Extraction and identification of flavonoids from parsley extracts by HPLC analysis

M. Stan, M. L. Soran, C. Varodi, and I. Lung

Citation: AIP Conference Proceedings 1425, 50 (2012); doi: 10.1063/1.3681964

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 Extraction And Identification Of Flavonoids From Parsley
Extracts By HPLC Analysis
M. Stan, M.L. Soran, C. Varodi and I. Lung

National Institute for Research and Development of Isotopic and Molecular Technologies, 65-103 Donath, 400293
Cluj-Napoca, Romania
Corresponding author: [email protected]

Abstract. Flavonoids are phenolic compounds isolated from a wide variety of plants, and are valuable for their multiple
properties, including antioxidant and antimicrobial activities. In the present work, parsley (Petroselinum crispum L.)
extracts were obtained by three different extraction techniques: maceration, ultrasonic-assisted and microwave-assisted
solvent extractions. The extractions were performed with ethanol-water mixtures in various ratios. From these extracts,
flavonoids like the flavones apigenin and luteolin, and the flavonols quercetin and kaempferol were identified using an
HPLC Shimadzu apparatus equipped with PDA and MS detectors. The separation method involved a gradient step. The
mobile phase consisted of two solvents: acetonitrile and distilled water with 0.1 % formic acid. The separation was
performed on a RP-C18 column.
Keywords: Flavonoids, Petroselinum crispum L., Extraction, HPLC analysis
PACS: 82.80.Bg

INTRODUCTION compounds more water soluble and combinations of

the solvents previously mentioned with water are thus
Flavonoids are one of the most diverse groups of better solvents for glycosides. In contrast, less polar
phenolic substances isolated from a wide range of aglycones such as isoflavones, flavonones and highly
vascular plants, with over 8000 individual compounds methoxylated flavones and flavonols tend to be more
known. They act in plants as antioxidants, soluble in non-aqueous solvents [6]. There are
antimicrobials, photoreceptors, visual attractors, multiple methods for extracting phenolic compounds
feeding repellants, and for light screening. Many for analytical purposes from their plant sources, but
studies have suggested that flavonoids exhibit three principal techniques are generally used:
biological activities including antiallergenic, antiviral, extraction using solvents, solid-phase extraction and
anti-inflammatory, and vasodilating actions [1]. These supercritical extraction.
compounds possess at least one phenol moiety, an Many of the experimental procedures for
aromatic ring with one or more hydroxyl substituents. ultrasonic-assisted and microwave-assisted solvent
Parsley (Petroselinum crispum L.) was selected for the extractions presented in literature require high
present work, as one of the most common culinary temperatures and high extraction times that could lead
herbs consumed globally and one of the richest sometimes to degradation of flavonoid compounds to
sources of commonly occurring phenolic aglycone, be studied [7, 8]. Our experimental procedures for
apigenin. these two unconventional modern extraction
The first step in the analysis is extraction of the techniques necessitate different experimental
analyte from the source material. Extraction of parameters that enable the obtaining of an amount of
phenolic compounds from plant material is influenced flavonoids comparable to classical extraction
by various parameters such as solvent polarity, particle techniques like maceration. The experimental
size, extraction procedures and conditions. Several procedure for our microwave-assisted solvent
solvents such as methanol, ethanol, acetone, water, extraction technique requires an extraction time of 1
ethyl acetate and, to a lesser extent, propanol, min per sample, which is a great advantage. Moreover,
dimethylformamide, dimethyl sulfoxide and their all extractions using the extraction techniques selected
combinations have been used for the extraction of for the present work were performed at low
different classes of phenolic compounds [2-5]. Most temperature of 35°C.
flavonoids occur naturally as glycosides. Soluble The main goal of our investigations was to
phenolic compounds are generally extracted using compare the extraction efficiency by using various
water, methanol, ethanol or acetone. The presence of techniques for extraction of flavonoids from parsley.
attached sugars tends to render the phenolic The parsley extracts obtained were analyzed by
Processes in Isotopes and Molecules (PIM 2011)
AIP Conf. Proc. 1425, 50-52 (2012); doi: 10.1063/1.3681964
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 HPLC-MS in order to identify the flavonoid that maceration with ethanol-water (50:50, v/v) gave
components using standard stock solutions of some the highest quantity of flavonoids as shown in Figure
flavonoid compounds. 1. The most efficient extraction technique was
maceration, which unfortunately is more time
EXPERIMENTAL PART consuming. By using the two modern extraction
methods (ultrasonic-assisted and microwave-assisted
Commercial plant material of Romanian origin was solvent extractions), an amount comparable to that
used. Parsley extracts were obtained by three different obtained by maceration was acquired with low
extraction techniques: maceration, ultrasonic-assisted extraction times, which is a great advantage.
and microwave-assisted solvent extractions. 0.5 grams The analysis of the absorption spectra carried out to
of dried parsley leaves and 20 mL of solvent mixture analyze the parsley extract samples showed absorption
were used for the extraction process. After filtration peaks characteristic of flavonoids. Because of their
and washing, the final volume of extracts was adjusted chemical structure, flavonoids possess characteristic
to 25 mL with the appropriate solvent mixture. The absorption spectra in the UV region, determined by the
flavonoid standards were obtained from Fluka benzopyrone core and with two peaks of absorption.
(Germany). All the solvents were purchased from Flavones present maximum absorption in the range of
Chimopar (Bucharest, Romania). All chemicals were 335-350 nm, and flavonols between 360-380 nm.
of analytical grade. Stock solutions were prepared in
ethanol at 100 µg mL-1.
The extraction time for maceration was 14 days at
35°C. Ultrasonic-assisted solvent extraction was 0.2

