Seasonal variation of the main individual phenolics and juglone in walnut (Juglans regia) leaves

Abstract

Context: Walnut [Juglans regia L. (Juglandaceae)] is a rich source of phenolic compounds, including phenolic acids, naphtoquinones and flavonoids. The increasing interest in the powerful biological activities of plant phenolics has outlined the necessity of determining their content in leaves of different walnut cultivars.

Objective: In this study, walnut leaves from walnut cultivars, originating from the same orchard and from the same year of production, were analyzed for their content in ellagic acid, rutin, myricetin and juglone. In addition, the seasonal variation of these major individual phenolics from June to August was determined.

Materials and methods: An HPLC method was used for identification and quantification of ellagic acid, rutin, myricetin and juglone contained in the methanol extract of walnut leaves in nine different cultivars grown under the same agricultural, geographical and climatic conditions.

Results: Cultivars and sampling date had statistically significant influence on the phenolics contents in walnut leaves. The results showed that ellagic acid, rutin, myricetin and juglone were more abundant in July 15th samples (average content is 84.62 mg/100 g FW, 98.9 mg/100 g FW, 178.09 mg/100 g FW and 73.81 mg/100 g FW, respectively). Their contents increases similarly in all the cultivars; therefore, the walnut leaves should preferentially be collected until early August, when phenolics content is higher.

Discussion and conclusion: The results reported here show that genotype and its interaction with the environment could make significant differences in leaf polyphenols. Walnut leaves may become a noticeable source of compounds with health protective potential.

• Ellagic acid
• HPLC-DAD
• myricetin
• rutin

Introduction

Phytochemicals, such as phenolic compounds, have received considerable attention for being potentially protective factors against cancer and heart diseases (Geleijnse et al., 2008) owing to their antioxidative properties (Cosmulescu & Trandafir, 2012; Oliveira et al., 2008) and their ubiquity in a wide range of commonly consumed foods of plant origin (Cartea et al., 2011). These compounds have been isolated from walnut leaves (Amaral et al., 2004; Cosmulescu & Trandafir, 2011a,b; 2012; Nour et al., 2013). Walnut is rich in phenolic compounds, including phenolic acids, naphtoquinones and flavonoids. Juglone (5-hydroxynaphthoquinone) is known to be the characteristic compound of Juglans spp. (Juglandaceae) and is reported to occur in fresh walnut leaves (Cosmulescu et al., 2011; Nour et al., 2013), annual shoots (Solar et al., 2006) and fruits (Colaric et al., 2005; Cosmulescu et al., 2010). Quantification of phenolic compounds in walnut leaves was performed by Amaral et al. (2004) using HPLC-DAD which revealed that quercetin 3-galactoside was always the major compound while 4-p-coumaroylquinic acid was the minor one. Walnut leaves are assessed to be a source of healthcare compounds and have been widely used in traditional medicine for treatment of skin inflammations, hyperhidrosis and ulcers and for their antidiarrheic, antihelmintic, antiseptic and astringent properties. Dry walnut leaves are frequently used to prepare infusions (Amaral et al., 2004; Pereira et al., 2007). There are many studies regarding pharmaceutical effects of walnut leaf (Kaumar et al., 2003); therefore it can be a good candidate for using it as antimicrobial agent against bacteria responsible for human gastro-intestinal and respiratory tract infections (Pereira et al., 2007) and antiproliferative effects (Salimi et al., 2012). Eidi et al. (2013) demonstrate that walnut extract acts as a good hepatoprotective and antioxidant agent in attenuating hepatocellular damage. Ellagic acid, a phenolic acid, is a major component of walnuts, and it has been suggested that it exerts antiatherogenic, anticarcinogenic and antioxidative properties (Anderson et al., 2001; Mertens-Talcott et al., 2003; Narayanan et al., 1999).

