Determination of the content in usnic acid and polyphenols from the extracts of Usnea barbata L. and the evaluation of their antioxidant activity

Abstract

The aim of this study was the identification and quantification of the main active compounds from different Romanian Usnea barbata L. extracts and evaluation of their antioxidant activity. The vegetal product Usneae lichen was harvested from Călimani Mountains, Suceava County, Romania, in March 2016. We prepared 10% extracts in: acetone, 96% ethanol and water. The quantitative analysis of the identified constituents was performed using an HPLC method. The antioxidant activity was evaluated by the scavenger DPPH (2,2-diphenyl-1-picrylhydrazyl) method. For this analysis, we prepared 20% extracts in: acetone, 96% ethanol and water. For each extract, we prepared two dilutions: 1:2 and 1:4. Our results showed that each extract contains usnic acid and polyphenols. The intensity of the antioxidant activity varied with the different solubility of polyphenols in each solvent. - Accession Number: WOS:000431275100022

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ORIGINAL ARTICLE
DETERMINATION OF THE CONTENT IN USNIC ACID AND
POLYPHENOLS FROM THE EXTRACTS OF USNEA BARBATA L.
AND THE EVALUATION OF THEIR ANTIOXIDANT ACTIVITY
VIOLETA POPOVICI 1#*, LAURA BUCUR 2#, ANTOANELA POPESCU 2#, AURELIANA
CARAIANE 3#, VICTORIA BADEA 1#
¹Department of Microbiology, Faculty of Dental Medicine, Ovidius University Constanța, 7 Ilarie Voronca Street, Constanța, Romania
²Department of Pharmacognosy, Faculty of Pharmacy, Ovidius University Constanța, University Street 1, Campus, Building
B, Constanța, Romania
3Department of Dental Medicine, Faculty of Dental Medicine, Ovidius University Constanța, 7 Ilarie Voronca Street,
Constanța, Romania
*corresponding author: popovicivioleta@gmail.com
#All authors contributed equally to this work
Manuscript received: October 2017
Abstract
The aim of this study was the identification and quantification of the main active compounds from different Romanian Usnea
barbata L. extracts and evaluation of their antioxidant activity. The vegetal product Usneae lichen was harvested from
Călimani Mountains, Suceava County, Romania, in March 2016. We prepared 10% extracts in: acetone, 96% ethanol and
water. The quantitative analysis of the identified constituents was performed using an HPLC method. The antioxidant activity
was evaluated by the scavenger DPPH (2,2-diphenyl-1-picrylhydrazyl) method. For this analysis, we prepared 20% extracts
in: acetone, 96% ethanol and water. For each extract, we prepared two dilutions: 1:2 and 1:4. Our results showed that each
extract contains usnic acid and polyphenols. The intensity of the antioxidant activity varied with the different solubility of
polyphenols in each solvent.
Rezumat
Lucrarea prezintă identificarea și determinarea conținutului în principii active cu acțiune terapeutică din extractele obținute
din Usnea barbata L. din Romania, în diferiți solvenți, precum și evaluarea acțiunii antioxidante a acestora. Produsul vegetal
Usneae lichen a fost recoltat din munții Călimani, județul Suceava, România, în martie 2016. Au fost preparate extracte 10%
în diferiți solvenți: apă, acetonă și etanol 96%. Identificarea și determinarea conținutului în acid usnic și polifenoli s-a realizat
prin metoda HPLC. Determinarea activității antioxidante s-a realizat prin metoda de captare a radicalilor liberi DPPH (2,2-
difenil-1-picrilhidrazil). Pentru aceasta determinare, am preparat extracte 20% in apă, acetonă și alcool etilic 96%. Pentru
fiecare extract am realizat 2 diluții, 1:2 și 1:4 și am determinat activitatea lor antioxidantă. Rezultatele obținute au demonstrat
faptul că toate extractele conțin acid usnic și polifenoli, în concentrații diferite, în funcție de solubilitatea lor în solvenții
utilizați. Activitatea antioxidantă, datorată în special polifenolilor, a fost prezentă la toate extractele luate în lucru, dar a
variat, în mod corespunzător cu solubilitatea polifenolilor în solvenții utilizați la extracție.
