ANTIOXIDANT POTENTIAL OF Juglans regia BARK: QUANTIFICATION OF SEVEN PHENOLIC COMPOUNDS BY RP-HPLC

Walnut (Juglans regia L., Juglandaceae) is traditionally used for the treatment of various common ailments including tuberculosis, tuberculosis of the cervical glands, and cancer [1, 2]. The bark of J. regia contains juglone along with numerous bioactive polyphenols [3, 4]. Although there have been many reports on several biological activities of J. regia bark [2–5], there has been only one report on its antioxidant activity [3]. The objectives of this work were to determine the antioxidant potential and bioactive compounds of the methanol extract (MOH) and its fractions obtained by partitioning from J. regia bark with hexane (HEX), chloroform (CHL), ethyl acetate (EA), and butanol (BU), and the water-insoluble part (INS). Methanol is able to extract out a significant amount of compounds (11.36%) from the walnut bark. The yields of fractions were: EA-3.18, BU-1.87, CHL-0.21, and HEX-0.11%, suggesting that ethyl acetate is a more effective solvent for fractionation. Some phenolic compounds also remained trapped in the water-insoluble part (INS, 1.4%). Total phenol content (TPC), determined using Folin–Ciocalteau reagent [6], was highest in the EA (470.3 ± 8.5) followed by BU (360.5 ± 4.8), MOH (343.5 ± 5.5), INS (193.4 ± 2.8), CHL (100.2 ± 3.5), and HEX (32.3 ± 2.3), all in mg g–1 dry extract. In a study, Labuckas and co-workers [7] has reported high total phenol content in aqueous-methanolic extract of hull of J. regia. The antioxidant activity of the extract and fractions were determined by DPPH (2,2'-diphenyl-1-picrylhydrazyl) [8], ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) [9] radical scavenging and FRAP (ferric reducing/antioxidant power) [10] assays, and the results are presented in Table 1. The EA showed highest antioxidant potential in three in vitro test systems. Both EA and BU were better DPPH scavengers than the positive control quercetin; however, none of the extract/fractions were as effective as butylated hydroxytoluene (BHT) or gallic acid. ABTS∙+ scavenging of extract/fractions was also much lower than gallic acid, BHT, and quercetin. In contrast to DPPH∙ scavenging, ABTS∙+ scavenging ability of the marker compound juglone was much higher than the extract or fractions. The reducing power of EA was found comparable to the positive controls quercetin and BHT. However, it was much lower than gallic acid. The reducing power of juglone was significantly lower than that exhibited by the extract or fractions except HEX and CHL. It was also noticed that the order of DPPH∙ scavenging and ferric reducing properties of the extract and fractions was similar to the order of TPC. This indicates that there is a significant correlation between antioxidant properties and TPC. The correlation coefficients (r2) of total phenolic content, DPPH∙ and ABTS∙+ scavenging, and FRAP activities were 0.975, 0.798, and 0.960, respectively. In order to quantify phenolic compounds in the extract/fractions, HPLC was carried out using a LiChrospher RP-18 column (250 mm х 4.6 mm, 5 μm) and Waters 2996 photodiode array detector at 30 degree C. a) Acetonitrile–methanol (70:30, v/v) and b) 0.05% trifluoro acetic acid were used as the mobile phase with a gradient programmed as follows: a) 80–0% in 0–10 min and back to 80% in 20 min with a flow-rate of 1 mL/min. Seven phenolic compounds, namely gallic acid (1), caffeic acid (2), quercitrin (3), rutin (4), myricetin (5), quercetin (6), and juglone (7), were quantified. Among all, quercitrin and gallic acid were observed in the highest amount in the MOH and its fractions (Table 2). In particular, the content of each phenolic compound was higher in EA in comparison to others, which corroborated with the observed trends in total phenol content and antioxidant activity. The marker compound juglone was detected only in the methanol extract and not in the fractions. The results of this study clearly indicate that walnut bark extract/fractions, especially the ethyl acetate fraction, may be used as a potential source of natural antioxidant