PHYTOTHERAPY RESEARCH

Phytother. Res. 15, 127130 (2001)

DOI: 10.1002/ptr.687

Screening of Brazilian Plant Extracts for

Antioxidant Activity by the Use of DPPH Free
Radical Method

Luciana L. Mensor1, Fabio S. Menezes1, Gilda G. Leitao2, Alexandre S. Reis1, Tereza C. dos
Santos2, Cintia S. Coube1 and Suzana G. Leitao1*
1
Departamento de Produtos Naturais e Alimentos, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias de
Saude, Bl. A, Ilha do Fundao, Rio de Janeiro, 21941-590 RJ, Brazil.
2
Nucleo de Pesquisas em Produtos Naturais, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude, Bl. H, Ilha do Fundao,
Rio de Janeiro, 21941-590 RJ, Brazil.

Brazilian plant extracts belonging to 16 species of 5 different families (71 extracts) were tested against
the stable DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free-radical. The ability to scavenge DPPH
radical was measured in these experiments by the discoloration of the solution. Ginkgo biloba and rutin,
commonly used as antioxidants for medical purposes, were used as standards. Based on our results, we
can say that as a general rule the ethanol extracts of plants belonging to the Verbenaceae family showed
lower EC50 values than the other plant extracts. Among the partitions, the more polar ones (ethyl acetate
and n-butanol) are those that generally have higher antioxidant activity (AA). Copyright # 2001 John
Wiley & Sons, Ltd.
Keywords: antioxidant activity; DPPH; free-radical; Acanthaceae; Lamiaceae; Leguminosae; Moraceae; Verbenaceae.

INTRODUCTION and Ginkgo biloba extract (EGb 761) from Tebonin

It has long been recognized that naturally occurring
substances in higher plants have antioxidant activity.
Recently, there has been increased interest in oxygen-
containing free-radicals in biological systems and their
implied roles as causative agents in the aetiology of a
variety of chronic disorders. Accordingly, attention is
being focused on the protective biochemical functions of
naturally occurring antioxidants in the cells of the
organisms containing them (Larson, 1988).
In our screening project for the search of antioxidative
agents from natural sources, we studied 71 Brazilian
plant extracts belonging to 16 species of 5 different
families, in order to ascertain their inhibitory effect
against free radicals. Antioxidant activity was measured
using the DPPH (2,2-diphenyl-1-picryl-hydrazyl-
hydrate) radical photometric assay in a process guided
by its discoloration (Xiong et al., 1996). The antioxidant
activity of these extracts was compared with standard
solutions of rutin and Ginkgo biloba.
MATERIAL AND METHODS
General. Commercial rutin was obtained from Merck1
* Correspondence to: Dr S. G. Leitao, Departamento de Produtos Naturais e
Alimentos, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro,
Centro de Ciencias da Saude, Bl. A, Ilha do Fundao, Rio de Janeiro, 21941-
590 RJ, Brazil.
E-mail: sgleitao@pharma.ufrj.br
Contract/grant sponsor: CAPES Brazil.
Contract/grant sponsor: FUJB.
Contract/grant sponsor: FAPERJ.
Copyright # 2001 John Wiley & Sons, Ltd.
40 mg oral solution. DPPH radical was purchased from
Sigma. Recordings were made in a UV-VIS Spectrometer
Shimadzu UV-2200. All reagents were of analytical
grade and obtained from Merck.
Plant Material. All plants were collected in Brazil:
Lantana trifolia L. and Lantana camara L. were collected
at Quirino, Rio de Janeiro; Vitex polygama Cham. and
Bouchea fluminensis (Vell.) Mold. were collected at
Marica, Rio de Janeiro; Vitex cymosa Bertero was
collected at Corumba, Mato Grosso do Sul; Verbena
litoralis H.B.K. was collected at Teresopolis, Rio de
Janeiro; Brillantaisia palisatii Lind. was collected at
Jardim Botanico, Rio de Janeiro; Marsypianthes cha-
maedrys (Vahl.) Kuntze was collected at Fortaleza,
Ceara; Hyptis elegans (Briquet) Briquet ex Micheli and
Hyptis tetracephala Bordignon were collected at Campo
Belo, Porto Alegre, Rio Grande do Sul; Platypodium
elegans Vog., Anadenanthera peregrina (Linn.) Speg.,
Apuleia leiocarpa (Vog.) Macbr., Pseudopiptadenia
contorta and Brosimum guianense (Aull.) Huber were
collected at Mata Boa Vista, district of Levy Gasparian,
Rio de Janeiro and Raphiodon echinus (Nees et Mart.)
Schauer was collected in the district of Santa Rita, Joao
Pessoa, Paraba.
Sample preparation. Plants were exhaustively extracted
with ethanol and evaporated to dryness under reduced
pressure. Aliquots of the extracts were solubilized in
ethanol to a final concentration of 1.0 mg/mL. L. trifolia,
B. fluminensis, H. elegans, R. equinus, B. palisatii, V.
cymosa and V. polygama ethanol extracts were parti-
tioned between water and hexane, dichloromethane, ethyl
acetate and n-butanol. These were evaporated to dryness
Received 28 July 1999
Revised 13 December 1999
Accepted 10 February 2000
128 L. L. MENSOR ET AL.