Keywords: Usneae lichen extracts, usnic acid, polyphenols, antioxidant activity
Introduction
Phytotherapy research aims to find herbal products
with bioactive compounds for the treatment of
various diseases. Usnea barbata L. is a lichen with
a large habitat, used in traditional medicine [13-15].
A lichen is a symbiotic organism, the symbiosis
being realized between a fungus (Ascomycetes or
Bazidiomycetes) and an algae or a cyanobacteria,
[3, 9, 10]. The most important natural compounds
from Usnea spp are secondary metabolites, especially
usnic acid (dibenzofuran derivative) and poly-
phenols [9, 13-16]. Usnic acid is the most prominent
secondary lichen metabolite in all approximately
500 species of Usnea genus [12-14]. Usnic acid has
many pharmacological activities: antibacterial, anti-
fungal, antiviral, antioxidant, antiinflammatory,
analgesic-antipyretic, anticancer, antigenotoxic, anti-
mutagenic, antiplatelet/antithrombotic, antiulcerogenic
[9, 10, 14-16] with important effects in various
diseases [14-16]. Polyphenols have a significant
antioxidant action [2, 7, 8-15] which can be used in
the treatment of oro-dental diseases [2, 3].
Materials and Methods
General methodology included work methods of
natural compounds from medicinal plants presented
in Romanian Pharmacopoeia Xth Edition and
European Pharmacopoeia 9th Edition [20, 21].

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Obtaining the vegetal product Usneae lichen
Usneae lichen, the Usnea barbata L. dry thallus,
was harvested from Călimani Mountains, România,
in March 2016, when the lichen presents the highest
content in bioactive compounds [9,13]. The lichen
was cleaned and dried at a constant temperature
below 25°C in an airy room, protected from the
sunlight and brought to the degree of crushing
required for loose tissues (sieve 3) [20].
HPLC analysis of phenolic compounds
For the separation, identification and quantification
of phenolic compounds, a standardized HPLC
method described by the USP 30-NF25 monograph,
has been adapted [24]. Apparatus: Agilent 1200
HPLC, quaternary pump, DAD detector, thermostat,
degassing system, auto sampler.
Working conditions: C18 column, 150 mm 4.6 mm;
5 µm (Zorbax XDB). Mobile phase: solution A:
0.1% phosphoric acid, solution B: acetonitrile, with
gradient elution. Temperature: 35ºC; flow: 1.5 mL/
min; detection: UV 310 nm; injection volume: 20 µL;
analysis time: 22 min.
Extracts preparation
Three samples of 10 g vegetal product were extracted
each with 100 mL solvent (water, acetone and 96%
ethanol). The three resulting extractive solutions
were filtered and then made up to 100 mL volumetric
flask with each solvent used in the extraction.
The reference substances (70% methanolic solutions)
were: E-resveratrol = 0.37 mg/mL, Z-resveratrol = 0.22
mg/mL (obtained by exposing the E-resveratrol
solution to UV radiation at λ = 254 nm for 12 h),
caffeic acid = 0.36 mg/mL, chlorogenic acid = 0.37
mg/mL, cinnamic acid = 0.58 mg/mL, vanillin = 0.42
mg/mL, gallic acid = 0.39 mg/mL, ferulic acid = 0.38
mg/mL, 3-methyl gallic acid = 0.51 mg/mL, ellagic
acid = 0.40 mg/mL. The reference substances were
injected 6 times (20 µL) in the chromatographic
system. The identification and quantitative determination
of the natural compounds of the solution to be
analysed, was performed by comparing the
chromatogram of the standards and the solution to
be analysed.
HPLC analysis of usnic acid
For the separation, identification and quantification
of usnic acid, a standardized HPLC method was
adapted [22].
Working conditions: C18 column chromatograph,
150 mm/4.6 mm; 5 µm (Zorbax XDB); mobile phase:
methanol:water:acetic acid = 80:15:5, detection:
UV = 282 nm, flow rate = 1.5 mL/min; temperature =
25ºC, injection volume = 20 µL; analysis time: 6
minutes.
