content per Cultivar (mg/100 g Dry Weight)
‘Fernor’‘Fernette’‘Franquette’‘Sava’‘Krka’‘Rubina’
Naphthoquinones
Juglone 251.0 ± 31.2 ac185.8 ± 8.6 ab281.2 ± 16.6 bc169.4 ± 10.2 a219.4 ± 24.1 ac299.8 ± 35.6 c
Hydrojuglone72.8 ± 2.2 bc89.9 ± 9.6 c50.7 ± 7.2 ab65.9 ± 5.5 bc21.3 ± 2.4 a66.9 ± 9.8 bc
Hydrojuglone β-D-glucopyranoside239.8 ± 13.0 a366.1 ± 18.7 b342.3 ± 17.8 ab285.5 ± 26.2 ab255.1 ± 11.9 ab523.4 ± 52.7 c
Hydrojuglone rutinoside47.6 ± 4.8 ab30.9 ± 3.1 a40.6 ± 5.5 a34.9 ± 2.8 a45.1 ± 3.8 ab60.9 ± 2.1 b
Hydrojuglone derivative 216.6 ± 5.1 a10.5 ± 4.7 a156.3 ± 21.1 b32.0 ± 9.2 a143.9 ± 9.4 b143.3 ± 22.0 b
Hydrojuglone derivative 41258.0 ± 73.5 c877.8 ± 53.9 b639.1 ± 83.0 ab471.6 ± 35.5 a417.4 ± 17.0 a535.2 ± 78.9 a
Hydrojuglone derivative pentoside 1566.3 ± 25.2 b757.7 ± 34.4 c401.4 ± 14.1 a699.1 ± 35.5 bc363.2 ± 21.9 a394.7 ± 42.7 a
Hydrojuglone derivative pentoside 26579.7 ± 402.1 b7608.0 ± 301.1 b4768.3 ± 337.3 a7189.0 ± 266.6 b4672.8 ± 174.5 a4954.1 ± 494.4 a
Hydrojuglone derivative pentoside 3605.9 ± 24.3 bc743.9 ± 32.8 c487.3 ± 24.5 ab681.9 ± 36.8 c427.7 ± 16.0 a460.3 ± 49.8 ab
Hydrojuglone derivative rhamnoside2015.2 ± 110.6 a2332.2 ± 103.3 a2172.8 ± 157.9 a2039.4 ± 75.6 a2222.2 ± 123.3 a2230.2 ± 193.9 a
Dihydroxytetralone hexoside43.4 ± 3.0 a54.0 ± 4.7 a48.8 ± 2.8 a47.2 ± 6.8 a37.7 ± 5.7 a49.6 ± 3.5 a
Hydroxybenzoic acids
Gallic acid derivative 280.0 ± 5.3 ab89.9 ± 5.3 ab106.7 ± 4.9 b92.5 ± 7.0 ab71.8 ± 3.5 a93.0 ± 8.9 ab
Gallic acid derivative 329.7 ± 3.1 bc31.1 ± 1.5 c15.6 ± 0.8 a35.2 ± 3.6 cd16.8 ± 2.3 ab44.1 ± 4.3 d
Ellagic acid derivative145.6 ± 12.6 b153.9 ± 8.9 b155.8 ± 9.7 b93.2 ± 9.4 a111.0 ± 7.1 ab123.0 ± 13.6 ab
Flavanols
Procyanidin dimer 2219.8 ± 18.8 a277.1 ± 20.2 a301.3 ± 25.6 a335.3 ± 24.5 a288.7 ± 14.6 a333.2 ± 45.3 a
Procyanidin dimer derivative 2200.9 ± 10.5 ab248.2 ± 21.8 b213.0 ± 17.1 ab245.4 ± 17.8 b149.8 ± 7.8 a178.4 ± 25.6 ab
(+)Catechin528.2 ± 56.6 a557.9 ± 40.9 a675.5 ± 42.0 a708.7 ± 49.5 a748.8 ± 53.0 a797.2 ± 103.9 a
Flavones
Santin14.4 ± 2.0 b6.3 ± 0.4 a7.7 ± 0.6 a10.6 ± 2.2 ab6.6 ± 0.3 a7.8 ± 0.1 a
5,7-Dihydroxy-3,4-dimetoxyflavone2.2 ± 0.6 ab0.9 ± 0.1 a4.3 ± 0.3 b4.4 ± 0.9 b2.4 ± 0.2 ab3.6 ± 0.5 b
Flavonols
Myricetin pentoside21.4 ± 1.3 a26.9 ± 1.5 a29.4 ± 1.0 a29.1 ± 2.7 a23.1 ± 3.2 a23.1 ± 3.2 a
Myricetin-3-rhamnoside129.4 ± 9.0 a156.3 ± 8.2 a162.7 ± 3.9 a152.1 ± 12.5 a121.0 ± 6.8 a133.4 ± 12.9 a
Quercetin-3-galactoside210.4 ± 10.7 a261.9 ± 19.1 ab248.4 ± 9.7 ab322.1 ± 28.4 b224.9 ± 6.5 a228.0 ± 25.0 a
Quercetin-3-glucoside141.0 ± 6.2 ab159.0 ± 5.1 ab124.3 ± 11.1 ab165.4 ± 9.2 b115.7 ± 4.2 a133.3 ± 17.6 ab