Juglone is commercially extracted from husks of walnuts, but fresh leaves of J. regia L. contain significant amounts of juglone and are deemed a good source of chemical compounds (Thakur, 2011). Juglone variation in fresh leaves was found within the range of 13.1–1556.0 mg/100 g dry weight on the basis of 1121 trees leaf samples (Thakur & Cahallan, 2011). Juglone is known for its antimicrobial effect (Clark et al., 1990), and it was also reported to decrease the incidence of tumors (Sugie et al., 1998). Phenolics contents in walnut depend on many environmental conditions, genotype of different cultivars (Colarič et al., 2005; Solar et al., 2005), as well as sampling date (Cosmulescu & Trandafir, 2011a,b; Solar et al., 2006). The increasing interest in the powerful biological activities of plant phenolics has outlined the necessity of determining their content in leaves of different species. In this study, walnut leaves from nine different walnut cultivars, originating from the same orchard and the same production year, were analyzed for their content in ellagic acid, rutin, myricetin and juglone. In addition, seasonal variation of these major individual phenolics from June to August was determined.

Materials and methods

Studies were carried out on walnut leaves from nine cultivars (Fernette, Vina, Muscelean, Fernor, Pedro, Valrex, Orastie, Hartley, Valcor). Fresh leaves of different cultivars were collected in five sampling dates in the year 2012 (June 1st, June 15th, July 1st, July 15th, August 15th) from the experimental plantation in Ramnicu Valcea (Romania) research station (located at 45 °07′N/24 °22′E), and they were preserved by freezing at −40 °C. The average annual temperature in Ramnicu Valcea was 13.3 °C; the absolute minimum temperature recorded was −22.7 °C and the absolute maximum temperature 40.4 °C. The annual average of rainfall dropped to 678 mm. Leaf samples were collected randomly from collection orchards, and 10 trees for each cultivar (10 leaves per tree). Walnut leaves were finely chopped and phenolics were extracted from 1000 mg sample in an DK 102 p Bandelin ultrasonic bath with 20 ml methanol and 1% BHT (2,6-di-tert-butyl-4-methylphenol) at 25 °C for 40 min. The extracts were centrifuged at 1200 g and the supernatants were filtered through 0.2 μm polyamide membrane and then stored at −20 °C.

Standards of ellagic acid, rutin, myricetin and juglone (Sigma-Aldrich, Germany), methanol (HPLC gradient grade, Baker, Netherlands), acetic acid (HPLC grade, Merck, Germany) and BHT (Sigma-Aldrich, Germany) were used in this experiment.

HPLC analyses were performed on a Finningan Surveyor Plus system (Thermo Electron Corporation, San Jose, CA) including a vacuum degasser, a Surveyor Plus LCPMPP pump, a Surveyor Plus ASP autosampler, a PDA5P diode array detector with 5 cm flow cell and a Chrom Quest 4.2 system manager as data processor. Separation was performed by a reversed-phase Hypersil Gold C18 column (5 µm particle size, 250 mm × 4.6 mm) provided by Thermo Electron Corporation (Madison, WI). According to Nour et al. (2013), the mobile phase consisted of 1% aqueous acetic acid solution (A) and methanol (B). Samples were eluted with the following gradient: 90% A from 0 to 27 min, from 90 to 60% A in 28 min, 60% A for 5 min, from 60 to 56% A in 2 min, 56% A for 8 min, from 56 to 90% A in 1 min and 4 min 90% A to re-establish the initial conditions, before the injection of another sample. All gradients were linear. The flow rate was 1 mL/min and the injection volume was 5 mL. Column temperature was maintained at 20 °C.

Each compound was identified by its retention time and by spiking with standards under the same conditions. The identities of constituents were also confirmed with a photodiode array (PDA) detector by comparison with ultraviolet (UV) spectra of standards in the wavelength range of 220–450 nm. Each compound was quantified according to peak area measurements, which were reported in calibration curves of corresponding standards.

All samples were extracted and analysed in triplicate. The contents of investigated phenolic compounds were expressed in mg/100 g fresh weight (FW) as mean values ± standard deviations. Data were evaluated by one-way analysis of variance (ANOVA) using Statgraphics Centurion XVI software (StatPoint Technologies, Warrenton, VA). Differences in content levels among the cultivars were estimated with a multiple range test using the least significant difference (LSD) at p < 0.05. Data were subjected to Pearson correlations.