Table 1. EC50 values of ethanolic plant extracts. Rutin and Ginkgo biloba were used as standards
a
Family Plant species Plant part/organ EC50  SD

Acanthaceae Brillantaisia palisatii Flowers and fruits 158.87  4.30

Hyptis elegans Aerial parts 89.91  5.16
Hyptis tetracephala Aerial parts 36.06  3.43
Lamiaceae Marsypianthes chamaedrys Branches 63.83  0.23
Raphiodon echinus Aerial parts 61.58  0.24
Anadenanthera peregrina Leaves 11.56  0.38
Apuleia leiocarpa Leaves 155.49  2.11
Leguminosae Platypodium elegans Leaves 184.92  2.69
Pseudopiptadenia contorta Leaves 13.84  0.30
Moraceae Brosimum guianense Leaves 119.31  5.03
Lantana camara Leaves 40.99  1.87
Bark 114.40  3.74
Lantana trifolia Leaves 25.84  0.67
Bark 123.00  5.31
Fruits 51.05  3.61
Flowerings 35.91  0.71
Verbenaceae Verbena litoralis Leaves 31.08  0.29
Vitex cymosa Leaves 98.27  2.15
Vitex polygama Leaves 21.94  0.82
Rutin 14.16  0.20
Ginkgoaceae Ginkgo biloba (EGb 761) Standardized extract of the leaves 40.72  0.19
a
Values obtained from regression lines with 95% of condence level. EC50 is dened as the concentration sufcient to obtain
50% of a maximum effect estimate in 100%, SD standard deviation.

