Antioxidant activity of phenolic compounds in methanolic extracts of some Macedonian medicinal plants collected on Jablanica Mountain

Abstract

Recently, research activities in plant secondary metabolites are focused on their properties
as a new source of natural antioxidants. In this study, nonenzymatic antioxidant acitvity (NEAA)
of 20 Macedonian medicinal plants was determined using -carotene/linoleic acid oxidation
method. Plant species were collected on the Jablanica Mt. at the altitude from 1450 to 2000
meters and different plant parts (leaves, stems and flowers) were used for antioxidant analyses.
Results from this study shown that some plant species such as Geum coccineum L., Stachys
alpina L., Achillea millefolium L. were identified among the best sources of antioxidant compounds.
The values for NEAA were highly correlated to the concentration of total phenolics (r ! 0.56)
in plant extracts. The highest significant correlation between NEAA and phenolic amount was
noticed in methanolic extracts of leaves (r=0.689), confirmed that phenolic compounds were
dominant antioxidant components in these plant parts. Therefore, traditional Macedonian me -
di cinal plants could be used as a potential available source of products with antioxidant
properties in cosmetics, pharmaceutics and food industry.

Full text

INTRODUCTION
Medicinal plants have been used in many domains
including medicine, nutrition, flavoring, beverages, dyeing,
fragrances, cosmetics and other industrial purposes
(Exarchou et al. 2002). A large number of plant species
have been recognized to have medicinal properties and
beneficial impact on health, e.g. antioxidant activity,
digestive stimulation action, antiinflammatory, antimicro-
bial, hypolipidemic, antimutagenic and anticarcinogenic
effects (Aaby et al. 2004; Luo et al. 2004). Phar ma co lo -
gical application of medicinal plants is manly due to the
presence of antioxidative compounds in their extracts
(Hirasa and Takemasa 1998).
Reactive oxygen species (ROS), such as hydrogen
peroxide (H2O2), superoxide anion (O2·-), hydroxyl (HO·)
and hydroperoxyl (HO2·-) radicals are produced as a part
of normal plant metabolic processes. All cellular compo-
nents (proteins, polyunsaturated fatty acids, nucleic acids
and carbohydrates) are biological targets of ROS, giving
rise to metabolic and cellular disturbances. Fortunately,
within biological systems, there are antioxidant enzymatic
103
Билт. Истраж. друш. студ. биол. год. 4, 103 - 111, 2010
Antioxidant activity of phenolic compounds in methanolic
extracts of some Macedonian medicinal plants collected on
Jablanica Mountain
Oliver Tusevski2, Mirko Trpevski1, Ivana Lozanovska1, Aleksandra Talevska1, Dragana
Ugurovska1and Sonja Gadzovska-Simic2
1Biology Students Research Society (BSRS), Institute of Biology, Faculty of Natural Sciences and Mathematics,
Ss. Cyril and Methodius University, P.O. Box 162, 1000 Skopje, Macedonia.
2Institute of Biology, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University, P.O. Box
162, 1000 Skopje, Macedonia.
e-mail: sonjag@pmf.ukim.mk
Abstract
Recently, research activities in plant secondary metabolites are focused on their properties
as a new source of natural antioxidants. In this study, nonenzymatic antioxidant acitvity (NEAA)
of 20 Macedonian medicinal plants was determined using -carotene/linoleic acid oxidation
method. Plant species were collected on the Jablanica Mt. at the altitude from 1450 to 2000
meters and different plant parts (leaves, stems and flowers) were used for antioxidant analyses.
Results from this study shown that some plant species such as Geum coccineum L., Stachys
alpina L., Achillea millefolium L. were identified among the best sources of antioxidant compounds.
The values for NEAA were highly correlated to the concentration of total phenolics (r ≥ 0.56)
in plant extracts. The highest significant correlation between NEAA and phenolic amount was
noticed in methanolic extracts of leaves (r=0.689), confirmed that phenolic compounds were
dominant antioxidant components in these plant parts. Therefore, traditional Macedonian me -
di cinal plants could be used as a potential available source of products with antioxidant
properties in cosmetics, pharmaceutics and food industry.
Key words: Antioxidant activity, flavonoids, medicinal plants, phenolic compounds,
reactive oxygen species.
Извод
Истражувањата за фитохемискиот состав на лековитите растенија се насочени кон
идентификацијата на секундарните метаболити и нивните антиоксидантни својства. Во
овој труд се презентирани резултатите за неензимска антиоксидативна активност (НЕАА)
кај 20 лековити растенија колектирани на планината Јабланица на надморска височина
од 1450 до 2000 m. Кај растенијата беше анализирана НЕАА во метанолни екстракти од
листот, стеблото и цветот. Според добиените резултати Geum coccineum L., Stachys alpina
L., Achillea millefolium L., беа селектирани како растенија со највисока НЕАА. Сигнификантно
позитивна корелација помеѓу НЕАА и содржината на феноли беше забележана во ек с -
трактите од листот, што покажува дека овие секундарни метаболити претставуваат главни
ан тиоксидантни соединенија во растителните екстракти. Лековитите растенија од флората
на Република Македонија претставуваат богат извор на антиоксидантни соединенија кои
можат да имаат примена во подготовната на различни препарати во козметичката,
фармацевтската и прехранбената индустрија.
Клучни зборови: aнтиоксидантна активност, флавоноиди, лековити растенија,
феноли, слободни кислородни радикали.

systems (superoxide dismutase, glutathione peroxidase,
catalase) and nonenzymatic chemical scavengers (α-
tocopherol, β-carotene, ascorbic acid, glutathione, uric
acid). All of these antioxidant systems are able to remove
oxygen free radicals formed in cells and thus protect
against oxidative damage (Martinez-Cayuela 1995).
