Content of different groups of phenolic compounds in microshoots ...

Acta Physiol Plant (2013) 35:443–450 DOI 10.1007/s11738-012-1087-7

ORIGINAL PAPER

Content of different groups of phenolic compounds in microshoots of Juglans regia cultivars and studies on antioxidant activity Monireh Cheniany • Hassan Ebrahimzadeh Kourosh Vahdati • John E. Preece • Ali Masoudinejad • Masoud Mirmasoumi

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Received: 30 September 2011 / Revised: 2 August 2012 / Accepted: 29 August 2012 / Published online: 12 September 2012 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2012

Abstract Phenolic and other compounds were extracted from micropropagated axillary shoots (microshoots) of the walnut (Juglans regia L.) cultivars ‘Chandler’, ‘Howard’, ‘Kerman’, ‘Sunland’, and ‘Z63’. Among cultivars, microshoots showed differences in phenolic compounds, phenolic acids, flavonoids, and proanthocyanidins. All cultivars contained the phenolics acids chlorogenic acid, gallic acid, p-coumaric acid; the naphthoquinone juglone; and the flavonoid quercetin. The phenolic acids syringic acid and vanillin were present only in microshoots of ‘Howard’. Microshoot extracts had different antioxidant activity with ‘Kerman’ the highest and ‘Chandler’ the lowest in each of three antioxidant assays: the phosphomolybdenum assay (PPM), reducing power assay, and 2,2diphenyl-1-picrylhydrazyl-scavenging effect. There was a strong linear relationship between total phenolic compound content of microshoots and increasing antioxidant activity.

Communicated by E. Lojkowska. M. Cheniany (&) H. Ebrahimzadeh M. Mirmasoumi Plant Physiology Laboratory, Department of Botany, School of Biology, College of Sciences, University of Tehran, 14155-6455, Tehran, Iran e-mail: [email protected]; [email protected] K. Vahdati Department of Horticulture, College of Abooraihan, University of Tehran, Tehran, Iran J. E. Preece National Clonal Germplasm Repository, USDA ARS, One Shields Drive, University of California, Davis, CA 95616, USA A. Masoudinejad Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

Keywords In vitro Juglone Micropropagation Quercetin Walnut Abbreviations BAP 6-Benzyl aminopurine BHT Butylated hydroxytoluene DKW Driver and Kuniyuki Walnut DPPH 2,2-Diphenyl-1-picrylhydrazyl DW Dry weight FW Fresh weight IBA Indolebutyric acid

Introduction Walnuts (Juglans spp.) are widely distributed throughout the world and are valuable for their nuts and wood. Since 1970s, research has focused on walnut micropropagation and has resulted in rootstock and scion cultivars that are produced in commercial micropropagation laboratories. These laboratories use a nodal segment axillary shoot multiplication system. However, little is known about the chemical composition of J. regia microshoots. Walnuts are rich in phenolics compounds. These compounds have been isolated from walnut green husks (Cosmulescu et al. 2010), green walnut fruit (Jakopic et al. 2007), mature walnut seed (Colaric et al. 2005; Fukuda et al. 2003), leaves (Amaral et al. 2008), the inner sapwood (Dehon et al. 2001), and rejuvenated shoot tissues (Claudot et al. 1997; Solar et al. 2006). Hybrid walnut microshoots have been reported to contain the naphthoquinones hydrojuglone glucoside and juglone, and the flavonoids myricitrin and quercitrin (JayAllemand et al. 1993). The naththoquinones increased and flavonoids were variable during root induction.

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El Euch et al. (1998) transformed J. nigra 9 J. regia somatic embryos with an antisense chalcone synthase (chs) gene. Transgenic lines had very low chalcone synthase activity and no detectable myricitrin, quercitrin or proanthocyanidins whereas these were high in nontransgenic lines. The decreased flavonoid content of transgenic stems was associated with better rooting of microshoots compared to nontransgenic lines. To our knowledge, there is one published report that measured total phenolic content (TPC) of J. regia ‘Howard’ and ‘Sunland’ microshoots over 56 days and found variability over time that was not related to rooting response (Cheniany et al. 2010). However, there are no published reports on detailed analysis of J. regia microshoots for phenolics compounds and antioxidant activity. The objectives of this study were to extract walnut microshoots of five cultivars to (1) determine phenolics, phenolic acids, flavonoids, and proanthocyanidins, (2) isolate and identify the content of some specific phenolic compounds, and (3) evaluate and compare the total antioxidant activity, DPPH scavenging capacity, and reducing power of microshoot extracts as new active free radical scavengers from natural resources.

