SUPPLEMENTARY MATERIAL
Comparison of phenolic content and antioxidant activity of Actaea
racemosa L. and Actaea cordifolia DC.
Grażyna Szymczaka, Magdalena Wójciak – Kosiorb*, Ireneusz Sowab, Karolina Zapałab and
Anna Bogucka-Kockac
a
Botanical Garden of Maria Curie - Skłodowska University in Lublin, Sławinkowska 3, 20-810 Lublin, Poland.
b
Department of Analytical Chemistry, Medical University of Lublin, Chodźki 4a, 20-093, Lublin, Poland.
c
Department of Pharmaceutical Botany, Medical University, Chodźki 1, 20-093 Lublin, Poland.
Abstract
A. racemosa is used as a component of drugs or dietary supplements to alleviate the
menopause symptoms. Its biological activity is associated, i.a. with the presence of phenolic
compounds. In our work, the analysis of isoflavones and phenolic acids: caffeic, ferulic and
isoferulic, both free and bonded in two species of Actaea was conducted using HPLC-PAD
technique. Moreover, the antioxidant effect of extracts from different parts of investigated
plants was determined on the basis of DPPH assay.
Significant variation of caffeic and isoferulic acids content was observed. The highest
content of caffeic acid was found in A. racemosa while A. cordifolia contained the highest
amount of isoferulic acid. Isoflavones were not found in investigated plants. The antioxidant
activity assay showed the high ability the extracts obtained from different parts of plant to free
radical scavenging.
Keywords: Actaea racemosa; Actaea cordifolia; isoflavones; phenolic acids; antioxidant
activity
*Corresponding author: M. Wójciak – Kosior email: kosiorma@wp.pl
3. Experimental
3.1 Plant material
Two species: A. racemosa L. and A. cordifolia DC. were collected during autumn season in
2013 year in Botanical Garden of UMCS in Lublin, identified in Department of
Pharmaceutical Botany Medical University of Lublin and deposited in Botanical Garden of
UMCS (voucher specimen number: 2299 and 3275, respectively). The plants were divided on
roots (R1, R2), rhizomes (Rh1, Rh2) and shoots (S1, S2), dried at 40°C and pulverized.
3.2 Chemicals and reagents
Methanol, HPLC-grade acetonitrile and trifluoroacetic acid (TFA) were purchased from
Merck (Darmstadt, Germany). Water for HPLC was purified by ULTRAPURE Milipore
Direct-Q® 3UV–R (Merck, Darmstadt, Germany). Standards: caffeic (CA), ferulic (FA),
isoferulic acids (iFA), daidzein (D), genistein (G), formononetin (F), biochanin A (BA),
trolox and 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) were from Sigma-Aldrich (St. Louis, MO,
USA).
3.3 Extraction and Standard Preparation
1 g of plant material was accurately weighed and extracted with 20 mL of methanol in
ultrasonic bath during15 min. The procedure was repeated three times with a fresh portion of
solvent. The combined extracts were concentrated to 10 mL.
Phenolic acid standards: 1.5 mg of caffeic and ferulic acid, and 5 mg of isoferulic acid
were dissolved in 25 mL of methanol (final concentration was 60 µg mL-1 and 200 µg mL-1,
respectively). Standard solutions were prepared by dilution of stock solutions in methanol to
appropriate concentrations.
3.4 HPLC condition
Chromatographic determination was performed on VWR Hitachi Chromaster 600
chromatograph (Merck, Darmstadt, Germany) with pump (5160), an degasser, thermostat
(5310), autosampler (5260), DAD detector (5430) and EZChrom Elite software.
The extracts were analysed on C18 reversed-phase column LiChrospher 100 (Merck) (25
cm × 4.0 mm i.d., 5 μm particle size), at temperature of 25°C. Twenty µL (R1,R2, Rh1 and
Rh2) or forty µL (S1 and S2) of sample was injected. Mixture of acetonitrile (solvent A) and
water with 0.025% of trifluoroacetic acid (solvent B) was used as a mobile phase. Phenolic
acids were separated by gradient elution: A 10%, B 90% during 0-15 min, and next A 15%, B
85 % during 15.5-35 min. The gradient elution program for isoflavones analysis was as
follow: A 30%, B 70% during 0-10 min, A increased 30-50% during 15.5-35 min. The flow
rate of eluent was 1.0 mL min-1. The data were collected in the range of wavelength from 200
to 400 nm. The quantitative analysis of phenolic acids were performed at λ=320 nm.
3.5 DPPH test
Methodology of DPPH assay was established on the basis of literature (Paduch et al., 2007;
Tirillini et al., 2013). 10 µL of the extract sample was diluted with methanol up to 1 mL and 1
mL of DPPH solution at concentration of 0.1 mg mL-1 was added. After 30 minutes, the
absorbance was measured at 515 nm. The percentage of inhibition activity was calculated
from equation: [(A0-A1)/A0] x 100, where A0 is absorbance of control and A1 is absorbance
of the extract. Trolox was used as a positive control.
References
Paduch, R., Wójciak-Kosior, M., & Matysik, G. (2007). Investigation of biological activity of
Lamii albi flos extracts. Journal of Ethnopharmacology 110, 69-75.
Tirillini, B., Menghini, L., Leporini, L., Scanu, N., Marino, S. & Pintore G. (2013).
Antioxidant activity of methanol extract of Helichrysum foetidum Moench. Natural Product
Research. 27, 1484-1487.
Figure S1. The chromatographic analysis of isoflavones: 1- mixture of standards: daidzein
(D), genistein (G), formononetin (F) and biochanin A (BA); Rh1 - extract from rhizome, R1extract from root and S1 – extract from shoot of A. racemosa.
Figure S2. The chromatographic analysis of phenolic acids: 1- mixture of standards: caffeic
acid (CA), ferulic acid (FA), isoferulic acid (iFA), Rh1 - extract from rhizome, R1- extract
from root and S1 – extract from shoot of A. racemosa.
Figure S3. The content of caffeic (CA), ferulic (FA) and isoferulic acid (iFA) in root (R),
rhizome (Rh) and shoot (S) in A. racemosa (1) and A. cordifolia (2) unbundled after acidic
hydrolysis.