Results and discussion

Walnut (Juglans regia) is rich in phenolic compounds, including phenolic acids, naphtoquinones and flavonoids. Seasonal variation of the main individual phenolics and juglone in walnut fresh leaves were analyzed. Studies were carried out on walnut leaves from nine cultivars and the results are given in Tables 1–4. Many significant differences in individual phenolic compounds were observed among sampling dates and cultivars (p ≤ 0.05).

Table 1. The content of ellagic acid (mg/100 g FW) in leaves of walnut cultivars.

	Sampling dates
Cultivars	June 1st	June 15th	July 1st	July 15th	August 15th
Fernette	7.5 ± 1.05^a	16.1 ± 1.10^b	30.3 ± 2.55^c	55.1 ± 1.92^c	37.3 ± 2.34^c
Fernor	30.4 ± 1.45^e	47.3 ± 2.20^f	60.9 ± 2.05^f	129.9 ± 2.78^g	49.3 ± 1.62^e
Pedro	23.7 ± 1.75^c	31.1 ± 1.35^d	68.7 ± 1.76^g	134.5 ± 3.08^h	63.1 ± 1.22^g
Vina	26.4 ± 0.85^d	40.0 ± 1.15^e	43.7 ± 1.22^e	44.8 ± 1.45^b	31.8 ± 0.95^b
Hartley	13.1 ± 0.80^b	15.1 ± 0.70^b	16.2 ± 0.93^b	38.5 ± 0.85^a	19.1 ± 0.90^a
Valcor	8.2 ± 0.55^a	4.03 ± 0.52^a	13.2 ± 0.75^a	91.6 ± 1.34^f	35.3 ± 1.24^c
Valrex	44.5 ± 1.20^g	46.6 ± 1.62^f	65.9 ± 1.71^g	128.6 ± 2.44^g	58.2 ± 1.68^f
Orastie	22.0 ± 1.10^c	47.6 ± 1.75^f	41.1 ± 1.76^e	70.9 ± 1.45^e	32.2 ± 1.05^b
Muscelean	37.6 ± 1.30^f	25.3 ± 1.20^c	34.3 ± 1.55^d	67.3 ± 1.76^d	40.6 ± 1.38^d
Mean ± SD	23.7 ± 12.72	30.3 ± 16.20	41.6 ± 20.42	84.6 ± 38.06	40.7 ± 13.88

Values in the same column followed by different uppercase letters are significantly different at p < 0.05.

Table 2. The content of rutin (mg/100 g FW) in the leaves of walnut cultivars.

	Sampling date
Cultivars	June 1st	June 15th	July 1st	July 15th	August 15th
Fernette	19.8 ± 0.95^b	18.9 ± 0.75^a	57.8 ± 0.72^d	67.9 ± 1.61^d	54.6 ± 1.78^g
Fernor	33.6 ± 1.04^d	47.2 ± 1.38^c	47.3 ± 1.64^b	50.6 ± 1.48^b	37.1 ± 1.51^d
Pedro	64.6 ± 1.62^h	48.1 ± 2.12^c	52.0 ± 1.25^c	78.7 ± 2.28^e	58.4 ± 1.34^h
Vina	59.8 ± 1.35^g	67.6 ± 1.74^d	63.1 ± 1.76^e	150.2 ± 3.43^g	48.5 ± 0.95^f
Hartley	37.9 ± 0.64^e	46.8 ± 1.05^c	143.3 ± 2.04^h	153.9 ± 3.20^g	56.9 ± 0.90^h
Valcor	51.4 ± 1.66^f	66.4 ± 1.24^d	80.9 ± 1.80^f	186.7 ± 3.48^h	34.9 ± 1.04^c
Valrex	35.4 ± 0.82^d	87.3 ± 1.37^e	96.6 ± 1.14^g	110.0 ± 2.37^f	32.6 ± 0.55^b
Orastie	24.3 ± 0.75^c	28.1 ± 0.96^b	31.0 ± 1.02^a	33.9 ± 0.96^a	27.4 ± 0.64^a
Muscelean	17.7 ± 0.82^a	27.7 ± 1.75^b	46.9 ± 1.12^b	57.7 ± 2.40^c	39.9 ± 1.27^e
Mean ± SD	38.3 ± 17.02	48.6 ± 25.53	68.8 ± 34.04	98.9 ± 53.74	43.4 ± 11.47

Values in the same column followed by different uppercase letters are significantly different at p < 0.05.