Acta Physiol Plant (2013) 35:443–450

Phenolic compound extraction and determination Phenolic compounds were extracted from 0.5 g samples of whole fresh microshoots in 20 ml of 80 % methanol based on procedure described by Waterman and Mole (1994) and quantities were estimated using Folin–Ciocalteu reagent at 760 nm with Shimadzu UV-160 Spectrophotometer (Japan). TPC expressed as mg phenolics g-1 FW (fresh weight) was calculated from a standard curve established using known concentrations of gallic acid and expressed as gallic acid equivalents (GAE). Bound phenolic acids extraction and determination

Materials and methods

Three 0.5 g FW samples of microshoots per cultivar were extracted under reflux in boiling 80 % methanol for 6 h. The extracts were combined, evaporated in vacuo to dryness, and dissolved in methanol for investigation. Total phenolic acid compounds (TPACs) were determined with the Matkowski et al. (2008) method. The 1 mg ml-1 extracts were added to 6 ml water, followed by 1 ml of 0.1 M HCl, 1 ml Arnow reagent (10 % w/v sodium molybdate and 10 % w/v sodium nitrate in distilled water), 1 ml M NaOH, filled up to 10 ml in a volumetric flask and the absorbance read immediately at 490 nm. The results were expressed as caffeic acid equivalents (CAE).

Plant materials and in vitro culture

Flavonoid compound extraction and determination

Microshoot cultures of five Persian walnut (J. regia L.) cultivars, ‘Chandler’, ‘Howard’, ‘Kerman’, ‘Sunland’, and ‘Z63’ that were maintained on DKW medium (Driver and Kuniyuki 1984) for more than 15 years by the University of Tehran, were used as a source of explants. These microshoot tips of walnut were originated from tree sample of walnut collection in Kamalshahr research station of seed and plant improvement institute of Iran. Axillary shoot proliferation was maintained through regular subcultures every 3 weeks in 500 ml glass jars with 38 ml medium. Four 3 cm long microshoots per jar were cultured on DKW medium supplemented with 2.1 g L-1 gellan gum (Phytagel, Sigma Co., USA), 0.01 mg L-1 indolebutyric acid (IBA), and 1 mg L-1 benzylaminopurine (BAP) and the pH was adjusted to 5.5 (Vahdati et al. 2004). Cultures were incubated at 26 ± 1 °C under cool white fluorescent lamps that provided a photon flux of 75 lmol m-2 s-1 and a 16 h photoperiod. The experiments were repeated thrice using 40 explants per cultivar. At the end of the multiplication cycle, suitable microshoots were excised and destructively analyzed. The data for growth determination including fresh mass, shoot diameter, shoot length, and lateral bud (per explants) were scored after five subcultures.

Flavonoid compounds were extracted from 0.5 g FW microshoot samples in 5 ml of methanol. The solvent was removed under the vacuum; the remainder was redissolved in 5 ml of methanol, filtered through a 0.2 lm cellulose acetate filter (Minisart, Sartorius), and stored for analysis. An aluminum chloride colorimetric method was used for flavonoids determination (Chang et al. 2002). From each cultivar, 0.5 ml of 1 g flavonoid extract per 10 ml methanol was mixed with 1.5 ml methanol, 0.1 ml 10 % aluminum chloride in distilled water, 0.1 ml 1 M potassium acetate in distilled water, and 2.8 ml distilled water. It remained at 25 °C for 30 min; the absorbance of the reaction mixture was measured at 415 nm. The calibration curve was generated by preparing quercetin solutions of different concentrations and the results are expressed as quercetin equivalents (QE).