Quercetin-3-arabinopyranoside69.0 ± 2.3 a93.5 ± 3.6 ab91.5 ± 6.1 ab100.1 ± 8.4 b74.1 ± 1.8 ab78.1 ± 9.7 ab
Quercetin-3-arabinofuranoside69.6 ± 1.9 ab100.4 ± 7.4 bc96.7 ± 7.9 ac123.4 ± 6.7 c64.9 ± 2.4 a92.4 ± 14.0 ac
Quercetin-3-rhamnoside223.8 ± 11.5 ab320.1 ± 13.3 bc255.5 ± 33.4 ab373.9 ± 19.0 c171.7 ± 5.5 a315.1 ± 47.4 bc
Quercetin galoyll hexoside82.7 ± 5.8 ab97.3 ± 4.9 ab82.6 ± 7.0 ab101.6 ± 6.5 b69.5 ± 3.0 a76.9 ± 10.0 ab
Kaempferol-7-hexoside 1150.5 ± 9.7 bc237.1 ± 8.6 d113.0 ± 18.3 ab195.3 ± 11.7 cd63.5 ± 6.3 a107.7 ± 23.5 ab
Kaempferol-7-hexoside 230.2 ± 1.3 b42.2 ± 1.5 c32.2 ± 3.6 bc26.6 ± 2.4 b14.8 ± 2.3 a34.1 ± 2.7 bc
Total naphthoquinones11696.3 ± 654.3 bc13056.9 ± 527.9 c9388.8 ± 596.2 ab11716.0 ± 488.3 bc8825.9 ± 339.7 a9718.4 ± 919.8 ab
Total hydroxycinnamic acidsndndndndNdnd
Total hydroxybenzoic acids255.3 ± 15.9 a274.9 ± 15.4 a278.2 ± 14.1 a220.8 ± 19.8 a199.6 ± 11.4 a260.2 ± 25.9 a
Total flavanols948.9 ± 84.2 a1083.3 ± 78.6 a1189.8 ± 81.3 a1289.3 ± 88.6 a1187.3 ± 72.3 a1308.7 ± 174.5 a
Total flavones16.6 ± 2.0 c7.2 ± 0.4 a12.0 ± 0.8 ac15.0 ± 2.9 bc9.0 ± 0.5 ab11.4 ± 0.6 ac
Total flavanonesndndndndNdnd
Total flavonols1128.0 ± 17.3 ab1494.5 ± 63.9 bc1236.2 ± 97.9 ac1589.5 ± 89.0 c943.2 ± 16.5 a1222.2 ± 160.8 ac
Total phenolics content (summation; relevant stadnards) x14045.2 ± 757.1 ac15916.8 ± 676.0 c12104.9 ± 774.8 ab14830.6 ± 673.2 bc11165.1 ± 422.7 a12520.9 ± 1270.9 ac
Total phenolics content (total extracts; mg gallic acid equivalents/100 g dry weight) y1956.2 ± 77.6 a2236.4 ± 251.1 a2544.4 ± 251.3 a1898.2 ± 121.7 a1979.3 ± 38.7 a2740.0 ± 440.6 a
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.
Table 8. Overview of the 83 phenolics identified for the walnut inner and outer husks, buds and bark.
Table 8. Overview of the 83 phenolics identified for the walnut inner and outer husks, buds and bark.
PhenolicHuskBudsBark
InnerOuter
Naphthoquinones
1,4-Naphthoquinone++
Juglone ++++
Hydrojuglone++++
Hydrojuglone β-D-glucopyranoside ++++
Hydrojuglone rutinoside++++
Hydrojuglone dihexoside +
Hydrojuglone derivative 1 +
Hydrojuglone derivative 2 ++
Hydrojuglone derivative 3 +
Hydrojuglone derivative 4 +
Hydrojuglone derivative 5++
Hydrojuglone derivative pentoside 1 ++
Hydrojuglone derivative pentoside 2++++
Hydrojuglone derivative pentoside 3 +
Hydrojuglone derivative rhamnoside++++
Hydrojuglone pentose galloyl derivative +
Hydrojuglone hexoside derivative++
bis-Juglone++
p-Hydroxymetoxybenzobijuglone++
Regiolone++
5-Hydroxy-2,3-dihydro-1,4-naphthalenedione +
