Bioresource Technology 100 (2009) 65926598

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Antioxidant activities of avonol derivatives from the leaves and stem bark
of Scutia buxifolia Reiss
Aline Augusti Boligon a, Romaiana Picada Pereira b, Andrili Cassel Feltrin a, Michel Mansur Machado b,
Vanessa Janovik a, Joo Batista Teixeira Rocha b, Margareth Linde Athayde a,*
a
b

Departamento de Farmcia Industrial, Programa de Ps-Graduao em Cincias Farmacuticas, Universidade Federal de Santa Maria, Santa Maria, RS 97105-900, Brazil
Departamento de Qumica, Programa de Ps-Graduao em Bioqumica Toxicolgica, Universidade Federal de Santa Maria, Santa Maria, RS 97105-900, Brazil

a r t i c l e

i n f o

Article history:
Received 6 January 2009
Received in revised form 23 March 2009
Accepted 24 March 2009
Available online 8 August 2009
Keywords:
Scutia buxifolia
Flavonols
HPLC-DAD
TBARS
DPPH

a b s t r a c t
This study evaluated the antioxidant activities in the leaves and stem bark fractions of Scutia buxifolia.
Cerebral lipid peroxidation (TBARS) was induced by Fe(II) and radical-scavenging activity was determined by DPPH method. FolinCiocalteu was used to determine phenolic contents. Quercetin, quercitrin,
isoquercitrin and rutin were isolated from leaf ethyl acetate fraction and their levels were measured by
high performance liquid chromatography.-photodiode array detector.IC50 (DPPH) varied from 4.35 1.30
to 29.55 0.54 lg/mL for stem bark and from 6.50 0.40 to 30.54 1.14 in the leaves. Ethyl acetate and
butanolic fractions caused a sharp fall in TBARS production with IC50 from 2.93 2.17 to 40.46 2.51 lg/
mL for the leaves and 0.66 0.17 to 27.3 1.23 for the stem bark. Results obtained indicated that S. buxifolia has a great potential to prevent disease caused by the overproduction of free radicals and also it
might be used as a potential source of natural antioxidant agents.
2009 Published by Elsevier Ltd.

1. Introduction
The consumption of natural antioxidants, such as avonoids
and other phenolic compounds present in most plants, has been
associated with a lower incidence of diseases related to oxidative
stress. The interest in antioxidant agents is due to the currently
growing demand from the pharmaceutical industry for natural
antiaging and anticarcinogenic bioactive compounds that possess
health promoting benets. Special attention has been given to
the association between neurodegenerative diseases such as Parkinsons (PD) and Alzheimers (AD) with oxidative stress (Behera
et al., 2008; Papetti et al., 2006).
The World Health Organization (WHO) estimates that 6580%
of the population of the developing countries depends on medicinal plants for basic pharmaceutical care (WHO, 1998). Accordingly,
the WHO has been stimulating studies involving medicinal plants,
with the aim of evaluating the potential benets of using them.
There is still a lack of knowledge of the clinical efcacy and safe
use of many of these remedies. Brazil has a great diversity of
plants, which increases the chances of identication of substances
with pharmacological activities. Moreover, plants are known to offer excellent perspectives for the discovery of new therapeutic
products.

* Corresponding author.
E-mail address: magareth@smail.ufsm.br (M.L. Athayde).
0960-8524/$ - see front matter 2009 Published by Elsevier Ltd.
doi:10.1016/j.biortech.2009.03.091

In recent years, there has been a great interest in nding natural antioxidants from plant materials. Numerous crude extracts
and pure natural compounds from plants were reported to have
antioxidant and radical-scavenging activities (Barla et al., 2007;
Chang and Sung, 2008; Chua et al., 2008; Nascimento et al.,
2006). Within the antioxidant compounds, avonoids and phenolics, with a large distribution in nature, have been studied more
comprehensively (Amico et al., 2008; Maksimovic et al., 2005;
Tung et al., 2007).
Scutia buxifolia Reissek belongs to the Rhamnaceae family and is
popularly known as coronilha. It is a native plant from South
America, with a dispersion area that include Rio Grande do Sul
state in Brazil, Argentina and Uruguay. Infusions of the root bark
are popularly used as a cardiotonic, antihypertensive and diuretic
(Wasicky et al., 1964). In spite of the popular use, little is known
about its secondary metabolites, with the exception of some cyclopeptide alkaloids isolated by Morel et al. (2005) which demonstrated antimicrobial activities.
Preliminary phytochemical investigations of our group concerning the leaves and the stem bark of S. buxifolia indicated the presence of a large number of phenolic compounds, including
avonoids. This feature, allied to the importance of the oxidative
stress in the pathogenesis of various diseases, and the continuous
use popularly recommended as a remedy, prompted us to better
evaluate the potential antioxidant properties of this plant. At the
same time, phytochemical procedures led to the isolation and the
identication by NMR spectroscopy of four quercetin-derived

A.A. Boligon et al. / Bioresource Technology 100 (2009) 65926598

avonols, which may play a special role in the antioxidant activities described in this study.