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Proanthocyanidin compound extraction and determination Proanthocyanidins were extracted based on the methods of Ksouri et al. (2008) and measured using the modified vanillin assay described by Sun et al. (1998). Aliquots of 250 ll of 0.5 g FW microshoot samples in 5 ml of


methanol were added to 3 ml methanol vanillin solution (1 % w/v) and 2.5 ml 9 N H2SO4. The samples were incubated at 38 °C for 15 min. The absorption was measured at 500 nm against the extract solvent as a blank. The amount of total condensed tannins is expressed as mg catechin equivalent (CE) g-1 FW. Analysis of phenolic compounds by HPLC Standards The following standards were used for quantification of phenolic compounds: juglone (5-hydroxy-1,4-naphtoquinone) and syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid) were obtained from Merk Co. (Darmstadt, Germany), Chlorogenic acid (3-caffeoylquinic acid), Gallic acid (3,4,5-trihydroxybenzoic acid), quercetin (2-[3,4-dihydroxyphenyl]-3,5,7-trihydroxy-4H-chromen-4-one), p-coumaric acid (4-hydroxycinnamic acid), and vanillin (4hydroxy-3-methoxybenzaldehyde) were from Sigma Co. (St. Louis, USA). These standards were prepared in HPLC grade methanol. Sample preparation The samples were prepared according to Fernańdez-Lorenzo et al. (2005) with some modification. Oven-dried microshoots from each cultivar were ground to a fine powder and 0.1 g quantities were extracted at 25 °C with 3 ml of HPLC grade methanol and then centrifuged at 3,000 rpm for 30 min. The supernatants were collected and evaporated to dryness in a speed-vac, then the residue was redissolved in 2 ml HPLC grade methanol, which was centrifuged at 3,000 rpm for 30 min and transferred to a vial and stored at -20 °C prior to analysis by HPLC. Each final extract was filtered with a 0.2 lm cellulose acetate filter (Minisart) before injection. These extracts were also used for antioxidant activities assays. Separation conditions Phenolic compounds were analyzed using an HPLC (Shimadzu SIL-6A) with SPD detection (Shimadzu-SPD-6AV). A C-18 (Hamilton) reverse phase column (4.1 9 150 mm, 5 lm particle size) was used. Between each analysis, a gradient elution was performed by gradually adding HPLC grade methanol to the 2 % acetic acid in double distilled water solvent to obtain 30 % methanol at 10 min, 70 % at 50 min, and 75 % at 75 min. The total run time was 75 min, with 30 min of equilibration treatment (100 % methanol). The injection volume of a sample was 20 ll, and the flow rate was 0.7 ml per min. The column temperature was 30 °C. Phenolic compounds were detected at

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a wavelength of 280 nm. The identification of phenolics was achieved through the following: comparison of the retention times of standard solutions with the retention times of compounds in samples, absorption maxima of compounds in the scanned spectrum, and the addition of standards to samples. The concentrations of specific phenolic compounds were calculated by referencing peak areas from corresponding external standards, including the phenolic acid compounds chlorogenic acid, p-coumaric acid, gallic acid, syringic acid, and vanillin; the flavonoid quercetin; and the naphthoquinone juglone. Total antioxidant assay The total capacity of the extract was evaluated by the phosphomolybdenum assay (Prieto et al. 1999) based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of a green phosphate-Mo (V) complex in acidic condition. A 0.3 ml extract (25 mg ml-1) was combined with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate in distilled water, and 4 mM ammonium molybdate in distilled water). The test tubes containing the reaction mixtures were incubated at 95 °C for 90 min. Then the absorbance of the solution was measured at 695 nm against a blank containing 0.3 ml of methanol mixed with 3 ml reagent solution after cooling to 25 °C. The antioxidant capacity was expressed as vitamin C equivalent (mg VCEAC 100 g-1 DW). In order to make comparison, butylated hydroxytoluene (BHT) was also tested under the same conditions as a standard antioxidant compound. Reducing power assay The reducing power of the methanolic extracts described above under ‘‘Sample preparation’’ was determined according to the method of Yen and Chen (1995) and Koleva et al. (2002) with some modifications. Briefly, the methanolic extracts from the microshoots were diluted further with 0.2, 0.4, 0.6, 0.8, or 1 ml methanol. A 1 ml aliquot of sample solutions were then added to 2.5 ml of a 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 10 mg ml-1 potassium ferricyanide distilled water solution and incubated at 50 °C for 20 min. After incubation, an equal volume of 1 % trichloroacetic acid was added to each mixture prior to centrifuging at 3,000 rpm for 10 min at 4 °C. The supernatant was separated and mixed with 2.5 ml of distilled water and 0.5 ml of 0.1 % ferric chloride. The mixture was incubated at 25 °C for 15 min. The absorbance was measured at 700 nm against an aliquot blank. The extract concentration providing 0.5 of absorbance (EC50) was calculated from the graph of absorbance registered at 700 nm. BHT and ascorbic acid were used as reference compounds.

