HPLC Determination of Phenolic Acids, Flavonoids and Juglone in Walnut Leaves

Abstract

A high-performance liquid chromatographic method with gradient elution and diode-array detection was developed to quantify free phenolic acids (gallic, vanillic, chlorogenic, caffeic, syringic, p-coumaric, ferulic, sinapic, salycilic, elagic and trans-cinnamic), flavonoids (catechin, epicatechin, rutin, myricetin and quercetin) and juglone in walnut leaves. Chromatographic separation was performed on a Hypersil Gold C18 column (5 µm particle size, 250 × 4.6 mm) and detection was conducted at three different wavelengths (254, 278 and 300 nm) according to the absorption maxima of the analyzed compounds. Validation procedures were conducted and the method was proven to be precise, accurate and sensitive. The developed method has been applied to analyze walnut leaves samples from nine different cultivars, with the same agricultural, geographical and climatic conditions. The experimental results revealed high concentrations of myricetin, catechin hydrate and rutin, and low concentrations of quercetin and epicatechin aglycones. Ellagic acid was established as the dominating phenolic acid of walnut leaves, followed by trans-cinnamic, chlorogenic and caffeic acids. Juglone content varied between 44.55 and 205.12 mg/100 g fresh weight. Significant differences were detected among cultivars for the concentration levels of phenolics.

Optimization of ultrasound-assisted hydroalcoholic extraction of phenolic compounds from walnut leaves using response surface methodology

“…As previously reported by Nour et al (2013b), ellagic acid was the most abundant phenolic acid in walnut leaves (272.29 mg/100 g DW), while vanillic acid was found in the lowest content (1.69 mg/100 g DW). High contents of ellagic acid were found also in other walnut products.…”

“…HPLC analyses were performed on a Finningan Surveyor Plus HPLC system (Thermo Electron Corporation, San Jose, CA) according to the method developed by Nour et al (2013b). Separation was achieved by a reversed-phase Hypersil Gold C18 column (5 mm particle size, 250 Â 4.6 mm) provided by Thermo Electron Corporation (San Jose, CA).…”

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

“…Separation was performed by a reversed-phase Hypersil Gold C18 column (5 mm 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.…”

“…These compounds have been isolated from walnut leaves (Amaral et al, 2004;Cosmulescu & Trandafir, 2011a,b;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 and fruits 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-pcoumaroylquinic acid was the minor one.…”