and aliquots were solubilized to a final concentration of
1.0 mg/mL. Ginkgo biloba extract and rutin were used as
standards and samples were prepared using the same
dilution procedures. Rutin was solubilized in methanol.
DPPH photometric assay. Sample stock solutions
(1.0 mg/mL) were diluted to final concentrations of
250, 125, 50, 25, 10 and 5 mg/mL, in ethanol. One mL of
a 0.3 mM DPPH ethanol solution was added to 2.5 mL of
sample solutions of different concentrations, and allowed
to react at room temperature. After 30 min the absorbance
values were measured at 518 nm and converted into the
percentage antioxidant activity (AA) using the following
formula:
AA% 100 fAbssample Absblank  100=Abscontrol g
Ethanol (1.0 mL) plus plant extract solution (2.5 mL) was
used as a blank. DPPH solution (1.0 mL; 0.3 mM) plus
ethanol (2.5 mL) was used as a negative control. The
positive controls were those using the standard solutions.
The EC50 values were calculated by linear regression
of plots where the abscissa represented the concentration
of tested plant extracts and the ordinate the average
percent of antioxidant activity from three separate tests.
Statistical analysis. The experiments were done in
triplicate. The results are given as mean  standard
deviation (SD). Students t-test was used for comparison
between two means and a one-way analysis of variance
(ANOVA) was used for comparison of more than two
means (Runyon and Haber, 1984). A difference was
considered statistically significant when p  0.05.
RESULTS
Tables 1 and 2 report the EC50 values for all plant extracts
Copyright # 2001 John Wiley & Sons, Ltd.
assayed. EC50 values obtained from regression lines
showed a good coefficient of determination (r2  0.80)
and statistical treatment (ANOVA) of data from the three
separate tests shows that all the experiments made for
each extract assayed are statistically equivalent
(p = 0.05).
DISCUSSION
DPPH is a free radical, stable at room temperature, which
produces a violet solution in ethanol. It is reduced in the
presence of an antioxidant molecule, giving rise to
uncoloured ethanol solutions. The use of DPPH provides
an easy and rapid way to evaluate antioxidants.
Based on results obtained from data in Tables 1 and 2
as well as those of statistical analysis, we can say that as a
general rule the ethanol extracts of plants belonging to
the Verbenaceae family showed lower EC50 values than
the other plant extracts.
Although the plants belonging to the Verbenaceae
family produce extracts very active against DPPH free
radical, the most active ethanol extracts were those
from the Leguminosae-Mimosoideae, A. peregrina
(EC50 = 9.82  0.49 mg/mL) and P. contorta (EC50 =
13.60  0.40 mg/mL).
Also, they were demonstrated to be better antioxidants
than Egb 761 (44.72  0.19 mg/mL). It is noteworthy that
the markedly high activity observed for these latter
extracts are in the range of activity of a pure compound
such as rutin (EC50 = 14.16  0.20 mg/mL), a single
substance well recognized as an antioxidant. Indeed,
they are more active than Egb 761 and rutin itself. Those
two plant extracts were the most active of all the ethanol
and partition extracts tested, except for the L. trifolia
ethyl acetate partition (EC50 = 8.82  0.22 mg/mL).
Except for the H. tetracephala ethanol extract, all
Lamiaceae extracts showed a similar pattern of activity,
Phytother. Res. 15, 127130 (2001)
 ANTIOXIDANT ACTIVITY IN BRAZILIAN PLANTS 129

Table 2. EC50 values of partitions. Rutin and Ginkgo biloba were used as standards
a
Family Sample Extractant EC50 Value

Verbenaceae Lantana trifolia Leaves Hexane

Dichloromethane 61.22  3.00
Ethyl acetate 8.82  0.22
n-Butanol 16.95  0.50
Lantana trifolia Flowerings Hexane
Dichloromethane 56.82  0.48
Ethyl acetate 12.30  0.06
n-Butanol 18.20  0.49
Vitex cymosa Leaves Hexane
Dichloromethane 105.12  2.07
Ethyl acetate 34.12  1.48
n-Butanol
Vitex cymosa Bark Hexane
Dichloromethane 152.20  2.07
Ethyl acetate 34.12  1.48
n-Butanol
Vitex polygama Leaves Hexane
Dichloromethane 38.35  0.48
Ethyl acetate 13.37  0.06
n-Butanol 17.24  0.49
Vitex polygama Bark Hexane
Dichloromethane 51.28  1.73
Ethyl acetate 13.21  0.57
n-Butanol 26.08  1.27
Vitex polygama Fruits Hexane
Dichloromethane 164.13  5.91
Ethyl acetate 35.23  2.29
n-Butanol
Bouchea uminensis Leaves Hexane
Dichloromethane
Ethyl acetate 91.53  0.87
n-Butanol
Bouchea uminensis Bark Hexane
Dichloromethane 149.92  2.45
Ethyl acetate 56.83  8.99
n-Butanol 130.77  1.63
Acanthaceae Brillantaisia palisatii Leaves Hexane
Dichloromethane 115.60  2.30
Ethyl acetate 45.68  0.85
n-Butanol 113.80  5.00
Brillantaisia palisatii Stems Hexane
Dichloromethane 17.40  1.00
Ethyl acetate 34.00  2.80
n-Butanol 51.08  1.20
Lamiaceae Hyptis elegans Aerial parts Hexane
Dichloromethane 231.13  0.50
Ethyl acetate 14.67  1.03
n-Butanol 58.67  4.17
Raphiodon echinus Aerial parts Hexane
Dichloromethane 98.87  2.19
Ethyl acetate 10.64  0.83
n-Butanol 95.90  2.46
Rutin 14.16  0.20
Ginkgoaceae Ginkgo biloba (EGb 761) Standardized extract of the leaves 40.72  0.19
a
Values obtained from regression lines with 95% of condence level.
Not calculated (very low activity).