Plant extracts also contain a wide variety of free radical
scavenging molecules, such as phenolics, flavonoids,
anthocyanins and other secondary metabolites which
are defined as antioxidant compounds (Larson 1988).
The antioxidant activity of plant extracts caused
mainly by phenolic compounds has been demonstrated
in many studies over recent years (Cai et al. 2004).
Singlet oxygen quenching and thus decreasing of local
oxygen concentrations has been suggested to the pres-
ence of phenolic compounds (Luiz et al. 2002). Phenolic
compounds are also thought to be capable of regenerating
endogenous α-tocopherol in the phospholipid bilayer of
lipoprotein particles back to its active antioxidant form
(Rice-Evans and Miller 1996). Namely, phenolics are
also known to inhibit various types of oxidizing enzymes
(Cos et al. 1998). These multiple potential mechanisms
of antioxidant action make the diverse group of phenolic
compounds an interesting target in the search of health-
beneficial phytochemicals (Hudson and Lewis 1983).
Flavonoids belong to a large family of phenolic com-
pounds with a common diphenylpropane structure
(C6C3C6) with different degrees of hydroxylation, oxidation
and substitution. These compounds are also called
polyphenols and commonly occur as glycosides in plants
(Pietta 2000). As antioxidants, flavonoids have been
reported to be able to interfere with the activities of
enzymes involved in ROS generation, quenching free
radicals, chelating transition metals and rendering them
redox inactive (Heim et al. 2002).
The main objectives of this study were: 1) to evaluate
the nonenzymatic antioxidant activity in methanolic
extracts from 20 medicinal plants collected from Jablanica
Mt., Republic of Macedonia; 2) to evaluate total phenolic
and flavonoid contents in these plant extracts; 3) to show
statistical relationships between phenolic content and
antioxidant activity of the extracts. This evaluation is
related to the antioxidant activity of various phenolic
compounds in order to find out new potential sources of
natural antioxidants. Results from this preliminary study
will provide a better understanding of the antioxidant
properties of these plants and allow the identification of
plants with high antioxidant activity for further research
activities and development into value-added foods and
nutrients.
MATERIAL AND METHODS
Plant material
Twenty medicinal plant species from nine families
(Tab. 1) were collected in July 2006 on the Jablanica
Mt. (in the vicinity of village Gorna Belica), Republic of
Macedonia at the altitude from 1450 to 2000 m.
Collected plants were used for determination of antiox-
104 Bull. Biol. Stud. Res. Soc. Vol 4, 103 - 111, 2010
O. Tusevski et al.: Antioxidant activity of phenolic compounds in methanolic extracts of some Macedonian medicinal plants
collected on Jablanica Mountain
Tab. 1 Ethnopharmacologic data of the studied plants.
Таб. 1 Етнофармаколошки податоци за испитуваните растенија.
Plant species
(растителен вид)
Family
(фамилија)
Active ingredient (активна
компонента)
Medicinal properties (леко-
вити својства)
References
(референци)
Achillea abrotanoides L.
Achillea atrata L.
Achillea chrysocoma L.
Achillea holosericea L.
Achillea millefolium L.
Asteraceae Asparagin, Coumarins,
Essential oils, Flavonoids,
Isovalerianic acid, Salicylic
acid, Sterols, Tannins
antiseptic, antispasmodic, ast ri -
ngent, carminative, cholago gue,
diaphoretic, digesti ve, emme -
nagogue, odontalgic, stimulant,
vasodilator, vulnera ry
Candan et al. 2003
Atropa belladonna L. Solanaceae Alkaloids; Asparagine, Cho -
line, Hyoscyamine (atropine),
Phy to sterol, Scopolamine,
Sco po letin, Scopolin, Tannins
analgesic, antidote, antispa -
smodic, diuretic, hallucinogenic,
mydriatic, narcotic, se dative
Kursinszki et al.
2005
Echium vulgare L. Boraginacea Kaempferol-3-neohesperido-
side, Uridine, Dihy dro xy phe -
nyllactic acid, Rosmarinic acid
antimicrobial, antitussive, aphro-
disiac, demulcent, diaphoretic,
diuretic, pectoral, vulnerary
Kuruüzüm-Uz et
al. 2004
Epilobium angustifolium
L.
Onagraceae Caffeic acid, Chlorogenic acid,
Ci nnamic acid, Ferulic acid,
Gallic acid, Gentisic acid, Pro -
to catechuic acid, Vitamin
A,Vitamin C
antimicrobial, antispasmodic,
astringent, demulcent, emolli-
ent, hypnotic, laxative, tonic
Battinelli et al.
2001
Galium verum L. Rubiaceae Anthraquinones, Coumarin,
Iridoid glucosides, Tannins
antispasmodic, astringent, diu -
re tic, foot care, lithontripic, vulne -
rary
Skovgaard
Rasmussen et al.
2006
Gentiana verna L. Gentianacea Gentisein, Isoorientin,
Loganin, Secologanin,
Sweroside, Xanthones
analgesic, antibacterial, anticho -
li nergic, anti-inflammatory, an ti -
ps ychotic, antiviral, hepatop ro -
te ctive, hypoglycemic, sedat ive
Jensen and
Shripsema 2002
Geum coccineum L. Rosaceae Ellagic acid, Glycosides,
Proanthocyanidins, Tannins,
Triterpenoids
antioxidant, vasorelaxant,
hypotensive
Oszmianski et al.
2007

idant acitivity, phenol and flavonoid contents.
Phytochemical analyses were made in different plant
parts such as stems, leaves and flowers. The ethnophar-
macologic data (scientific names, family names, active
ingredients and medicinal properties) of the plant species
are detailed in Tab. 1.
Plant extraction
The collected plant material was allowed to air dry
and afterwards was ground to fine powder. The ground
samples (0.2-0.5 g) were extracted with 80% (v/v)
methanol for 24 hours at 4ºC. After centrifugation (15
min. at 13000 rpm), supernatant was used for determi-
nation of antioxidant activity of plant extracts and quan-
tification of phenol and flavonoid contents.
