Identification and Quantification of the Major Phenolic Constituents in Castanea sativa and Commercial Interspecific Hybrids (C. sativa x C. crenata) Chestnuts Using HPLC-MS/MS.

Due to the lack of studies on chestnut metabolites, this study was conducted to identify and quantify the major phenolic constituents in chestnuts. Data were compared with the three most commonly grown interspecific hybrids of C. sativa and C. crenata ('Bouche de Betizac', 'Marsol', and 'Maraval') and three "native" accessions of C. sativa. High-performance liquid chromatography coupled with mass spectrometry was used to identify and quantify these compounds. Four dicarboxylic acid derivatives, five hydroxybenzoic acids, nine hydroxycinnamic acids, and three flavanols were identified and quantified, most of them for the first time. Hydroxybenzoic acids were the major phenolic compounds in all chestnut cultivars/accessions, followed by flavanols, dicarboxylic acid derivatives, and hydroxycinnamic acids. Of all the compounds studied, the (epi)catechin dimer was the most abundant in chestnut. The assumption that cultivars from commercial hybrids have a better and different metabolic profile than "native" accessions was refuted.

Novel roles for the polyphenol oxidase enzyme in secondary metabolism and the regulation of cell death in walnut.

The enzyme polyphenol oxidase (PPO) catalyzes the oxidation of phenolic compounds into highly reactive quinones. Polymerization of PPO-derived quinones causes the postharvest browning of cut or bruised fruit, but the native physiological functions of PPOs in undamaged, intact plant cells are not well understood. Walnut (Juglans regia) produces a rich array of phenolic compounds and possesses a single PPO enzyme, rendering it an ideal model to study PPO. We generated a series of PPO-silenced transgenic walnut lines that display less than 5% of wild-type PPO activity. Strikingly, the PPO-silenced plants developed spontaneous necrotic lesions on their leaves in the absence of pathogen challenge (i.e. a lesion mimic phenotype). To gain a clearer perspective on the potential functions of PPO and its possible connection to cell death, we compared the leaf transcriptomes and metabolomes of wild-type and PPO-silenced plants. Silencing of PPO caused major alterations in the metabolism of phenolic compounds and their derivatives (e.g. coumaric acid and catechin) and in the expression of phenylpropanoid pathway genes. Several observed metabolic changes point to a direct role for PPO in the metabolism of tyrosine and in the biosynthesis of the hydroxycoumarin esculetin in vivo. In addition, PPO-silenced plants displayed massive (9-fold) increases in the tyrosine-derived metabolite tyramine, whose exogenous application elicits cell death in walnut and several other plant species. Overall, these results suggest that PPO plays a novel and fundamental role in secondary metabolism and acts as an indirect regulator of cell death in walnut.

Effect of Different Walnut and Hazelnut Leaf Compost Treatments on Yield and Phenolic Composition of Lactuca sativa L.

The use of compost made from the leaves of Juglans regia has long been controversial because of its inhibitory effect due to the presence of juglone. Therefore, the aim of our study was to replicate the typical habits of farmers and gardeners, where the dried leaves are collected at the end of the season and placed in a composter. Then, the effects of the different treatments on the yield of the plant (lettuce), secondary metabolism, and possible toxicity of the compost of the grown plant were evaluated. The lowest yield of lettuce was obtained in soil with composted walnut and hazelnut leaves, while the highest yield was recorded in in soil with compost control, soil with composted walnut leaves and grass with the addition of composting agent and soil with composted walnut leaves with addition of composting agent. Some allelochemicals were still present in the compost but at such low levels that they did not affect yield. We suggest that dry walnut leaves and cut grass can be used for composting, while dry hazelnut leaves still contain some allelochemicals after two years that significantly inhibit plant growth and thus yield, so we would not recommend their use for composting.

The response of Corylus avellana L. phenology to rising temperature in north-eastern Slovenia.