Table 3. The content of myricitin (mg/100 g FW) in the leaves of walnut cultivars.

	Sampling dates
Cultivars	June 1st	June 15th	July 1st	July 15th	August 15th
Fernette	55.7 ± 0.90^a	59.1 ± 1.52^a	117.6 ± 1.63^b	143.7 ± 3.10^c	94.4 ± 2.42^d
Fernor	74.9 ± 2.51^b	80.1 ± 2.55^c	115.3 ± 2.22^b	153.2 ± 3.96^d	66.6 ± 1.71^a
Pedro	100.2 ± 2.58^d	100.2 ± 2.10^d	111.7 ± 3.20^b	129.5 ± 2.17^b	86.7 ± 2.70^c
Vina	128.0 ± 2.40^e	134.1 ± 2.39^e	141.0 ± 2.88^c	156.3 ± 2.91^d	106.1 ± 2.52^e
Hartley	83.1 ± 1.71^c	104.5 ± 3.06^d	160.6 ± 3.44^e	221.0 ± 4.21^f	124.7 ± 3.12^f
Valcor	178.2 ± 3.49^f	194.9 ± 3.12^f	230.8 ± 5.10^f	248.9 ± 4.27^h	149.1 ± 3.18^g
Valrex	130.8 ± 2.58^e	218.8 ± 4.38^g	238.3 ± 4.62^g	231.2 ± 4.16^g	175.1 ± 4.12^h
Orastie	72.9 ± 1.90^b	78.4 ± 1.22^c	80.9 ± 2.11^a	114.2 ± 2.18^a	77.0 ± 1.83^b
Muscelean	54.1 ± 1.22^a	70.2 ± 1.53^b	148.6 ± 3.49^d	204.2 ± 2.62^e	123.1 ± 3.17^f
Mean ± SD	97.5 ± 41.09	115.6 ± 56.49	149.4 ± 53.64	178.0 ± 48.83	111.4 ± 35.17

Values in the same column followed by different superscript letters are significantly different at p < 0.05.

Table 4. The content of juglone (mg/100 g FW) in the leaves of walnut cultivars.

	Sampling dates
Cultivars	June 1st	June 15th	July 1st	July 15th	August 15th
Fernette	35.2 ± 0.91^c	32.7 ± 0.65^ab	45.0 ± 0.49^c	60.5 ± 1.05^d	35.1 ± 0.81^b
Fernor	56.7 ± 1.52^f	56.7 ± 1.05^f	64.3 ± 1.35^f	80.6 ± 1.31^e	49.0 ± 1.12^f
Pedro	32.7 ± 0.84^b	41.7 ± 1.28^d	87.5 ± 1.24^g	108.0 ± 1.85^h	74.5 ± 1.01^g
Vina	43.1 ± 1.26^e	31.9 ± 0.65^a	45.4 ± 1.02^c	55.3 ± 0.75^c	35.8 ± 1.01^bc
Hartley	32.7 ± 0.72^b	34.6 ± 1.05^c	41.5 ± 0.86^b	51.9 ± 0.95^b	45.0 ± 1.04^e
Valcor	16.2 ± 0.54^a	34.6 ± 0.95^c	56.5 ± 1.02^e	88.2 ± 1.21^g	26.7 ± 0.46^a
Valrex	55.1 ± 1.14^f	45.3 ± 1.06^e	54.4 ± 0.75^d	85.1 ± 0.88^f	38.1 ± 0.60^d
Orastie	40.5 ± 0.90^d	63.6 ± 1.38^g	65.5 ± 1.31^f	86.2 ± 1.43^f	37.4 ± 0.81^d
Muscelean	34.1 ± 1.26^bc	33.8 ± 0.76^bc	32.2 ± 0.86^a	48.2 ± 0.68^a	37.2 ± 0.74^cd
Mean ± SD	38.5 ± 12.36	41.6 ± 11.50	54.7 ± 16.39	73.8 ± 20.49	42.1 ± 13.64

Values in the same column followed by different superscript letters are significantly different at p < 0.05.