Antioxidant assay with β-carotene/linoleic acid
method
Antioxidant properties of soluble methanolic extracts
were estimated by the β-carotene/linoleic acid oxidation
method adapted from Marron et al. (2002). This method
was based on the ability of plant extracts to decrease
oxidative losses of β-carotene in a β-carotene/linoleic
acid emulsion. During oxidation, an atom of hydrogen is
abstracted from the active bis-allylic methylene group of
linoleic acid located on carbon-11 between two double
bonds (Frankel 1998). The pentadienyl free radical so
formed then attacks highly unsaturated β-carotene mol-
ecules in an effort to reacquire a hydrogen atom. As the
β-carotene molecules lose their conjugation, the
carotenoids lose their characteristic orange colour.
Fortunately, this process can be monitored spectropho-
tometrically. The presence of a phenolic antioxidant can
hinder the extent of β-carotene destruction by „neutral-
izing“ the linoleate free radical and any other free radicals
formed within the system. Hence, this forms the basis
by which plant extracts can be screened for their antiox-
idant potential (Amarowitz 2003). The reaction mixture
was prepared as follows: methanolic extract was adjusted
with 80 % (v/v) methanol and linoleic acid/β-carotene
emulsion was added. The mixture was heated to 50°C.
The control consisted of 80 % (v/v) methanol and β-
carotene/linoleic acid emulsion. Absorbance was meas-
ured at 470 nm every 15 minutes for 45 minutes. Results
were calculated as the ratio of β-carotene protection of
the extract to the control (80 % methanol).
Extraction of antioxidant compounds in different plant
organs (leaves, stems and flowers) of plant material was
made with 80% methanol. Several intermediate extrac-
tions (acetone, ethanol, methanol) have been used to
ensure a maximum extraction of the available antioxidants
(Kahkonen et al. 1999; Mantle et al. 2000). According to
Gadzovska et al. (2007), antioxidant activities in 80%
methanolic extracts were stable for several days stored
at 4ºC. In this study, as an antioxidant test reaction heat-
induced oxidation of an aqueous emulsion system of β-
carotene and linoleic acid was used. In this particular
model, β-carotene undergoes rapid discoloration in the
absence of an antioxidant.
Phenolic compounds assay
Phenolic quantification was performed as described
by Gadzovska et al. (2007). The method used to deter-
mine total soluble phenolic contents was based on the
reduction of phospho-molybdene/phospho-tungstate
present in the Folin-Ciocalteau reagent. Total phenol
content was determined as follows: methanolic extract
105
Билт. Истраж. друш. студ. биол. год. 4, 103 - 111, 2010
О. Тушевски и сор.:Антиоксидантна актив. на фенолни соед. во метанолни екстракти кај некои македонски леко-
вити растенија колектирани на планината Јабланица
Plant species
(растителен вид)
Family
(фамилија)
Active ingredient (активна
компонента)
Medicinal properties (лекови-
ти својства)
References
(референци)
Lotus corniculatus L. Fabaceae Flavonoids, Glycosides, Phy to -
alexins (Sativan and Ves titol),
Polyphenols, Proa nt ho cyanidins,
Tannins
anti-inflammatory, antispasmodic,
cardiotonic, carminative, feb ri fuge,
hypoglycaemic, restorati ve, seda-
tive, tonic, vermifuge
Hedqvist et al.
2000
Mentha longifolia L. Lamiaceae Essential oils (Carvone, Men t hol,
Menthone, Menthofuran, Pipe r i -
to ne, Pinene, Sabinene)
abortifacient, anodyne, antiseptic,
antispasmodic, carminative, chol-
agogue, diaphoretic, refrige rant,
stomachic, tonic, vasodilator
Ruiz del Castillo
et al. 2004
Myosotis sylvatica L. Boraginaceae Essential oils, Flavonoids,
Tannins
astringent, ophthalmic Winkworth et al.
2002
Onobrychis scardica
Griseb.
Fabaceae Flavonoids, Flavanols,
Isoflavones, Glycosides,
Proanthocyanidines
stomachic, tonic Lu et al., 2000
Salvia verticillata L. Lamiaceae Cryptotanshinone, Essential oils
(Thujone, Cineol, Camphor, Bor -
ne ol, Sabinyl acetate), Glyco si -
des, Lithospermic acid B, Ros -
ma rinic acid, Tannins
antihydrotic, antiseptic, antispas-
modic, astringent, carminative,
cholagogue, galactofuge, stimulant,
tonic, vasodilator
Tepe et al. 2007
Sideritis raeseri
Boiss.
Lamiaceae Essential oils, Flavonoids,
Flavones, Glycosides,
Terpenoids
analgesic, antibacterial, antioxi-
dant, antiinflammatory
Gabrieli et al.
2005
Stachys alpina L.
Stachys jacquinii L.
Stachys sylvatica L.
Lamiaceae Betulinic-acid, Camphor, Del phi -
nidin, Hyperoside, Oleanolic acid,
Rosmarinic-acid, Rutin, Ursolic
acid, Tannins
alterative, antibacterial, antipyretic,
antiseptic, antispasmodic, astrin-
gent, carminative, diuretic, feb ri fu -
ge, hypotensive, stomachic, styptic,
tonic, vermifuge, vulnerary
Skaltsa et al.
2003

The highest percent of NEAA in leaves was detected
in Geum coccineum L. and the lowest in Salvia verticillata
L. (Fig. 1). Мethanolic extracts from leaves with higher
antioxidant activity than 50% can be arranged as follows:
Geum coccineum > Myosotis sylvatica > Epilobium
angustifolium > Achillea millefolium > Gentiana verna
> Echium vulgare > Stachys alpina
Achillea abrotanoides was the unique plant species
with NEAA higher than 50% in stems. In the opposite,
the lowest antioxidant activity in stems was noticed in
Galium verum (Fig. 1).