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Scavenging effect assay The capacity to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was monitored according to a method by Hatano et al. (1988). Various concentrations of 0.1 ml sample extracts were mixed with 3 ml methanol containing 1 ml 100 lM DPPH solution in methanol. The mixture was shaken vigorously and left to stand at 20 °C in darkness until stable absorption values were obtained (30 min). For the control, 1 ml 100 lM DPPH solution was mixed with 3 ml methanol. The reduction of the DPPH radical was measured by monitoring the decrease of absorption at 517 nm against the blank. The DPPH scavenging effect was calculated as percentage of DPPH discoloration using the equation: % scavenging effect = [(ADPPH – AS)/ADPPH] 9 100, where AS is the absorbance of the solution containing a specific concentration of the sample extract and ADPPH is the absorbance of the DPPH solution. The extract concentration providing 50 % inhibition (EC50) was calculated from the graph of scavenging effect percentage against extract concentration. The DPPH radical scavenging activity of phenolic compounds was also expressed as mg 100 g-1 vitamin C equivalent in 30 min. The radical solution was prepared daily. BHT and ascorbic acid were used as positive controls.

Acta Physiol Plant (2013) 35:443–450 Table 1 Growth factors (fresh weight, shoot diameter, shoot length, and lateral bud number) analyzed in Persian walnut microshoots at the end of multiplication cycle Shoot length (cm)

Lateral bud number (per explant)

2.3 ± 0.10 ab

3.1 ± 0.09 c

3.2 ± 0.08 b

2.0 ± 0.32 c

4.7 ± 0.14 a

3.2 ± 0.06 b

0.89 ± 0.054 a

2.3 ± 0.41 ab

3.7 ± 0.13 b

3.1 ± 0.06 b

Sunland

0.37 ± 0.054 d

3.0 ± 0.21 a

3.2 ± 0.48 c

3.4 ± 0.11 ab

Z63

0.35 ± 0.003 d

2.2 ± 0.26 ab

3.3 ± 0.10 c

3.8 ± 0.60 a

Cultivar

Fresh weight (g)

Shoot diameter (cm)

Chandler

0.60 ± 0.005 c

Howard

0.75 ± 0.066 b

Kerman

Data reported as mean ± SEM (n = 40); values marked with different letters are significantly different at P \ 0.05

Statistical analysis Data were collected on microshoots at the end of the multiplication phase (every 21 days). Data were evaluated statistically by analysis of variance (ANOVA) followed, when significant by generation of standard errors of means using the SPSS data analysis software package (version 16 for windows). All results presented are mean ± standard error of at least three independent replicates from three separate experiments.

Results and discussion Growth determination Growth analyses show that the microshoots of walnut cultivars grow differently in vitro (Table 1). Although the highest fresh mass belongs to ‘Kerman’, the most value of shoot diameter and shoot length are in ‘Sunland’ and ‘Howard’ cultivars, respectively. Lateral bud outgrowth during multiplication period shows ‘Z63’ has the lowest apical dominance while ‘Kerman’ has the highest one. These data confirm that although walnut cultivars grow well in vitro, they differ based on genotype in studied growth factors.

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Fig. 1 a Total phenolic compounds (GAE), b total phenolic acid compounds (CAE), c total flavonoid compounds (QE), d total proanthocyanidin compounds (CE). Data presented are mean ± SEM (n = 3)

Total phenolics, phenolics acid, flavonoids, and proanthocyanidins in walnut microshoots There were significant differences among cultivars for microshoot content of total phenolics (GAE), phenolic


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Table 2 Flavonoid, phenolic acid, and naphthoquinone compounds in microshoots of J. regia cultivars as determined by HPLC Name of compound