Knowledge of plant-weather relationships can improve crop management, resulting in higher quality and more stable crop yields. The annual timing of spring phenophases in mid-latitudes is largely a response to temperature, and reflects the thermal conditions of previous months. The effect of air temperature on the variability of hazelnut (Corylus avellana L.) phenophases (leafing, flowering) was investigated. Meteorological and phenological data for five cultivars were analysed over the periods 1969-1979 (P1) and 1994-2007 (P2) in Maribor, Slovenia. Phenological data series were correlated strongly to the temperature of the preceding months (R(2): 0.64-0.98) and better correlated to daily maximum and mean temperatures than to daily minimum temperatures. About 75% of phenophases displayed a tendency towards earlier appearance and a shorter flowering duration during P2, which could be explained by the significant temperature changes (+0.3°C/decade) from December to April between 1969 and 2007. An increase in air temperature of 1°C caused an acceleration in leafing by 2.5-3.9 days, with flowering showing higher sensitivity since a 1°C increase promoted male flowering by 7.0-8.8 days and female flowering by 6.3-8.9 days. The average rate of phenological change per degree of warming (days earlier per +1°C) did not differ significantly between P1 and P2. An estimation of chilling accumulation under field conditions during 1993-2009, between 1 November and 28 February, showed that all four of these months contributed approximately similar amounts of accumulated chilling units. The growing degree days (GDD) to flowering were calculated by an estimated base temperature of 2°C and 1 January as a starting date, given the most accurate calculations. In general, thermal requirements were greater in P2 than in P1, although this difference was not significant. Longer-time series data extended to other agricultural and wild plants would be helpful in tracking possible future changes in phenological responses to local climate.

The Effects of the Cultivar and Environment on the Phenolic Contents of Hazelnut Kernels.

Different climatic conditions are known to affect the synthesis of primary and secondary metabolites. Therefore, the phenolic contents in new growing areas could affect the quality and flavor of hazelnuts. The aim of this study was to determine the variability of the phenolic contents of the kernels in different commercial hazelnut cultivars depending on their growing area. Five cultivars ('Tonda Gentile delle Langhe', 'Merveille de Bollwiller', 'Pauetet', 'Tonda di Giffoni', and 'Barcelona' (syn. 'Fertile de Coutard')) grown in different European collection orchards were included in the study. High-performance liquid chromatography coupled with mass spectrometry was used to identify and quantify the phenolic compounds. Thirteen phenols were identified in the hazelnut kernels, including 7 flavanols, 2 hydroxybenzoic acids, 3 flavonols, and one dihydrochalcone. Catechin and procyanidin dimers were the main phenolic compounds found in the hazelnut kernels. The highest contents of catechin and total flavanols were determined in cultivars cultivated in Spain and northern Italy, and the lowest in Slovenia and France. Flavanols were the major phenolic groups independent of the place of cultivation, as they accounted for more than 50% of all phenolic compounds identified. The flavanols were followed by hydroxybenzoic acids, flavonols, and dihydrochalcones. Higher contents of flavanols and flavonols were found in kernels from areas characterized by higher natural irradiation, which stimulates their accumulation. The contents of hydroxybenzoic acids correlated with altitude, which stimulated phenolic acid synthesis. A negative correlation was observed between the dihydrochalcone content and annual rainfall, probably due to hydric stress.

Characterisation of selected hazelnut cultivars: phenology, growing and yielding capacity, market quality and nutraceutical value.

BACKGROUND: Sixteen hazelnut cultivars growing in the continental climate of Slovenia were analysed over 15 years for their phenology, growth habit, yield potential, susceptibility to hazelnut weevil and the pomological traits and phenolic content of their nuts in order to obtain a complex value of these cultivars for growers, the confectionary industry and consumers. RESULTS: Blooming occurred over an interval of 10-23 days for female (pistillate) flowers and 11-22 days for male (staminate) flowers. Nocchione, Romai, Pauetet, ID and Daria were the most productive cultivars, with a nine-year cumulative yield ranging from 31.8 to 44.7 kg per plant. Pauetet, F. Coutard, Nocchione and Segorbe were less susceptible to unfavourable weather conditions during blooming and fertilisation, in terms of maintaining acceptable yields with limited blank production. Under integrated pest management, less than 2% of the nuts of Romai, Daria, TGDL and Nocchione were affected by hazelnut weevil, compared with an average of 5.5% for the other cultivars. Daria, Pauetet and T. Giffoni performed best with regard to kernel percentage and blanching ratio. The results suggest that raw kernels are a good source of the natural antioxidants gallic acid and epicatechin. CONCLUSION: The results of this study provide direction when choosing hazelnut cultivars for planting, consuming or processing. They can be applied not only in Slovenia and nearby countries but also in other parts of central and northern European countries with similar climates and growing conditions.

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

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.

Identification and quantification of the major phenolic constituents in Juglans regia L. peeled kernels and pellicles, using HPLC-MS/MS.