Ellagic acid is a natural antioxidant phenolic compound found in numerous fruits and vegetables. Previous experimental results have indicated that ellagic acid is a major component of walnuts (Nour et al., 2013; Papoutsi et al., 2008). The antiproliferative and antioxidant properties of ellagic acid have spurred preliminary research into the potential health benefits of ellagic acid consumption (Anderson et al., 2001; Festa et al., 2001; Mahmoodi et al., 2011; Mertens-Talcott et al., 2003). The results showed that ellagic acid was more abundant in July 15th samples, followed by July 1st and August 1st samples (Table 1). The mean ellagic acid content ranged from 23.78 to 84.62 mg/100 g FW, which represents a 3.55-fold difference between sampling dates. In August 15th, the ellagic acid content was only 48.2% (mean values) of the July 15th concentrations. The highest content was recorded on July 15th, when it ranged from 38.56 to 134.54 mg/100 g FW, which represents a 3.48-fold difference between Pedro and Hartley cultivars. Statistically significant differences (p < 0.05) were found between the analyzed cultivars. Differences between ellagic content were found by other authors and are assumed to be due to cultivar, sampling dates, climatic or technological impact, geographical origin, cultivation techniques. Stampar et al. (2006) found that the content of ellagic acid ranged from 3.9 to 98.3 mg/100 g dry weight (DW) after analyzing walnut husks at different sampling dates (July 18th and May 30th, respectively). Colaric et al. (2005) found ellagic acid to be predominant in walnut kernel and pellicle (average values of 5.90 and 128.98 mg/100 g, respectively). The amount of ellagic acid increased continuously from the 1st sampling to the 4th sampling in most of cultivars then they decreased during August, with the minor exception of Orastie sample.

Flavonoids are polyphenolic compounds existing in plants that have been widely investigated in recent years, because of their potential antioxidant activity, and their possible beneficial effects on human health (Geleijnse et al., 2008; Hirano et al., 2001; Spencer et al., 2008). Rutin (quercetin-3-rutinoside), a flavonoid which is reported to have beneficial effects on health (Kreft et al., 2003; La Casa et al., 2000), was quantified in walnut leaves of the nine cultivars (Table 2). The results showed that the highest rutin content was recorded on July 15th sampling date, when the rutin content ranged from 33.96 to 186.76 mg/100 g FW, which represents a 5.49-fold difference between Orastie and Valcor cultivars. There were also significant differences (p < 0.05) due to cultivars. The average value for this sampling date is 98.9 mg/100 g FW, higher average values being recorded for Vina, Hartley, Valcor and Valrex cultivars. The lowest average content was recorded on June 1st, i.e., 2.58-times lower than the one registered on July 15th. The rutin content increased throughout the growing season, reaching its maximum value in July 15th (average content 98.9 mg/100 g FW) in all cultivars (Table 2). Similar seasonal variation of rutin content was observed in many other horticultural crops. Usenik et al. (2004) reported a similar variation in apple leaves, where the peak occurred in August, and it was followed by a continuous decline thereafter. Colaric et al. (2007) reported that the highest rutin content in pear leaves among samplings was recorded in July, while Petkovsek et al. (2009) reported that the rutin content in apple leaves increased throughout the growing season, reaching its maximum value in August.