Family

Retention time (min)

mg 100 g-1 DW Chandler

Howard

Kerman

Sunland

Z63

Gallic acid

PAC

7.55

4.489 ± 0.19

4.882 ± 0.13

4.532 ± 0.17

7.242 ± 0.20

7.121 ± 0.21

Chlorogenic acid

PAC

21.57

18.201 ± 0.35

15.241 ± 0.14

25.296 ± 0.47

25.248 ± 0.22

19.855 ± 0.27

Quercetin

FC

22.35

65.827 ± 0.40

67.602 ± 0.27

73.634 ± 0.31

72.474 ± 0.19

48.491 ± 0.44

Syringic acid

PAC

32.69

ND

0.688 ± 0.11

ND

ND

ND

p-Coumaric acid

PAC

37.49

1.604 ± 0.11

5.566 ± 0.24

4.183 ± 0.13

2.656 ± 0.15

3.446 ± 0.14

Vanillin

PAC

41.87

ND

38.475 ± 0.33

ND

ND

ND

Juglone

NQC

50.19

24.472 ± 0.23

24.282 ± 0.26

38.475 ± 0.11

28.311 ± 0.14

21.182 ± 0.32

ND not determined, FC flavonoid compounds, PAC phenolic acid compounds, NQC naphthoquinone compounds, DW dry weight Data reported as mean ± SEM (n = 3)

Table 3 Antioxidant activities of walnut microshoot extracts as determined by the phosphomolybdenum (PPM) assay, reducing power (RP) assay, and the DPPH scavenging assay Walnut cultivar

Antioxidant standard

PPM assay

RP assay

DPPH scavenging assay

VCEAC (mg 100 g-1 DW)a

50 % of Absorbance (mg ml-1)b

VCEAC (mg 100 g-1 DW)a

EC50 (mg ml-1)c

Chandler

1,713.1 ± 21.18

0.668 ± 0.02

155.3 ± 0.92

0.631 ± 0.01

Howard

1,766.7 ± 19.09

0.602 ± 0.02

194.7 ± 1.17

0.567 ± 0.02

Kerman

2,872.4 ± 28.26

0.374 ± 0.01

326.2 ± 1.55

0.312 ± 0.01

Sunland

2,501.6 ± 23.44

0.398 ± 0.01

301.8 ± 1.41

0.353 ± 0.01

Z63

2,026.7 ± 31.16

0.459 ± 0.01

283.7 ± 1.18

0.425 ± 0.01

BHT

207.81 ± 11.00

0.676 ± 0.01

–

0.660 ± 0.02

Ascorbic acid

–

0.146 ± 0.01

–

0.150 ± 0.02

a

VCEAC (mg VCEAC 100 g-1 DW): effective concentration at which the antioxidant activity is equal to vitamin C activity

b

50 % of absorbance (mg ml-1): effective concentration at which a half of absorbance is provided

c

EC50 (mg ml-1): effective concentration at which 50 % of DPPH radicals are scavenged

acids (CAE), flavonoids (QE), and proanthocyanidins (CE, Fig. 1). ‘Kerman’ microshoots contained the highest amount of total phenolics, phenolic acids, and flavonoids and the lowest amounts of these compounds were in extracts from ‘Chandler’ microshoots. Proanthocyanidins were the highest in ‘Z63’ and the lowest in ‘Kerman’. Claudot et al. (1997) reported that in nursery grown J. nigra 9 J. regia, flavonoids accumulated in shoots by the end of growth and that naphthoquinone metabolism was associated with rejuvenation. Solar et al. (2006) found that flavonoids in rejuvenated J. regia shoots increased throughout the season. Therefore, it is interesting that flavonoids are also present in actively growing microshoots and may be a sign of rejuvenation. In addition, although there are many researches showing a distinct influence of culture conditions on the contents of phenolic metabolites of explants (Kwiecien et al. 2010a, b), our results confirm that these contents could also be affected by the genotype of explants.