Antioxidant activity in flower extracts was found with
the highest value in Geum coccineum L. in comparison
with the lowest NEAA in Achillea atrata L. (Fig. 1). Flower
methanolic extracts with antioxidant activity higher than
50% can be arranged as follows: Geum coccineum >
Stachys alpina > Achillea millefolium > Achillea chryso-
coma > Sideritis raeseri Boiss.> Salvia verticillata
Relationship between antioxidant activity (NEAA),
phenolic and flavonoid contents
The correlation coefficient (r) between antioxidant
activity (NEAA), phenolic and flavonoid contents of 20
medicinal plant extracts was determined in different plant
organs (leaves, stems and flowers). Correlations between
phenolics and flavonoids are shown in Tab. 2. Phenolic
content was significantly highly correlated with flavonoid
was mixed with Folin-Ciocaltaeu reagent (Carlo-Erba
Reagenti, Rodano, Italy) and 0.7 M Na2CO3. Samples
were incubated for 5 min at 50°C and then cooled 5 min
at room temperature. Absorbance was measured spec-
trophotometrically at wavelength of 765 nm. The con-
centration of total phenolic compounds was calculated
using (+)-catechin (0-10 mg·mL-1) as a standard.
Flavonoid assay
Flavonoid contents were determined in methanolic
extracts with the method described by Markham (1993).
The extracts were filtered through Sep-pack C18 cartridges
to exclude chlorophyll and carotenoid pigments.
Spectrophotometric measurements of the absorbance
were made at 360 nm. Molar extinction coefficient of
quercetin (ε360=13.6 mM-1·cm-1) was used to determine
total flavonoid amounts.
Statistical analysis
The extractions were independently repeated twice
under the same conditions and all analyses were per-
formed in triplicate. The statistical analyses were per-
formed with the SPSS statistical software program (SPSS
version 11.0.1 PC, USA, IL).
RESULTS
In this study, 20 plant species were collected on the
Jablanica Mt., Republic of Macedonia and were selected
according to their traditional medicinal properties. Plant
species have been recognized for their therapeutic value
and/or usage for treatment of different disorders (Tab.
1). The results for phenolic and flavonoid contents were
presented in our previous study (Trpevski et al. 2007).
Correlations between antioxidant activity, phenol and
flavonoid contents in different extracts from leaves, stems
and flowers of the collected plant species are shown in
this study.
Antioxidant activity
Methanolic extracts from leaves, stems and flowers
of different medicinal plants were estimated for their
nonenzymatic antioxidant activities (NEAA), (Fig. 1). The
NEAA exhibited extremely large variation in different
plants, as well in plant organs. Percent of NEAA was
ranged from 19.04% to 68% in leaves, from 7.98% to
53% in stems and from 21.83% to 81.62% in flowers.
106 Bull. Biol. Stud. Res. Soc. Vol 4, 103 - 111, 2010
O. Tusevski et al.: Antioxidant activity of phenolic compounds in methanolic extracts of some Macedonian medicinal plants
collected on Jablanica Mountain
Fig. 1 Total nonenzymatic antioxidant activity (% NEAA) of extracts from studied plants.
Сл. 1 Вкупна неензимска активност (% НЕАА) на екстрактите од испитуваните растенија.
1. Achillea abrotanoides L., 2. Achillea atrata L., 3. Achillea chrysocoma L., 4. Achillea holosericea L., 5. Achillea
millefolium L., 6. Atropa belladonna L., 7. Echium vulgare L., 8. Epilobium angustifolium L., 9. Galium verum L.,
10. Gentiana verna L., 11. Geum coccineum L., 12. Lotus corniculatus L., 13. Mentha longifolia L., 14. Myosotis
sylvatica L., 15. Onobrychis scardica Griseb., 16. Salvia verticillata L., 17. Sideritis raeseri Boiss., 18. Stachys
alpina L., 19. Stachys jacquinii L., 20. Stachys sylvatica L.

107
Билт. Истраж. друш. студ. биол. год. 4, 103 - 111, 2010
О. Тушевски и сор.:Антиоксидантна актив. на фенолни соед. во метанолни екстракти кај некои македонски леко-
вити растенија колектирани на планината Јабланица
content (r=0.803, p<0.001) in flower extracts. Also, sig-
nificant correlation (r=0.509, p<0.01) was found between
phenolic and flavonoid content in stem extracts. In the
opposite, there was no correlation between phenolic
and flavonoid contents in leaf extracts.
The overall relationship between antioxidant activity
and total phenolic content for all tested extracts of different
plant organs (leaves, stems and flowers) was found sig-
nificantly positive and linear correlations between antiox-
idant activity and phenolic contents are shown in Tab. 2.
The highest significant correlation between antioxidant
activity and phenolic amount was noticed in leaf extracts
(r=0.689, p<0.001).
Tab. 2. Correlation coefficient (r) between antioxidant
ac tivity, phenol and flavonoid content in extracts from
di fferent plant organs of Macedonian medicinal plants
collected on the Jablanica Mt. (n=20).
Таб. 2 Коефициент на корелација (r) помеѓу ан ти ок -
си дантната активност, содржината на феноли и фла -
во ноиди во екстракти од различни растителни органи
кај некои лековити растенија колектирани на пла ни -
на та Јабланица, Република Македонија (n=20).
Denoted values are significantly different (* p<0.05; **
p<0.01; *** p<0.001).
Values represent n=20±SE. ns - non significant chan-
ges.
Означените вредности се сигнификантно различни
(* p<0.05; ** p<0.01; *** p<0.001).
Вредностите претставуваат n=20±SE. ns -
несигнификантни промени.
