3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P37 ExTRACTION AND DETERMINATION OF
FLAVONOIDS IN PLANT MATERIALS
BARBORA HOHnOVá a,b , PAVEL KARáSEK b and
MILEnA VESPALCOVá a
a Faculty of Chemistry, Brno University of Technology, Purkyňova
118, Brno 612 00,
b Institute of Analytical Chemistry of the ASCR, v.v.i.,Veveří 97,
Brno 602 00,
hohnova@fch.vutbr.cz
Introduction
Flavonoids are one of the largest groups of secondary
metabolites and they are responsible for coloration of plants.
They are found in fruits, vegetables, beverages like tea,
wine, beer, and large amount of flavonoids was also found in
several medicinal plants. Flavonoids play important role in
human diet because of their antioxidant properties, estrogenic
action and wide range of antimicrobial and pharmaceutical
activities 1 .
Extraction of flavonoids from the samples is usually performed
by maceration with aqueous methanol 2 , solid-phase
extraction 3 , sonication 4 , and supercritical fluid extraction
(SFE) 5 . High-performance liquid chromatography (HPLC) 6 ,
capillary electrophoresis (CE) 7 , capillary electrochromatography
(CEC) 8 or gas chromatography (GC) 9 are commonly
used for their determination and quantification.
Since rapid and efficient extraction technique prior to
chromatographic analysis is nowadays of primary interest, the
liquid extraction at high temeperature and pressure – Pressurized
fluid extraction (PFE), was introduced. PFE offers several
advantages over conventional extraction procedures. The use
of higher temperature increases the diffusion rate, solubility
and mass transfer of the compounds and decreases the viscosity
and surfance tension of the extraction solvent. These
changes improve the contact of analytes with the solvent and
enhance extraction, which can be achieved more rapidly with
less solvent consumption as compared with conventional
extraction methods. The elevated pressure not only maintains
the extraction solvents in liquid state, but also improves the
contact of solvent with analytes trapped in matrix pores. The
absence of light and air significantly reduces degradation and
oxidation of compounds during extraction 10 .
In the last years, pressurized fluid extraction has been
succesfully applied to the extraction of flavonoids from plant
materials and foods. This application of PFE as powerful
sample preparation step was recently reviewed by Mendiola
et al. 11
Recently, intense attention has been paid to Stevia rebaudiana
Bertoni (Asteraceae) because of content of low-caloric
high intensity sweet diterpene glycosides (50–400 times
sweeter than sucrose) 12 . Besides sweet glycosides also flavonoids
have been isolated from Stevia. Rajbhandari et al.
determined that Stevia contains apigenin, luteolin, quercetin,
kaempferol and quercitrin. The flavonoids were extracted by
s651
classical maceration and their structures were determined by
standard methods of uv, nmr, and ms spectroscopy 13 .
In this study, we focused on optimalization of liquid
chromatograpfic conditions after extraction step for rapid
and succesful determination and quantification of quercetin,
luteolin, apigenin and kaempferol contained in Stevia leaves.
Experimental
C h e m i c a l s a n d S t a n d a r d S o l u t i o n s
Stevia leaves were obtained from Ukraine. Acetonitrile,
methanol and formic acid, all HPLC-grade, were purchased
from Riedel-de Haën (Prague, Czech Republic). Water was
purified with a reverse osmosis system Ultra Clear UV
(Barsbüttel, Germany).
Flavonoid standards quercetin, kaempferol, luteolin and
apigenin were obtained from Sigma-Aldrich (Prague, Czech
Republic). Stock solution of all flavonoids (1 g dm –3 each)
was prepared in methanol and stored in the fridge at 5 ˚C.
P F E o f S t e v i a R e b a u d i a n a L e a v e s
A static PFE of Stevia leaves was performed using a
onePSE extractor (Applied Separations, Allentown, PA,
USA). A portion (1 g) of leaves was placed into 22 ml extraction
cell that contained inert material (glass beads (570–
700 µm)) at the bottom of the cell. The PFE parameters were
set as follows, temperature 60–120 ºC, pressure 15 MPa,
extraction time 3 × 5 minutes, rinsing time 20 s, and nitrogen
purge time 90 s after each cycle and 120 s after extraction
run. After PFE run, the extract was cooled to 5 ºC and stored
in the fridge until HPLC analysis.
C h r o m a t o g r a p h i c C o n d i t i o n s
HPLC apparatus was eqCipped with LC 1150 pump
(GBC Scientific Equipment Pty Ltd, Dandenong, Australia),
injection valve with 20-µl sample loop (Ecom, Praha, Czech
Republic), column oven LCO 101 (Ecom, Praha, Czech
Republic) and Linear UVIS-206 Multiple Wavelength detector
(Linear Instruments, Fremont, CA). The wavelength was
set at 360 nm. Mobile phase was composed of acetonitrile
(A) and water (B) adjusted at pH 2.3 by formic acid. Several
reversed-phase columns were tested: 1) 4.6 mm i.d., 150 mm
long Symmetry C18 5 µm stationary phase (Waters, USA);
2) 3.0 mm i.d., 150 mm long Xbridge C18 3.5 µm stationary
phase (Waters, USA); 3) 3.0 mm i.d., 150 mm long Atlantis
T3 C18 3 µm stationary phase (Waters, USA); 4) 3.0 mm
i.d., 150 mm long Xterra RP C18 3.5 µm stationary phase
(Waters, USA); and 5) 4.6 mm i.d., 250 mm long Supelcosil
LC-18 5 µm stationary phase (Supelco, USA). Gradient
programme was employed at flow rate 0.5 ml min –1 : 0 min
10 % A; 20 min 70 % A. When Xterra C18 column was used,
the flow rate was set to 0.3 ml min –1 because of high backpressure.
LC systems were connected to PC and controlled
by Clarity software (DataApex, Czech Republic).