Reference substance (solution in acetone): usnic
acid = 50 µg/mL (ppm). The reference substance
was injected 6 times (20 µL) in the chromatographic
system. The identification and quantitative determination
of the active principles in the assayed solutions was
performed by comparing the chromatogram of the
standard and the solution to be analysed. Unic acid
presented a retention time of 4.463 ± 0.008053 min,
with a correlation coefficient r2 of 0.9998.
Antioxidant activity assay - DPPH-method
The antioxidant capacity was determined on a Jasco
V630 UV-Vis spectrophotometer, using the DPPH
method [1, 4-7, 10-12, 14-19]. The principle of this
method is that antioxidants react with the free
radical DPPH (purple coloured solution) and by
hydrogen donating, transform it into a clear DPPH-
H, where the degree of discoloration indicates the
antioxidant potential [19]. The DPPH solution was
prepared by dissolving DPPH (Sigma Aldrich) in
methanol to obtain an absorbance value of 0.8 ±
0.02; 0.1 mL for each extract was vortexed for 30 s
with 3.9 mL of DPPH solution and left to react for
30 min, after which the absorbance at 515 nm was
recorded. The DPPH solution with no added extract
was used as control [4]. Gallic acid was used as
reference standard, dissolved in water, in order to
obtain a solution with a similar concentration as the
Usneae lichen solutions [4]. Methanol was used as
blank [4]. Four extractive solutions were analysed:
S1 = Usneae lichen extract 200 mg/mL in ethanol
96%, S2 = Usneae lichen extract 200 mg/mL in
water, S3 = Usneae lichen extract 200 mg/mL in
acetone, S4 = reference usnic acid solution 50
µg/mL (0.05 mg/mL). For each extract 200 mg/mL
(S1, S2, S3) 1:2 (100 mg/mL) and 1:4 (50 mg/mL)
dilutions were obtained.
The scavenger activity was calculated as follows:
% scavenger DPPH = [(Acontrol - Asample)/Acontrol] x 100,
where: Acontrol and Asample are the absorbances at 515
nm for DPPH methanol solution and samples [4, 13].
Results and Discussion
The quantitative determination of the bioactive
compounds:
Using different extraction methods and different
solvents, extracts with different content of bioactive
compounds were obtained.
HPLC analysis of phenolic compounds
Retention times for the reference substances and
correlation coefficients of the calibration curves are
shown in Table I.
In order to determine the polyphenolic compounds
from Usnea barbata L. extracts, an optimised
HPLC method was used. We identified 6 poly-
phenols in the extracts: caffeic acid, p-coumaric
acid, ellagic acid, chlorogenic acid, gallic acid,
cinnamic acid. We found polyphenols especially in
the aqueous and ethanolic extracts.
Three polyphenols were found in the ethanolic and
the aqueous extracts: chlorogenic acid, gallic acid
and p-coumaric acid, with higher amounts in
aqueous extract. The amounts of polyphenols

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339
identified in the analysed extracts, expressed as
mg% are presented in Table II.
Table I
Retention times of phenolic compounds (reference
substances) and square of correlation coefficients
No
Phenolic
compound
Retention time
(min)
r2
1.
Gallic acid
0.990 ± 0.025
0.99537
2.
3-methyl gallic acid
2.606 ± 0.008
0.99563
3.
Chlorogenic acid
3.501 ± 0.015
0.99999
4.
Caffeic acid
4.598 ± 0.036
0.99619
5.
Vanillin
6.919 ± 0.051
0.99691
6.
p-Coumaric acid
7.187 ± 0.019
0.99798
7.
Ferulic acid
8.565 ± 0.058
0.99863
8.
E-Resveratrol
14.467 ± 0.017
0.99965
9.
Ellagic acid
15.303 ± 0.027
0.99885
10.
Z-resveratrol
15.751 ± 0.058
0.99729
11.