The coefficient of determination (r2) between NEAA,
phenolic and flavonoid contents in leaf, stem and flower
extracts are shown in Fig. 2.
Coefficient of correlation (r)
Leaves Phenolic content (mg/g)
Flavonoid content (mol/g) 0.157 ns
Antioxidant activity (%) 0.689***
Stems Phenolic content (mg/g)
Flavonoid content (mol/g) 0.509**
Antioxidant activity (%) 0.564**
Flowers Phenolic content (mg/g)
Flavonoid content (mol/g) 0.803***
Antioxidant activity (%) 0.569**
А
B
C
D
E

Fig. 2 Coefficient of determination (r2) between antioxidant
activity (NEAA), phenolic and flavonoid contents in leaves
(A, B), stems (C, D) and flowers (E, F) in studied plants.
Сл. 2 Коефициент на детерминација (r2) помеѓу
содржината на феноли (x-оска) и антиоксидантната
активност – NEAA (B, D, F) и флавоноиди (A, C, E) во
листови (A, B), стебла (C, D) и цветови (E, F) кај
испитуваните растенија.
DISCUSSION
The relationships between nonenzymatic antioxidant
activity (NEAA), phenolic and flavonoid contents in 20
medicinal plants collected on Mountain Jablanica are
presented in this study. Phenolic and flavonoid contents
in different organs of evaluated plant species have been
presented in our previous study (Trpevski et al. 2007).
Results for NEAA in this experiment showed a large
variability among the plant species, as well in different
plant organs. NEAA in methanolic extracts of all studied
plants ranged from 6% to 82%. This can be apparently
explained by the influences of different environmental
factors such as location, weather conditions, altitude
etc., which could affect the level of antioxidant compounds
presented in plant extracts. However, the efficiency of
NEAA in plant extracts differs depending on the particular
assay methodology, reflecting the complexity of the
mechanisms involved in total antioxidant capacity. There
are many methods to determine NEAA of plant extracts
which differ in terms of their principle assay and exper-
imental conditions; consequently, in different methods
particular antioxidant compounds have varied total antiox-
idant potential (Cao and Prior 1998). So far as plant
phenolics constitute one of the major groups of com-
pounds acting as primary antioxidants or free radical
scavengers, it was reasonable to determine their total
amount and NEAA in the selected plant extracts.
Flavonoids as one of the most diverse and widespread
group of natural compounds are the most important nat-
ural phenolics (Agrawal 1989). These compounds pos-
sess a broad spectrum of chemicals and biological activ-
ities including radical scavenging properties.
According to our previous study (Trpevski et al.
2007), phenolic and flavonoid compounds were found
in higher amounts in leaf extracts than in flower and
stem. Mentha longifolia, Geum coccineum and Sideritis
raeseri should be considered as plants rich in soluble
phenolics. Flavonoids were the main phenolic subclass
in extracts of Lotus corniculatus. Actually, Geum coc-
cineum, was noticed as a plant reaches with phenolic
metabolites and NEAA in leaf and flower extracts, indi-
cating that phenolics are the major contributors to antiox-
idant activity in these plant organs. Harborne and Williams
(2000) suggested that polyphenols are found in highest
contents in leaves which is in accordance with the results
of our study. These authors confirmed that the biosyn-
thesis of these metabolites is accelerated by light expo-
sure and serves as a filtration mechanism against UV-
B radiation.
Results from our study showed the highest correlation
(r=0.803, p<0.001) between phenolic and flavonoid con-
tents in flower extracts which confirmed that flavonoid
metabolites are the main phenolic compounds in plant
flowers. The accumulation of high amount of phenolic
compounds such as flavonoids in flowers could suggest
the adaptive strategy of plants using antioxidants for
protection of their own reproductive organs (Matkowski
2006). Although in all studied plant species considerable
amounts of phenolic compounds were determined, no
simple correlation between phenolic and flavonoid con-
tents in leaves was established. These results suggest
a great complexity of the involved mechanisms that can
vary even among the related plant species. The apparent
discrepancy between antioxidant properties assayed by
various techniques and lack of correlation to the widely
distributed polyphenols has been mentioned by Mantle
et al. (2000). However, due to the diversity and complexity
of the natural mixtures of phenolic compounds in studied
plant extracts, it is not easy to characterize every com-
pound and assess their antioxidant activities. Different
plant extracts contain generally various phenolic com-
pounds with different antioxidant activity (Djeridane 2006).
Significant correlation between NEAA and phenolic
content was found in leaf extracts (r2=0.475), although
no significant correlation between phenolic and flavonoid
contents was registered (r2=0.025). These results suggest
that 47.5% of total phenolic compounds in leaf extracts
are responsible for their antioxidant capacity. In addition,
antioxidant activity does not necessarily correspond with
total flavonoid content. Velioglu et al. (1998) found sig-
nificant relationships between total phenolics and antiox-
idant activity in 28 flaxseed and cereal products. These
authors suggested that besides the total contents of
phenolics, other individual active phenolic compounds
can play a major role in the antioxidant activity of plant
materials.
The unclear relationship between NEAA and phe-
nolics may be explained in numerous ways. In fact, total
phenolic content doesn’t incorporate all antioxidant com-
pounds. In addition, the synergism between antioxidants
in the mixture makes the NEAA not only dependent on
the concentration, but also on the structure and interaction
between antioxidants (Djeridane 2006). Furthermore,
the Folin–Ciocalteu assay gives a crude estimate of total
phenolic compounds presented in plant extracts. This
assay is not specific only to phenols, but many interfering
compounds may react with the reagent giving apparently
elevated phenolic concentrations (Prior et al. 2005).
Moreover, various phenolic compounds respond differ-
ently in this assay, depending on the number of phenolic
groups they have (Singleton and Rossi 1965). Therefore,
total phenolic content does not incorporate necessarily
all antioxidants that may be present in an extract.