Absorbance (a.u.)
performed with a Transsonic T 310 bath at 35 kHz and
an installed power of 95 W. The samples were first
soaked for 10 min with the extraction solvent mixture 0.0

and then subjected to 30 min sonication at 35°C. Sonication

Microwave-assisted solvent extraction was performed Microwave assisted solvent
using a home made apparatus [9]. Taking in account -0.2
Maceration
the specificity of plant material the following 300 400 500 600

parameters were selected: maximum temperature Wavelength (nm)

35°C, action time 1 min and duty coefficient of 40% at
an installed power of 900 W. FIGURE 1. Determination of total flavonoid content from
The extracts were analyzed by High Performance parsley extracts - a comparison of the extraction method
Liquid Chromatography (HPLC) technique using a efficiency.
Shimadzu HPLC equipped with PDA (Photodiode
Array) and MS (Mass Spectrometer) detectors. The mAU
Quercetin/8.384
/8.843

220nm,4nm (1.00)
3000
HPLC column utilized was Grace Alltima C18 (100 x 2750
3 mm, 3µm). Elution was carried out using gradient 2500

mode (A = acetonitrile and B = distilled water with 2250

formic acid 0.1 %) with 0.43 ml/min flow rate. The 2000

gradient elution program used was from 5% to 42 % A 1750

in 30 min, return to 5% and equilibration for 5 min. 1500

/8.529

A systematic variation of different proportions of 1250

ethanol-water solvent mixtures (100:0, 80:20, 70:30, 1000

Kaempferol/10.429

60:40, 50:50 and 40:60, v/v) was used to compare the 750
/10.065
Apigenin/9.412
/8.149

500
extraction efficiency of flavonoid compounds from
Luteolin/9.791

/10.298

250
parsley material by UV-Vis spectrophotometric 0
analysis using a UV 1800-Shimadzu apparatus. The
2.5 5.0 7.5 10.0 12.5 min
direct method of quantitative determination of
flavonoids needs aluminum chloride as reagent and
rutoside as standard [10]. FIGURE 2. The Chromatogram of Petroselinum crispum L.
extract obtained by maceration.
RESULTS AND DISCUSSIONS
The corresponding flavonoid standards were
The UV-Vis spectrophotometric data provided the employed for identification of flavonoid components
optimum extraction method. Thus, it was determined from parsley extract. The chromatogram presented in
Figure 2 indicates the flavonoids identified from

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 parsley extract: apigenin, luteolin, quercetin and parameters that enable the obtaining of an amount of
kaempferol. The chromatogram was obtained at 220 flavonoids comparable to classical extraction
nm, the wavelength for maximum intensities of peaks. techniques like maceration, which is more time
The mass spectrum of the flavone apigenin is consuming. In Petroselinum crispum L. extracts were
presented in Figure 3. identified flavonoids like the flavones apigenin and
luteolin, and the flavonols quercetin and kaempferol.

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