This study was designed to identify and quantify the major phenolic constituents in peeled kernel and pellicle of the walnut Juglans regia L. Data were compared across six different cultivars: 'Fernor', 'Fernette', 'Franquette', 'Sava', 'Krka' and 'Rubina'. High-performance liquid chromatography coupled with mass spectrometry was used to identify and quantify these compounds. Nineteen ellagitanins, 12 ellagic acid derivatives, 4 anthocyanins and 5 other phenols were identified in the pellicle of these walnuts, and 15 dicarboxylic acid derivatives and 1 phenol in the peeled walnut kernels. Thirteen previously unreported compounds were identified in the peeled walnut kernels, and 14 in the walnut pellicle. Hydrolysable tannins were the main phenolic compounds identified in the walnut kernels, and accounted for 31.0% to 35.1% of the total phenolics analysed. The highest intake of total phenolics content per walnut kernel can be obtained by consumption of 'Franquette' and 'Rubina', with the lowest for 'Krka'.

Changes in phenolic profiles of red-colored pellicle walnut and hazelnut kernel during ripening.

In studies of secondary metabolites in nuts, many constituents in the kernel remain unidentified due to a high content of phenolic compounds in the pellicle. In the present study, we focused on the investigation of the phenolic and dicarboxylic acid profiles of walnut and hazelnut pellicle-less kernels. High-performance liquid chromatography with diode array and mass spectrometric detection (HPLC-DAD-MSn) was used to carry out the determination of individual phenolics and dicarboxylic acids in brown and red-pellicle walnut and hazelnut. Results show that hexahydroxydiphenic acid (HHDP) di-galloyl hexose isomer, vanillic acid hexoside, quinic acid derivative and catechin are the main constituents of the phenolic profile of walnut, while galloylquinic derivative, caffeoyl hexoside and catechin are the main constituents of the hazelnut kernel. Even though both walnut and hazelnut kernels have a considerably lower content of phenolic compounds and dicarboxylic acids in comparison to the pellicles, when calculated as a percentage of the total mass, the kernel makes a significant contribution to the total phenolic content of the whole nut.

Red Walnut: Characterization of the Phenolic Profiles, Activities and Gene Expression of Selected Enzymes Related to the Phenylpropanoid Pathway in Pellicle during Walnut Development.

A rare walnut variant with a red seed coat (pellicle) was examined for alterations in its phenolic profile during development. The red-walnut (RW) pellicle was compared with two commonly colored walnut varieties: 'Lara' (brown) and 'Fernor' (light brown). Furthermore, the activities of selected enzymes of the phenylpropanoid- and flavonoid-related pathways and the relative expressions of the structural genes phenylalanine ammonia lyase ( PAL) and anthocyanidin synthase ( ANS) were examined in the pellicles of the three varieties. In the pellicles of the RWs, phenylalanine ammonia lyase (PAL) activity and related PAL expression was most pronounced in August, about one month before commercial maturity, suggesting a high synthesis rate of phenolic compounds at this development stage. The most pronounced differences between the red and light- and dark-brown varieties were the increased PAL activity, PAL expression, and ANS expression in RWs in August. The vibrant color of the RW pellicle is based on the presence of four derivatives of cyanidin- and delphinidin-hexosides.

Phenolic acids, syringaldehyde, and juglone in fruits of different cultivars of Juglans regia L.

Phenolic acids (chlorogenic, caffeic, p-coumaric, ferulic, sinapic, ellagic, and syringic acid) as well as syringaldehyde and juglone were identified in ripe fruits of 10 walnut cultivars: Adams, Cisco, Chandler, Franquette, Lara, Fernor, Fernette, Alsoszentivani 117 (A-117), Rasna, and Elit. Analyses were done using a high-performance liquid chromatograph equipped with a diode array detector. Significant differences in the contents of identified phenolics were observed among cultivars. Phenolics were determined separately in the kernel and in the thin skin of the walnut, termed the pellicle. Not only in the kernel but also in the pellicle did syringic acid, juglone, and ellagic acid predominate (average values of 33.83, 11.75, and 5.90 mg/100 g of kernel; and 1003.24, 317.90, and 128.98 mg/100 g of pellicle, respectively), and the contents of ferulic and sinapic acid (average values of 0.06 and 0.05 mg/100 g of kernel and 2.93 and 2.17 mg/100 g of pellicle, respectively) were the lowest in all cultivars. The highest differences in the sum of all identified phenolics were observed between Rasna and Fernette fruits; in Rasna there were >2-fold higher contents of identified phenolics in both kernel and pellicle. It was found that the walnut pellicle is the most important source of walnut phenolics. The ratio between the contents in pellicle and kernel varied by at least 14.8-fold for caffeic acid (cv. Adams) and by up to 752.0-fold for p-coumaric acid (cv. Elit).

HPLC-MSn identification and quantification of phenolic compounds in hazelnut kernels, oil and bagasse pellets.