Kahkonen et al. (1999) reported that no significant cor-
108 Bull. Biol. Stud. Res. Soc. Vol 4, 103 - 111, 2010
O. Tusevski et al.: Antioxidant activity of phenolic compounds in methanolic extracts of some Macedonian medicinal plants
collected on Jablanica Mountain
F

109
Билт. Истраж. друш. студ. биол. год. 4, 103 - 111, 2010
О. Тушевски и сор.:Антиоксидантна актив. на фенолни соед. во метанолни екстракти кај некои македонски леко-
вити растенија колектирани на планината Јабланица
relation was found between phenolic content and antiox-
idant activity in 92 plant extracts. These authors showed
that different phenolic compounds have different colori-
metric responses to Folin–Ciocalteu reagent. Thus, the
antioxidant activity of an extract can not be predicted on
the basis of its total phenolic content. In addition, Pokorny
(1987) reported that substituted monophenolic com-
pounds exhibited a greater antioxidant activity than
unsubstituted monophenolics. Hence, this may explains
the unequal correlation between phenolic content and
antioxidant activity in our tested plant species. Plant
species Galium verum and Echium vulgare with similar
contents of phenolics (28.6 mg·g-1 and 34.46 mg·g-1,
respectively) have varied antioxidant activities (7.98 %
and 36.97 %, respectively). In our study, extracts of two
plant species of Lamiaceae family (Stachys jacquinii
and Salvia verticillata) were compared for their antioxidant
activity. NEAA in Stachys jacquinii extracts was uniformly
distributed throughout the plant: leaves (48.15 %), stems
(42 %) and flowers (46.15 %). On the other hand, in
extracts of Salvia verticillata differential distribution of
antioxidant activity within the plant: leaves (19.04 %),
stems (34.31 %) and flowers (52.38 %) was noticed.
Therefore, it is not possible to predict theNEAA of a cer-
tain plant species on the basis of its taxonomic classifi-
cation.
In this study, β-carotene/linoleic acid assay was
used for determination of NEAA of plant extracts.
However, before undertaking any such systematic esti-
mation of antioxidant activity in plant extracts it is nec-
essary to develop alternative standard assay procedures
that gives opposite data for particular plant species
(Wong et al. 2006). This may in turn involve taking a
consensus of data obtained from a number of additional
methods for measuring NEAA.
Nowadays, there are no publications for antioxidant
properties of medicinal plants, traditionally used in
Macedonia. Research activities for the most ofexamined
plant species are very scarce in the literature and the
existing data refer usually to separate species or a select-
ed family. In the recent years, plant medicinal species
from Macedonian flora become an important source for
pharmaceutical industry. Therefore, Jablanica Mt. can
offer an important number of plant medicinal species
which could be used in phytotherapy, as well as a renew-
able source of natural remedies. There were identified
about 100 plants of pharmaceutical interest, among
which the 20 presented in this study have a particular
importance because of their antioxidant activity.
CONCLUSIONS
In this study, methanolic extracts of 20 medicinal
plants collected on the Jablanica Mt. were found to pos-
sess antioxidant activitiy as determined by a β-
carotene/linoleic acid model system. This model system
has revealed that a wide range of nonenzymatic antiox-
idant activity (NEAA) exists among these tested plants.
Due to the elevated values of NEAA, plant species Geum
coccineum L., Stachys alpina L., Achillea millefolium L.,
Myosotis sylvatica L., Achillea chrysocoma L., Epilobium
angustifolium L., Achillea abrotanoides L., may be con-
sidered as an interesting source of natural antioxidants
for medicinal and commercial uses.
A highly significant positive correlation between antiox-
idant activity and phenolic content in leaf extracts indicates
that phenolic compounds are the major contributor to
antioxidant activity in evaluated plant species. Antioxidant
capacities of plant extracts are influenced by chemical
composition of extracts and conditions of test system
which can not be fully described with a single method.
Therefore, it is necessary to perform more than one type
of antioxidant measurement to take into account the var-
ious mechanisms of antioxidant action.
ACKGNOWLEDGEMENTS
We acknowledge Dr Ljupco Melovski and Dr Slavco
Hristovski (Department of Plant Еcology), Institute of
Biology, Faculty of Natural Sciences and Mathematics,
Ss. Cyril and Methodius University, Skopje, Macedonia
for their help in plant collection and taxonomy.
REFERENCES
Aaby, K., Hvattum, E., Skrede, G. (2004): Analysis of
fla vonoids and other phenolic compounds using
high-performance liquid chromatography with
coulometric array detection: Relationship to antiox-
idant activity. J. Agric. Food Chem. 52: 4595–
4603.
Agrawal, P. K. (1989): Carbon-13 NMR of flavonoids.
New York: Elsevier.
Amarowicz, R., Pegg, R. B., Moghaddam, P. R., Barl,
B., & Weil, J. A. (2004): Free-radical scavenging
capacity and antioxidant activity of selected plant
species from Canadian prairies. Food Chem. 84:
551–562.
Battinelli L, Tita, B., Evandri, M.G., Mazzanti, G. (2001):
Antimicrobial activity of Epilobium spp. extracts.
Il Farmaco 56: 345-348.
Cai, Y. Z., Luo, Q., Sun, M., Corke, H. (2004): Antioxidant
activity and phenolic compounds of 112 traditional
Chinese medicinal plants associated with anti-
cancer. Life Sci. 74 (17): 2157–2184.
Candan F, Unlu, M., Tepe, B., Daferera, D., Polissiou,
M., Sökmen, A., Askin Akpulat, H. (2003):
Antioxidant and antimicrobial activity of the essen-
tial oil and methanol extracts of Achillea millefolium
subsp. millefolium Afan. (Asteraceae). J.
Ethnopharm. 87: 215-220.