Cinnamic acid
15.867 ± 0.007
0.99845
Table II
Total polyphenols contents in the extracts
Extract
Polyphenols
Contents (mg%)
Ethanolic
extract
Caffeic acid
0.46
p-Coumaric acid
0.35
Ellagic acid
255.99
Chlorogenic acid
0.56
Gallic acid
30.24
Cinnamic acid
19.87
Aqueous
extract
p-Coumaric acid
0.82
Chlorogenic acid
0.86
Gallic acid
66.18
In the acetonic extract we did not identified any
polyphenol from the reference mixture.
HPLC analysis of usnic acid
Usnic acid has the greatest content in acetonic
extract; we can observe that the usnic acid content
is the smallest in the aqueous extract from Usnea
barbata L. (Table III).
Table III
Usnic acid content in Usnea barbata L. extracts
Usnea barbata L. extract
Usnic acid content (mg%)
Acetonic extract
2115.29
Ethanolic extract
256.56
Aqueous extract
44.60
Cansaran D et al. studied six species of Usnea
genus (U. florida, U. barbata, U. longissima, U.
rigida, U. hirta and U. subflorida), collected from
different areas of Anatolia¸ Turkey [6]. The usnic
acid content in acetone extracts was determined by
HPLC and varied between 220 mg% and 6490
mg% [6]. The amount of usnic acid in the acetonic
extract of Usnea barbata L. from Anatolia, was
2160 mg% [6], comparable to the results obtained in
this study (2115.29 mg%).
Antioxidant activity assay
Usnea barbata L. extracts reduced DPPH radical
with different degrees of scavenging activity. It was
found that the ethanolic extract has the greatest
antioxidant activity, followed by the aqueous
extract and acetonic extract. The results of the
antioxidant activity evaluated by DPPH method are
presented in Figure 1.
Figure 1.
DPPH scavenger activity of the Usneae lichen different extracts (S1 - S3) and of the usnic acid standard solution
(S4), where S1 = Usneae lichen ethanolic extract; S2 = Usneae lichen aqueous extract; S3 =Usneae lichen acetonic
extract; S4 = usnic acid 50 µg/mL (0.05 mg/mL) in acetone
The scavenging effect was higher with the increase
of the extracts concentration. It was found that
Usneae lichen 200 mg/mL extract in ethanol had a
DPPH scavenger activity over 50%.
The IC 50 value was 189.94 mg/mL for the
ethanolic extract, calculated by linear interpolation,
using the generated equation:
inhibition = 0.1561 x concentration + 20.35.
The acetonic and aqueous extracts in the present study
presented a scavenger activity under 50%, IC 50
was not calculated.
Rankovic B et al. evaluated the antioxidant potential
of the Usnea barbata L. acetonic extract. They used
the DPPH method, with ascorbic acid as reference
substance and they showed an IC 50 value of
667.97 µg/mL, for the acetonic extract [16].
0%#
10%#
20%#
30%#
40%#
50%#
60%#
200MG/ML#
100MG/ML#
50MG/ML#
200MG/ML#
100MG/ML#
50MG/ML#
200MG/ML#
100MG/ML#
50MG/ML#
0.05MG.ML#
S1# S2# S3# S4#

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Zugic et al. evaluated the antioxidant activity of
Usnea barbata L. using the FRAP (ferric reducing
ability of plasma) assay for 4 extracts of Usnea
barbata L.: supercritical CO2 extract (SCE),
compared to the extracts (etheric, ethanolic and
aqueous) obtained with conventional techniques.
The obtained results showed that SCE had the best
antioxidant potential among the investigated
extracts with FRAP value, being almost twice
higher in comparison to the etheric fraction of
Soxhlet extract, followed by a lower antioxidative
potential of ethanolic fraction of Soxhlet extract
and, finally, aqueous extract [21].
Conclusions
Our research on Usnea barbata L. from Călimani
Mountains, Suceava County, România, showed a
2115.29 mg% usnic acid content in the acetonic
extract.
All the analysed extracts in different solvents of
Usnea barbata L. showed antioxidant activity. The
results indicate that Usnea barbata L. presents a
potential to be use in the treatment of oro-dental
conditions or other diseases related to oxidative
stress.
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