4,5,8-Trihydroxynaphthalene-5-D-glucopyranoside++
1,4,8-Trihydroxynaphthalene-1-D-glucopyranoside++
Dihydroxytetralone hexoside++++
Dihydroxytetralone galloyl hexoside++
Hydroxycinnamic acids
Neochlorogenic acid (3-caffeoylquinic acid)+++
3-p-Coumaroylquinic acid+++
p-Coumaric acid derivative 1 +
p-Coumaric acid derivative 2++
p-Coumaric acid derivative 3++
p-Coumaric acid derivative 4++
Caffeic acid hexoside derivative +
Caffeic acid derivative 1 +
Caffeic acid derivative 2+
Caffeic acid derivative 3+
Caffeic acid derivative 4++
Diferuoyl hexoside +
Ferulic acid derivative 1+
Ferulic acid derivative 2++
Ferulic acid derivative 3++
Hydroxybenzoic acids
Gallic acid derivative 1 +
Gallic acid derivative 2 ++
Gallic acid derivative 3++++
Gallic acid derivative 4++
Gallic acid derivative 5++
Gallic acid methyl ester +
bis-HHDP-glucose +
Ellagic acid derivative ++
Flavanols
Procyanidin dimer 1 +
Procyanidin dimer 2 ++
Procyanidin dimer derivative 1 +
Procyanidin dimer derivative 2 ++
Procyanidin dimer derivative 3 +
(+)Catechin++++
(−)Epicatechin+++
(epi)Catechin derivative 1 +
(epi)Catechin derivative 2 +
(epi)Catechin derivative 3 +
(epi)Catechin derivative 4 +
(epi)Catechin derivative 5 +
Galloyl-3-(epi)catechin +
Flavones
Santin++++
5,7-Dihydroxy-3,4-dimetoxyflavone++++
Flavanones
Naringenin +
Flavonols
Myricetin galactoside +
Myricetin pentoside ++
Myricetin-3-rhamnoside ++
Quercetin-3-galactoside++++
Quercetin-3-glucoside ++
Quercetin-3-arabinopyranoside ++
Quercetin-3-arabinofuranoside ++
Quercetin-3-rhamnoside++++
Quercetin galoyll hexoside ++
Quercetin hexoside derivative 1 +
Quercetin hexoside derivative 2 +
Quercetin +
Kaempferol pentoside 1 +
Kaempferol pentoside 2 +
Kaempferol pentoside 3 +
Kaempferol rhamnoside +
Kaempferol-7-hexoside 1 +
Kaempferol-7-hexoside 2 +
Kaempferol +
+, phenolic identified.
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Medic, A.; Jakopic, J.; Solar, A.; Hudina, M.; Veberic, R. Walnut (J. regia) Agro-Residues as a Rich Source of Phenolic Compounds. Biology 2021, 10, 535. https://doi.org/10.3390/biology10060535

AMA Style

Medic A, Jakopic J, Solar A, Hudina M, Veberic R. Walnut (J. regia) Agro-Residues as a Rich Source of Phenolic Compounds. Biology. 2021; 10(6):535. https://doi.org/10.3390/biology10060535

Chicago/Turabian Style

Medic, Aljaz, Jerneja Jakopic, Anita Solar, Metka Hudina, and Robert Veberic. 2021. "Walnut (J. regia) Agro-Residues as a Rich Source of Phenolic Compounds" Biology 10, no. 6: 535. https://doi.org/10.3390/biology10060535

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