with intermediate EC50 values. These values were just a
little higher than the EC50 value calculated for EGb 761
(Tables 1 and 2). Plants belonging to the Acanthaceae,
Leguminosae (subfamilies Papilionoideae and Caesalpi-
nioideae) and Moraceae were less active, using the DPPH
method. Brillantaisia palisatii (Acanthaceae) did not
show a good AA, in comparison with the other extracts
(EC50 = 168.18  9.72 mg/mL) (Table 1).
The EC50 values of the ethanol extracts of different
Copyright # 2001 John Wiley & Sons, Ltd.
plant parts of L. trifolia and L. camara showed that
extracts from the stems have lower antioxidant activity
(AA) than other plant parts such as leaves, fruits or
flowerings, the extracts from the leaves of L. trifolia
being the best of these.
Among the partitions, the more polar ones (ethyl
acetate and n-butanol) are those that generally have
higher antioxidant activity (AA). Therefore, the smaller
AA are found for the less polar partitions, the exception
Phytother. Res. 15, 127130 (2001)
130 L. L. MENSOR ET AL.
being the n-butanol partition of B. fluminensis, which
showed a very low AA.
The results obtained for the partitions of different plant
parts showed that the ethyl acetate ones exhibited the best
antioxidant results (Table 2). The dichloromethane
partition of B. palisatii (stems) was the only exception,
with an EC50 value of 17.40  1.00 mg/mL in contrast to
the value of 34.00  2.80 mg/mL obtained for its ethyl
acetate partition. In summary, it was clear that the
polarity of the extractants markedly influences the
antioxidant activity (AA).
Another feature that can be observed is that, despite the
general correlation observed between AA and polarity,
the n-butanol partitions of B. fluminensis (leaves), V.
cymosa (barks) and V. polygama (fruits) revealed a very
low activity, almost undetectable. Also, for all hexane
partitions no relevant activity could be detected.
Recent studies demonstrated that the interaction of a
potential antioxidant with DPPH depends on its structural
conformation. The number of DPPH molecules that are
reduced seems to be correlated with the number of
available hydroxyl groups (Brand-Williams et al., 1995).
It is strongly suggested that the DPPH free radical
abstracts the phenolic hydrogen of the electron-donating
molecule and this could be the general mechanism of the
scavenging action of antiperoxidative flavonols, for
example (Ratty et al., 1988).
Based on the mechanism of reduction of the DPPH
molecule extensively described in the literature (Basnet
et al., 1997; Burmistrov et al., 1997; Cotelle et al., 1996;
Fauconneau et al., 1997; Korounakis et al., 1997; Nanjo
et al., 1996; Sreejayan and Rao, 1996; Takao et al., 1994;
Touvay et al., 1986; Tseng et al., 1997) that is correlated
with the presence of hydroxyl groups on the antioxidant
REFERENCES
Basnet P, Matsumo T, Neidlein R. 1997. Potent free radical
scavenging activity of propol isolated from Brazilian
propolis. Z Naturforsh [C] 52 (1112): 828833.
Brand-Williams W, Cuvelier ME, Berset C. 1995. Use of a free
radical method to evaluate antioxidant activity. Leb-
ensmWiss A-Technol 28: 2530.
Burmistrov SO, Oparina TI, Prokopenko VM, Arutiunian AV.
1997. Antioxidant activity of the blood serum in pregnant
and nonpregnant women: a comparison of various
detection methods. Klin Lab Diagn 11: 1417.
Cotelle N, Bernier JL, Catteau JP, Pommery J, Wallet JC,
Gaydou EM. 1996. Antioxidant properties of hydroxy-
avones. Free Radic Biol Med 21: 3543.
Fauconneau B, Waffo Teguo P, Huguet F, Barrier L, Decendit
A, Merillon JM. 1997. Comparative study of radical
scavenger and antioxidant properties of phenolic com-
pounds from Vitis vinifera cell cultures using in vitro
tests. Life Sci 61: 21032110.
Gordon MH. 1996. Dietary antioxidants in disease prevention.
Nat Prod Rep 13: 265273.
