SUPPLEMENTARY MATERIAL
Quality evaluation of different preparations of dry extracts of birch (Betula pendula Roth)
leaves
Lina Raudonėa, Raimondas Raudonisa,b, Valdimaras Janulisa and Pranas Viškelisb
a
Department of Pharmacognosy, Lithuanian University of Health Sciences, Eivenių str. 4, LT-
50161, Kaunas, Lithuania, e-mail: farmakog@lsmuni.lt
b
Institute of Horticulture, Lithuanian Research Center for Agriculture and Forestry, Kauno str.
30, LT-54333, Babtai, Kaunas distr., Lithuania, e-mail: biochem@lsdi.lt
Abstract
Qualitatively prepared dry extracts are advantaged over conventional fluid extracts.
Extracts of Betula pendula Roth leaves have mild diuretic effects and anti-inflammatory
activity. In this work the spray-drying and lyophilization processes were evaluated for
producing dry extracts of birch leaves. The purpose was to identify the best drying method
that provides the highest content of phenolic compounds and possesses greatest antioxidant
activity. Predominant compounds hyperoside and chlorogenic acid were chosen as markers
for quantitative control of phenolic compounds. Their determined amounts in
hydroalcoholic birch leaf extract were 15.85 and 8.77 mg/g of dry residue, respectively.
Insignificant differences were determined in spray dried and freeze dried extracts. In the
antioxidant profile hyperoside (28.9 μmol TE/g dw) and chlorogenic acid (22.8 μmol TE/g
dw) possessed strongest ferric reducing activity. Also no significant differences of
antioxidant activity were determined in the in spray dried and freeze dried extracts.
Keywords: birch, FRAP, antioxidant, phenolic, Betula pendula, dry extract.
1. Experimental
1.1 Chemicals
HPLC grade acetonitrile was purchased from Sigma–Aldrich (Buchs, Switzerland).
Formic acid (98–100%), acetic acid (99.8%) and hydrochloric acid were obtained from Fluka
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Chemie (Buchs, Switzerland). Ethanol (96.3%) was provided by Stumbras (Kaunas, Lithuania).
Ultrapure water was prepared using a Millipore water purification system (Bedford, MA). The
following reagents were used: iron(III) chloride hexahydrate (FeCl3·6H2O), 2,4,6-tripyridyl-striazine (TPTZ) and sodium acetate trihydrate from Sigma–Aldrich Chemie (Steinheim,
Germany). The following standards were used were of HPLC grade: 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox), chlorogenic acid, hyperoside, avicularin,
quercitrin, quercetin were purchased from Fluka (Buchs, Switzerland), Roth (Karlsruhe,
Germany), Sigma–Aldrich (Buchs, Switzerland).
1.2 Plant material and preparation of hydroalcoholic extract
Fresh leaves of B. pendula Roth (Betulaceae) were collected during June 2013 from wild
populations located in different parts of Lithuania. The samples were botanically identified. B.
pendula leaves were air dried and milled. The powdered material (mean particle size of 355 μm)
was extracted by maceration with 70% ethanol at room temperature, initially for 48 h and
thereafter until exhaustion. The mass/volume ratio was of 1:10. The obtained hydroalcoholic
extract was filtered.
1.3 Preparation of dried extracts
The spray drying process of hydroalcoholic extract was performed in the Buchi Mini
Spray Dryer B-290 with Inert Loop B-295 (BUCHI, Switzerland) for solvent collection. The
experiments were carried out following conditions: extract feed rate of 6 mL/min, a drying air
inlet temperature of 140 °C and a spray nozzle nitrogen flow rate of 450 L/h.
The hydroalcoholic extract was concentrated under vacuum (at 50 °C) and then subjected
to freeze drying. Extract was frozen at –30 °C in a glass jar then freeze dried for 48 h using
Freeze Dryer with Shell Freezer FD8512S (IlShin, Lab Co. Ltd., Korea). Sublimation was
carried out at –80 °C condenser temperature and a pressure of 0.007 mbar.
1.4 HPLC-FRAP post-column assay
The HPLC equipment system applied consisted of Waters 2695 Alliance solvent manager
(Milford, MA) equipped with a Waters 2998 photodiode array detector. The chromatographic
separation was carried out using a 5-μm ACE C18 analytical column (250 × 4.6 mm) with guard
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column 5-μm ACE C18 (20 × 4.0 mm) (Aberdeen, Scotland) thermostated at 25 °C. The mobile
phase consisted of 1% (v/v) formic acid (solvent A) and acetonitrile (solvent B). The optimized
linear gradient elution program was determined to be 10–15% solvent B at 0–20 min, 15%
solvent B at 20–30 min, 15–30% solvent B at 30–45 min, 30–40% solvent B at 45–55 min. The
flow rate was set at 1 mL/min and the injection volume of all samples was 10 μL. The
confirmation of the chromatographic peaks identity was achieved by comparing the retention
times and spectral characteristics (λ = 200–600 nm) of the eluting peaks with those of reference
compounds. For quantitative analysis, a calibration curve was obtained by injection of known
concentrations (2.5–100 μg/ml) of different standard compounds. Chlorogenic acid was
quantified at 320 nm, hyperoside, avicularin, quercitrin and quercetin at 360 nm.
The working FRAP solution comprised TPTZ (0.01 M dissolved in 0.04 M HCl),
FeCl3·6H2O (0.02 M in water) and acetate buffer (0.3 M, pH 3.6) in the ratio of 1:1:25. On-line
post-column addition of working FRAP solution was performed by Waters 515 HPLC Pump
(Milford, MA). The flow rate was set at 0.5 mL/min. The post-column reactor was made of 1 mL
(PEEK, 0.3 mm i.d., 15 m) coil. The reactor temperature was set at 25 °C. The antioxidant
activity chromatograms after FRAP post-column reaction were registered at 593 nm using
Waters 2487 dual λ absorbance (UV/Vis) detector (Milford, MA).
The antioxidant activity of phenolic compounds was expressed as μmol Trolox equivalent
(TE) for 1 g of dry extract. TE was calculated by the following formula:
TE = c×V/m (μmol/g)
(1)
c – the concentration of Trolox established from the calibration curve, μM; V – the
volume of extract, L; m – the weight (precise) of dry extract, g.
Antioxidant profile enables the evaluation of contribution to antioxidant activity (CAA)
of individual compounds, because determined relevant peak areas correspond to relevant
antioxidant activity of sample mixture (Beekwilder et al. 2005).
CAA = Area(x)/Area(total)×100 (%),
(2)
Area(x) – the area of a peak of interest; Area(total) – the sum of the area of all peaks in
antioxidant profile.
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1.5 Statistical analysis
Statistical analysis was performed using SPSS version 15.0 (Chicago, USA) and
Microsoft Excel. All determinations were done in triplicate, and results are presented as mean ±
standard error (SE). Linear regression model was analysed. The Mann–Whitney U test was
performed for the hypothesis concerning equality of distributives. Level of significance α=0.05.
References
Beekwilder J, Jonker H, Meesters P, Hall RD, van der Meer IM, Ric de Vos CH. 2005.
Antioxidants in raspberry: On-line analysis links antioxidant activity to a diversity of
individual metabolites. J Agric Food Chem. 53:3313–3320.
Figure S1. Phenolic profile (A) and FRAP post-column assay antioxidant profile (B) of
hydroalcoholic extract of B. pendula Roth leaves. (1) chlorogenic acid, (2) hyperoside, (3)
avicularin, (4) quercitrin and (5) quercetin.
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