Cao, G., Prior, R. L. (1998): Comparison of different ana-
lytical methods for assessing total antioxidant
capacity of human serum. Clin. Chem. 44: 1309–
1315.
Cos, P., Ying, L., Calome, M., Hu, J. P., Cimanga, K.,
van Poel, B., Pieters, L., Vlietinck, A. J., Van den
Berghe, D. (1998): Structure-activity relationship
and classification of flavonoids as inhibitors of
xanthine oxidase and superoxide scavengers. J.
Nat. Prod. 61: 71–76.
Djeridane, A., Yousfi, M., Nadjemi, B., Boutassouna, D.,
Stocker, P., Vidal, N. (2006): Antioxidant activity
of some algerian medicinal plants extracts con-
taining phenolic compounds. Food chem. 97: 654-
660.
Exarchou, V., Nenadis, N., Tsimidou, M., Gerothanassis,
I. P., Troganis, A., Boskou, D. (2002): Antioxidant
activities and phenolic composition of extracts
from Greek oregano, Greek sage and summer

savory. J. Agric. Food Chem. 50(19): 5294–5299.
Frankel, E. N. (1998): Hydroperoxide formation. In Lipid
oxidation (pp. 23–41). Dundee: The Oily Press.
Gabrieli C. N, Kefalas, P. G., Kokkalou, E. L. (2005)
Antioxidant activity of flavonoids from Sideritis
raeseri. J. Ethnopharm. 96: 423-428.
Gadzovska, S., Maury, S., Delaunay, A., Spasenoski,
M., Joseph, C., Hagege, D. (2007): Jasmonic acid
elicitation of Hypericum perforatum L. cell sus-
pensions and effects on the production of phenyl-
propanoids and naphtodianthrones. Plant Cell
Tissue and Organ Culture, 89: 1–13.
Harborne, K. B. and Williams, C.A. (2000): Advances in
flavonoid research since 1992. Phytochemistry,
55: 481-504.
Hedqvist H, Mueller-Harvey, I., Reed, J. D., Krueger, C.
G., Murphy, M. (2000) Characterisation of tannins
and in vitro protein digestibility of several Lotus
corniculatus varieties. Anim. Feed Sci. Techn. 87:
41-56
Heim, K. E., Tagliaferro, A. R., Bobilya, D. J. (2002):
Flavonoid antioxidants: chemistry, metabolism
and structure–activity relationships. J. Nutr.
Biochem. 13: 572–584.
Hirasa, K., Takemasa, M. (1998): Spice science and
technology. New York: Marcel Dekker.
Hudson, B. J. F., Lewis, J.I. (1983): Polyhydroxy flavonoid
antioxidants for edible oils. Structural criteria for
activity. Food Chem. 10: 47–55.
Jensen S. R., Shripsema, J. (2002): Chemotaxonomy
and pharmacology of Gentianaceae. In L Struwe,
Albert, V.A., ed, Gentianaceae-systematics and
natural history. Cambridge University Press,
Cambridge, pp 573- 632
Kahkonen, M. P., Hopia, A. I., Vuorela, H. J., Rauha, J.
P., Pihlaja, K., Kujala, T. S. (1999): Antioxidant
activity of plant extracts containing phenolic com-
pounds. J. Agric. Food Chem. 47: 3954–3962.
Kursinszki L, Hank, H., László, I., Szoke, E. (2005)
Simultaneous analysis of hyoscyamine, scopo-
lamine, 6ß-hydroxyhyoscyamine and apoatropine
in Solanaceous hairy roots by reversed-phase
high-performance liquid chromatography. J.
Chromatography A 1091: 32-39
Kuruüzüm-Uz A, Güvenalp, Z., Ströch, K., Demirezer,
O., Zeeck, A. (2004): Phytochemical and antimi-
crobial investigation of Echium vulgare growing
in Turkey. Biochem. System. Ecol. 32: 833-836
Larson, R. A. (1988): The antioxidants of higher plants.
Phytochemistry 4: 969-978.
Lu, Y., Yan Sun, L., Foo, Y., McNabb, W. C., Molan, A.
L. (2000): Phenolic glycosides of forage legume
Onobrychis viciifolia. Phytochemistry 55: 67-75.
Luiz, M., Biasutti, A., Garcia, N. (2002): Micellar effects
on the scavenging of singlet molecular oxygen by
hydroxybenzenes. Red. Report, 7(1): 23–28.
Luo, Y., Cai, Q., Sun, M., Corke, H. (2004): Antioxidant
activity and phenolic compounds of 112 traditional
Chinese medicinal plants associated with anti-
cancer. Life Sci. 74: 2157–2184.
Mantle, D., Eddeb, F., Pickering, A. (2000): Comparison
of relative antioxidant activities of British medicinal
plant species in vitro. J. Ethnopharmacol. 72(1–
2): 47–51.
Markham, K. R. (1993): Flavones, flavonols and their
glycosides. In: Methods in Plant Biochemistry, ed.
J.B. Harborn, London: Academic Press, 197-235.
Marron, N., Delay, D., Petit, J., Dreyer, E., Kahlem, G.,
Delmotte, F., Brignolas, F. (2002): Physiological
traits of two Populus x euramericana clones, Luisa
Avanzo and Dorskamp, during water stress and
re-watering cycle. Tree Phys. 22: 849-858.
Martinez-Cayuela, M. (1995): Review: Oxygen free rad-
icals and human disease. Biochimie, 77: 147–
161.
Matkowski A. (2006): Plant phenolic metabolites as
antioxidants and antimutagens. In: Blume Y,
Durzan DJ, Smertenko P, editors. NATO Science
Series Vol. 371: Cell biology and Instrumentation:
UV Radiation, Nitric Oxide and Cell Death in
Plants. Amsterdam: IOS Press1-58603-574-6,
340 pp.