Korounakis AP, Rekka EA, Korounakis PN. 1997. Antioxidant
activity of guaiazulene and protection against paraceta-
mol hepatotoxicity in rats. J Pharm Pharmacol 49: 938
942.
Larson RA. 1988. The antioxidants of higher plants. Phyto-
chemistry 27: 969978.
Molgaard P, Ravn H. 1988. Evolutionary aspects of caffeoyl
ester distribution in dicotyledons. Phytochemistry 27:
24112421.
Nanjo F, Goto K, Seto R, Suzuki M, Sakai M, Hara Y. 1996.
Scavenging effects of tea catechins and their derivatives
on 1.1-diphenyl-2-picrylhydrazyl radical. Free Radic Biol
Med 21: 895902.
Copyright # 2001 John Wiley & Sons, Ltd.
molecule, we can infer that the very good activity of the
polar extracts is probably due to the presence of
substances with an available hydroxyl group (phenolic
or not). This structural requirement could be linked to the
presence of flavonols or condensed tannins, which are
known to occur in plant species belonging to the
Leguminosae-Mimosoideae family, for example. In
species from the Verbenaceae family, the antioxidant
activity could be due to flavonols and also phenylpropa-
noid glycosides such as verbascoside, which have been
detected in a large number of species in this family
(Molgaard and Ravn, 1988). Indeed, substances such as
verbascoside (Xiong et al., 1996) and flavonoids have
already been studied as antioxidants and demonstrated to
be very active (Gordon, 1996; Rapta et al., 1995;
Yokozawa et al., 1997).
The screening of plant extracts using the DPPH free
radical method proved to be effective for the selection of
those which could have an antioxidant activity. These
extracts may be rich in radical scavengers, such as
flavonoids, known as antioxidants. Further, more de-
tailed, studies on the chemical composition of those
extracts, as well as studies with other models, such as
lipid peroxidation and in vivo assays are essential to
characterize them as biological antioxidants.
Acknowledgements
One of us (LLM) is indebted to CAPES - Brazil for a fellowship. We
wish to thank Professor Maria Thereza Loureiro Lima (in memorian),
from Departamento de Medicamentos of the Faculty of Pharmacy
UFRJ for use of the UV spectrometer. We are also indebted to FUJB
and FAPERJ for financial support.
Rapta P, Misik V, Stasko A, Vrabel I. 1995. Redox inter-
mediates of avonoids and caffeic acid esters from
propolis: an EPR spectroscopy and cyclic voltammetry
study. Free Radic Biol Med 18: 901908.
Ratty AK, Sunamoto J, Das NP. 1988. Interaction of avonols
with 1.1-diphenyl-2-picrylhydrazyl free radical. liposomal
membranes and soybean lipoxygenase-1. Biochem
Pharmacol 37: 989995.
Runyon RP, Haber A. 1984. Fundamentals of Behavioral
Statistics. Addison-Wesley: Massachusetts.
Sreejayan N, Rao MN. 1996. Free radical scavenging activity
of curcuminoids. Arzneimittelforschung 46: 169171.
Takao T, Kitatani F, Watanabe N, Yagi A, Sakata K. 1994. A
simple screening method for antioxidants and isolation
of several antioxidants produced by marine bacteria from
sh and shellsh. Biosci Biotech Biochem 58: 17801783.
Touvay C, Vilain B, Etienne A, Sirois P, Borgeat P, Braquet P.
1986. Characterization of platelet activating factor (PAF)-
acether-induced contractions of guinea pig lung strips by
selected inhibitors of arachidonic acid metabolism and by
PAF-acether antagonists. Immunopharmacology 12: 97
104.
Tseng TH, Kao ES, Chu CY, Chou FP, Lin Wu HW, Wang CJ.
1997. Protective effects of dried ower extracts of
Hibiscus sabdariffa L. against oxidative stress in rat
primary hepatocytes. Food Chem Toxicol 35: 11591164.
Xiong Q, Kadota S, Tani T, Namba T. 1996. Antioxidative
effects of phenylethanoids from Cistanche deserticola.
Biol Pharm Bull 19: 15801585.
Yokozawa T, Dong E, Wu Liu Z, Shimidzu M. 1997.
Antioxidant activity of avones and avonols in vitro.
Phytother Res 11: 446449.
Phytother. Res. 15, 127130 (2001)