Specific phenolic compounds in walnut microshoots Microshoots differed significantly among cultivars for the content of seven phenolic compounds: gallic acid, chlorogenic acid, quercetin, syringic acid, p-coumaric acid, vanillin, and juglone as determined by HPLC (Table 2). The most prevalent compound was the flavonoid quercetin followed by the naphthoquinone juglone and the phenolic acid chlorogenic acid. ‘Howard’ microshoots were the only cultivar to contain the phenolic acids syringic acid and vanillin. Solar et al. (2006) identified eight phenolics in rejuvenated annual shoots of J. regia cultivars. They reported on seasonal variation in amounts of catechin, myricetin, vanillic acid, syringic acid, ellagic acid, and chlorogenic acid, juglone and 1,4-naphthoquinone. The quinones were present 3.5-fold higher than total flavonoids and 5.3-fold higher than the total phenolics acids. Claudot et al. (1997) found chalcone synthase (CHS) activity in bark and phloem of rejuvenated J. nigra 9 J. regia shoots. The

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Fig. 2 Linear regression analysis for demonstration of correlation coefficient between the antioxidant activity and total phenolic compounds (TPC), total phenolic acid compounds (TPAC), total flavonoid compounds (TFC) and total proanthocyanidin compounds (TAC)

Fig. 3 Linear regression analysis for demonstration of correlation coefficient between the DPPH scavenging activity and total phenolic compounds (TPC), total phenolic acid compounds (TPAC), total flavonoid compounds (TFC) and total proanthocyanidin compounds (TAC)

flavonoids myricitrin and quercitrin accumulated in shoots as did hydrojuglone glucoside. Jay-Allemand et al. (1993) reported the presence of quercitrin, myricitrin, juglone, and hydrojuglone glucoside in hybrid walnut shoots. Our present paper is the first time that phenolics have been reported in microshoots of J. regia cultivars. Their presence in hybrid and Persian walnut microshoots and in shoots on trees indicates that there are metabolic similarities between microshoots and macroshoots. Antioxidant activity Among cultivars, there were differences in the antioxidant activities of the microshoot extracts (Table 3). In all

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assays, the highest antioxidant activity was in microshoot extracts of ‘Kerman’ followed with ‘Sunland’. Walnut microshoots showed reducing powers at very low concentrations (less than 1 mg ml-1) that are in accordance with the results of Pereira et al. (2007). Results of DPPH scavenging of these walnut microshoots appeared to be concentration-dependent and increased with increasing concentration of each extract. These results showed that with regard to standard antioxidant compounds, antioxidant activities of microshoots are high (Table 3). There are strong linear correlations (R2 = 0.865; 2 R = 0.996, respectively) between total phenolic compound concentration and total antioxidant activity and DPPH scavenging (Figs. 2, 3). Total flavonoids compounds


were also correlated (R2 = 0.308; R2 = 0.411, respectively) with total antioxidant activity and DPPH scavenging. Other phenolics were weakly correlated or negatively correlated with both assays. ‘Kerman’ and ‘Sunland’ microshoots contained the highest amounts of total phenolics compounds (Fig. 1) and the highest amounts of chlorogenic acid, juglone, and quercetin (Table 1) and had the highest antioxidant activities. ‘Chandler’ microshoots contained the lowest amounts of total phenolics compounds and flavonoids (Fig. 1) and had the least antioxidant activity. Because of the high correlation between total phenolics and flavonoids and antioxidant activity, it is likely that these compounds play this antioxidant role endogenously in walnut microshoots. Walnut microshoots differ according to genotype in the amount and types of phenolics compounds. There is a strong correlation between phenolics compounds and antioxidant activity. Cultivars with the most total phenolics and one or more flavonoids had the highest antioxidant activity. The importance of that is the suggestion of Persian walnut as potential sources of natural antioxidants for medicinal and commercial uses. Authors contribution Hassan Ebrahimzadeh and Ali Masoudinejad conceived and designed the experiments. Monireh Cheniany performed the experiments. Monireh Cheniany, Kourosh Vahdati, and Masoud Mirmasoumi analyzed the data. Monireh Cheniany, John E. Preece, and Hassan Ebrahimzadeh contributed reagents/materials/ analysis tools. Monireh Cheniany, Kourosh Vahdati, and John E. Preece wrote the manuscript. Monireh Cheniany, Kourosh Vahdati, Masoud Mirmasoumi, and Hassan Ebrahimzadeh checked and validated the results. Acknowledgments This paper represents a portion of the first author’s dissertation presented to the College of Sciences, University of Tehran, in partial fulfillment of the requirements for the PhD degree. The authors also express sincere thanks to Dr. Hadi Zare (Faculty of Mathematics and Computer Science, Amirkabir University of Technology) for statistical suggestions and comments.

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