Oszmianski J, Wojdylo, A., Lamer-Zarawska, E., Swiader,
K. (2007): Antioxidant tannins from Rosaceae
plant roots. Food Chem. 100: 579-583.
Pietta, P. G. (2000): Flavonoids as antioxidants. J. Nat.
Prod. 63: 1035–1042.
Pokorny, J. (1987): Major factors affecting the autoxidaiton
of lipids. In H. W.-S. Chan (Ed.), Autoxidation of
unsaturated lipids (pp. 141–206). London:
Academic Press.
Prior, R. L., Wu, X., Schaich, K. (2005): Standardized
methods for the determination of antioxidant capac-
ity and phenolics in foods and dietary supplements.
J. Agric. Food Chem. 53: 4290–4302.
Rice-Evans, C. A., Miller, N. J. (1996): Antioxidant activities
of flavonoids as bioactive components of foods.
Biochem. Soc. Trans. 24: 790–795.
Ruiz del Castillo, M. L, Blanch, G. P., Herraiz, M. (2004):
Natural variability of the enantiomeric composition
of bioactive chiral terpenes in Mentha piperita. J.
Chromatography A 1054: 87-93.
Singleton, V. L., Rossi, J. A. (1965): Colorimetry of total
phenolics with phosphomolybdic-phosphotungstic
acid reagents. Am. J. Enol. Viticult. 16: 144–158.
Skaltsa, H. D., Demetzos, C., Lazari, D., Sokovic, M.
(2003) Essential oil analysis and antimicrobial
activity of eight Stachys species from Greece.
Phytochemistry 64: 743-752.
Skovgaard Rasmussen, L, Rank, C., Jensen, S. (2006)
Transfer of iridoid glucosides from host plant
Galium verum to hemiparasitic Euphrasia stricta.
Biochem. System. Ecol. 34: 763-765.
Te pe B , E mi na g ao g l u, O . , A k pu l at , H . A. , Ay d in , E . (2 0 07 )
Antioxidant potentials and rosmarinic acid levels
of the methanolic extracts of Salvia verticillata (L.)
subsp. verticillata and S. verticillata (L.) subsp.
amasiaca (Freyn & Bornm.) Bornm. Food Chem.
100: 985-989.
Trpevski, M., Lozanovska, I., Talevska, A., Ugurovska,
D., Spasenoski, M., Pavlova, V., Gadzovska, S.
(2007): Phenolic and flavonoid contents of some
medicinal plants from Jablanica Mt., Republic of
Macedonia. Proceedings of the 3rd Congress of
Ecologists of the Republic of Macedonia with
International Participation. Special Issues of the
Macedonian Ecological Society 8: 40-46.
Velioglu, Y. S., Mazza, G., Gao, L., Oomah, B. D. (1998):
Antioxidant activity and total phenolics in selected
fruit, vegetables and grain products. J. Agric. Food
Chem. 46 (10): 4113-4117.
Winkworth, R. C., Grau, J., Robertson A. W., Lockhart,
110 Bull. Biol. Stud. Res. Soc. Vol 4, 103 - 111, 2010
O. Tusevski et al.: Antioxidant activity of phenolic compounds in methanolic extracts of some Macedonian medicinal plants
collected on Jablanica Mountain

111
Билт. Истраж. друш. студ. биол. год. 4, 103 - 111, 2010
О. Тушевски и сор.:Антиоксидантна актив. на фенолни соед. во метанолни екстракти кај некои македонски леко-
вити растенија колектирани на планината Јабланица
P. J . ( 2 0 0 2 ) : T h e o r i g i n s an d e v o l u t i on o f t h e g e n u s
Myosotis L. (Boraginaceae). Mol. Phylog. Evol.
24: 180-193
Wong, S. P., Leong, L. P., Koh, J. H. W. (2006): Antioxidant
activities of aqueous extracts of selected plants.
Food Chem. 99: 775–783.
Антиоксидантна активност на
фенолни соединенија во
метанолни екстракти кај
некои македонски лековити
растенија колектирани на
планината Јабланица
Оливер Тушевски2, Мирко Трпевски1,
Ивана Лозановска1, Александра
Талевска1, Драгана Угуровска1и Соња
Гаџовска-Симиќ2
1Истражувачко друштво на студенти биолози,
Институт за Биологија, Природно-математички
факултет, 1000 Скопје
2Институт за Биологија, Природно-математички
факултет, 1000 Скопје
e-mail: sonjag@pmf.ukim.mk
РЕЗИМЕ
Антиоксидантната активност на метанолните
екстракти од 20 лековити растенија колкетирани на
планината Јабланица беше анализирана со примена
на β- каротен/линолеинска киселина методот.
Екстрактите од испитуваните органи (лист, стебло и
цвет) кај колектираните растенија покажаа активност
на неензимскиот антиоксидантен систем (НЕАА).
Geum coccineum L., Stachys alpina L., Achillea mille-
folium L., Myosotis sylvatica L., Achillea chrysocoma L.,
Epilobium angustifolium L. и Achillea abrotanoides L.
се растителни видови со релативно висока антиокси-
дантна активност.
Антиоксиднатна активност на испитуваните
екстракти беше во сингнификантно позитивна коре-
лација со содржината на феноли на листот, што пока-
жува дека фенолните соединенија учествуваат во
НЕАА кај испитуваните растителни видови.
Антиоксидантниот капацитет на растителните екс-
тракти зависи од хемискиот состав на екстрактите и
примената на различни методи за одредување на
неензимската антиоксидантна активност. Поради тоа,
неопходно е да се стандардизираат повеќе методи
за одредување на НЕАА во растителните екстракти,
со цел да се испитаат различни механизми на дејство
на антиоксидантите.
Резултатите од овој труд ќе дадат придонес
во примената на лековитите растенија како извор на
природни антиоксиданти во фармацевтската, пре-
хранбената и козметичката индустрија.