Myricetin is another important dietary flavonoid, which occurs mainly in blackcurrants, black grapes, cranberries, bilberries, broad beans, red wine and grape juice (Hertog et al., 1992) but walnuts too are a rich dietary source (Cosmulescu et al., 2010; Cosmulescu & Trandafir, 2011b; Stampar et al., 2006). Myricetin is reported to have beneficial effects on renal functions (Ozcan et al., 2012) or a promising agent for the chemoprevention of skin cancer (Kang et al., 2011). The highest levels of myricetin were recorded in July 15th samples of Valcor leaves (248.99 mg/100 g FW), while the lowest were found in samples taken on June 1st from the Muscelean cultivar (54.14 mg/100 g FW). There are significant differences (p < 0.05) between cultivars in terms of myricitin content (Table 3). According to literature myricetin content varies depending on cultivar, harvest date and biological material. Cosmulescu and Trandafir (2011b) reported that in walnut leaves, at the end of vegetation period, myricetin content was found in the highest concentration, followed by juglone. In walnut husk, Stampar et al. (2006) found myricetin content between 2.88 mg/100 g DW (in July 18th) and 25 mg/100 g DW (in May 30th). In general, in this study, the levels of myricetin were lower in early June samples as compared to the samples taken on July 15th from the same cultivar. This was also reported by Solar et al. (2006) in rejuvenated annual walnut shoots (catechin and myricetin that belong to the flavonoids group increased from the spring growth cycle in May to the summer flush of growth in August). In August 15th, the myricetin content was only 62.5% of the July 15th concentrations.

Juglone is a phenolic compound used in popular medicine as a phytotherapic in treating inflammatory and infectious diseases (Saling et al., 2011). Juglone, a well-known walnut component, is found in considerable amounts in all green and growing parts of trees and unripe hulls of the fruit (Prasad, 2003). In our study, the June leaves contained the lowest quantities of juglone (mean values between 38.53 and 41.68 32 mg/100 g FW). Its content increased to a maximum on July 15th (73.81 mg/100 g FW) and then decreased again on August 15th (42.12 mg/100 g FW). The results presented in Table 4 showed the highest juglone content on July 15th sampling date, when the juglone content ranged from 48.23 to 108.07 mg/100 g FW, which represents a 2.22-fold difference between Muscelean and Pedro cultivars. All cultivars showed statistically significant differences (p < 0.05) in contents on different sampling dates. Previous research (Cosmulescu et al., 2011) on juglone content in walnut leaves, by the end of vegetation period, showed that its content ranged between 5.42 and 22.82 mg/100 g in different cutivars. During growing season, there were oscillations of mean juglone content levels. In early June, the average juglone content was up to 1.91 times less than in samples from July 15th. Previous seasonal changes of juglone in black walnut leaves showed a linear decrease over the growing season (Coder, 1983). Measurements of seasonal distribution of juglone among various tissues of pecan revealed that the highest concentrations occurred in leaflets in June and in nuts in September (Borazjani et al., 1985). Stampar et al. (2006) found a juglone content of 1404 mg/100 g dry weight in fresh walnut husks on June 21st and only 218 mg/100 g dry weight on August 19th.

Seasonal variation of the main individual phenolics and juglone is following, in general, the same variation as the content of total phenols. Previous experimental results (Cosmulescu & Trandafir, 2011a) on the variation of total phenols show that they increase over the months of June and July, then they decrease over the month of August, then a new increase is recorded by the beginning of September. These differences in terms of total phenols at different sampling times are supposed to be the effect of change in ecological parameters.

In regard to the correlation (Pearson correlation) between the main individual phenolics and juglone, the results show a positive correlation coefficient between ellagic acid and juglone content (R²= 0.613), which is highly significant (p < 0.05). The other correlations have been positive, insignificant. Therefore, further research is required to reveal the possible relationship of various types of phenolics and the influence of environmental factors on phenolics content.

Conclusions

The phenolic content data in the present study indicated significant differences in phenolic contents between the nine walnut cultivars analyzed at the same harvest time. Bearing in mind that flavonoids and phenolic acids have been the subject of several studies owing to their antioxidant potential, the results obtained suggest that, for this purpose, walnut leaves should preferentially be collected in July, when phenolic content is higher. The results reported here show that genotype and its interaction with the environment could make significant differences in leaf polyphenols. There appears to be a general trend whereby polyphenols increase until the beginning of August and decrease in the summer; therefore, sampling leaves until early August is likely to produce larger amounts of polyphenols, but suitable cultivars must be selected for obtaining specific types of phenolic compounds. More research is needed to investigate the cause of location effects and extending the study over a period of several years. Genotype and environmental variation in phenolic content shall be investigated in future studies.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.