The paper reports the phenolic content of kernels, bagasse pellets (residue of oil pressing) and oils from five different cultivars and a mix of cultivars. Phenolic compounds were identified by high-performance liquid chromatography coupled with mass spectrometry. Two compounds, glansreginin A and glansreginin B, were detected for the first time in hazelnuts. The main polyphenolic subclass comprised of mono- and oligomeric flavan 3-ols, which accounted between 34.2 and 58.3% in hazelnut kernels and between 36.7 and 48.6% in pellets of the total phenolics detected. In hazelnut oils four compounds have been detected, their content levels ranged from 0.97 to 0.01µgg-1. Total phenolic content ranged from 491.2 to 1700.4mgGAEkg-1 in kernels, from 848.4 to 1148.5mgGAEkg-1 in pellets and only from 0.14 to 0.25mgGAEg-1 in oils. The percentage of radical scavenging activity ranged from 60.0 to 96.4% for kernels, 63.0 to 73.2% for pellets and from 17.7 to 29.9% for oil. The study provides clear evidence on high phenolic contents and similarly high antioxidant potential of hazelnut kernels and bagasse pellets. The latter could be used as ingredients in cooking and baking or even for production of plant-based pharmaceuticals.

Roasting affects phenolic composition and antioxidative activity of hazelnuts (Corylus avellana L.).

The potential effect of skin removal and roasting on individual and total phenolic content, and on antioxidative potential of 6 hazelnut cultivars were investigated. HPLC-MS identification of individual phenolics confirmed the presence of 7 flavan-3-ols (catechin, epicatechin, 2 procyanidin dimers, and 3 procyanidin trimers), 3 flavonols (quercetin pentoside, quercetin-3-O-rhamnoside, and myricetin-3-O-rhamnoside), 2 hydrobenzoic acids (gallic acid, protocatechulic acid), and 1 dihydrochalcone (phloretin-2'-O-glucoside). Flavonols were only detected in whole hazelnut kernels. The content of individual phenolics, with the exception of gallic acid, was always highest in whole unroasted hazelnuts and was significantly reduced after skin removal. Similarly, total phenolic content and antioxidative potential decreased when skin was removed. Roasting had a significant negative effect on individual phenolics but not on the total phenolic content and antioxidative potential of kernels. From a health promoting phytochemical composition of hazelnuts the consumption of whole unroasted kernels with skins should be preferential to peeled kernels either roasted or unroasted. Practical Application: A significant reduction in the antioxidative potential and total phenolic content is detected after hazelnut skin removal but not after roasting, suggesting that hazelnut kernels should be consumed whole. In hazelnut skin, many phenolic compounds are located, which are not present in flesh and, therefore, the health properties of hazelnuts are strongly affected by skin removal. Thermal processing and roasting conditions used in this study had a lesser effect on the individual phenolic composition of the kernel and thus roasted and unroasted hazelnuts without skin contain comparable amounts of health promoting compounds.

Genotypic differences in branching pattern and fruiting habit in common walnut (Juglans regia L.).

Architectural analysis of 840 Slovenian walnut (Juglans regia L.) genotypes was performed to determine the most typical and frequent morphological types and to evaluate their vegetative and generative potential. Four branching and fruiting patterns (I-IV) were detected. A 3-year-old fruiting branch, consisting of a 3-year-old shoot plus corresponding 2-year-old and 1-year-old shoots, was used as a structural unit for quantitative analysis. In the intermediate fruit-bearing types with mesotonic and acrotonic branching pattern (types II and III), the total lengths of 3-, 2- and 1-year-old shoots were 385 and 380 cm, respectively, compared with 275 and 253 cm in the terminal and lateral-fruiting types (types I and IV). In type I, 1-year-old shoots had significantly fewer nodes than in other types. In addition, they had a thinner basal diameter than types III and IV, and their angles were the most erect (39 degrees ). Only 0.4 out of 3.6 1-year-old shoots were flowering with one mixed bud with 1.9 female flowers. In type IV, 2-year-old shoots had significantly more nodes and a larger basal diameter than other types. One-year-old shoots in type IV are thicker than those in other types. Ratios between the number of flowering and the total number of 1-year-old shoots were 0.7 in type IV, 0.6 in type III, 0.5 in type II and 0.1 in type I. On 1-year-old shoots in type IV, 1.7 mixed buds with a mean of three female inflorescences per bud were counted. Consequently, the generative potential is highest in type IV and lowest in type I. In types II and III, growth and the ability to bear fruits are more balanced.