6593

2. Methods

eter. The total phenolic content was expressed in milligrams equivalents of gallic acid (GAE) per gram of each fraction. The equation
obtained for the calibration curve of gallic acid in the range of
0.0050.030 mg/mL was Y = 11.969  0.0454 (r = 0.9984).

2.1. Chemicals, apparatus and general procedures

2.5. Radical-scavenging activity DPPH assay

All chemicals were of analytical grade. Silica Gel 60, Silica Gel 60
F254 coated plates, solvents for the extractions and analytical procedures, dichloromethane, ethyl acetate, ethanol, methanol, nbutanol, acetonitrile, ascorbic acid, and gallic acid were purchased
from Merck (Darmstadt, Germany). FolinCiocalteau phenol reagent 2 N, DPPH radical (1,1-diphenyl-2-picrylhydrazyl), rutin
and quercetin were acquired from Sigma Chemical Co. (St. Louis,
MO, USA). NMR spectra were carried out on a Bruker AMX 400
spectrometer equipped with a broadband 5-mm probe, using a
spectral width of 10 ppm (parts per million). Chemical shifts were
expressed as ppm relative to the TMS. Deuterated methanol was
used as solvent for the samples.

The antioxidant activity of the fractions and the crude extracts

was evaluated by monitoring their ability in quenching the stable
free radical DPPH, according to a slightly modied method previously described by Choi et al. (2002). Spectrophotometric analysis
was used to measure the free radical-scavenging capacity (RSC)
and to determine the scavenging concentration or inhibitory concentration (IC50). The DPPH quenching ability was expressed as
IC50 (the extract concentration (lg/mL) required to inhibit 50% of
the DPPH in the assay medium).
Six different ethanol dilutions of each fractions and crude extracts at 250, 125, 62.5, 31.25, 15.62 and 7.81 lg/mL were mixed
with 1.0 mL of DPPH 0.3 mM in ethanol solution. After 30 min,
absorption was measured at 518 nm, where the radical DPPH
shows maximum absorption. A solution of DPPH (1 mL; 0.3 mM)
in ethanol (2.5 mL) was used as a negative control and ascorbic
acid in the same concentrations used for the fractions and the
crude extracts provided the positive control. Ethanol was used to
calibrate the spectrophotometer. The test was performed in triplicate and the calculation of the antioxidant activity followed the
equation:

2.2. Plant collection and extractions

Stem bark and leaves of S. buxifolia were collected in Dom Pedrito (Rio Grande do Sul State of Brazil) in October of 2007 (coordinates 30590 0900 S and 54270 4400 W). A dried voucher specimen is
preserved in the herbarium of the Department of Biology at Federal
University of Santa Maria by register number SMBD 10919.
The parts of the plant were dried at room temperature and powdered in a knife mill. The powder of stem bark (651.52 g) and
leaves (372.34 g) were macerated separately at room temperature
with ethanol 70% for a week with daily shake-up. After ltration,
the two extracts were evaporated under reduced pressure to
remove the ethanol. Each extract was suspended in water and
partitioned successively with dichloromethane, ethyl acetate and
n-butanol (3  200 mL for each solvent).

% inhibition 100 

Abssample  Absblank  100

Abscontrol

where Abssample is absorbance of each fraction; Absblank is absorbance of fractions without adding the DPPH; Abscontrol is absorbance
the solution of ethanol in DPPH. The percentage of inhibition was
calculated and a graphic of percentage of inhibition versus concentration was constructed. Correlation coefcients were optimised
(Tsimogiannis and Oreopoulou, 2006).

2.3. Phytochemical screening and characterization of the fractions

2.6. In vitro Fe(II)-induced lipid peroxidation in rat brains
Analytical thin-layer chromatography (TLC) were carried out
using
ethyl
acetate:methanol:water = 100:13.5:10
(v/v/v),
chloroform:ethanol:water = 80:40:5 (v/v/v), lower phase and
chloroform:ethanol:water = 60:40:5 (v/v/v), as systems to TLC
investigation of alkaloids, avonoids, tannins, sterols, and triterpenoids (Wagner et al., 1984; Schubert et al., 2007). The solvent
system: ethyl acetate:formic acid:acetic acid:water = 100:11:11:27
(v/v) was used for the TLC investigation of avonoid glycosides
(Wagner et al., 1984). The separated compounds were observed
under UV light (254 and 366 nm) and detection were performed
with anisaldehydeH2SO4/100 C, and Dragendorffs reagent.
Authentic samples of quercetin, rutin, chlorogenic and caffeic acids
were used as reference standards. Authentic samples of the cyclopeptides alkaloids adouetine-Y and scutianines-D and C isolated
from S. buxifolia obtained from Dr. A. Morel (Chemistry Department of Federal University of Santa Maria) were also used as reference standards.
2.4. Determination of total phenolics
The determination of total phenolic content was performed by
the FolinCiocalteu method (Chandra and Mejia, 2004). Briey,
0.5 mL of 2 N FolinCiocalteu reagent was added to a 1 mL of each
sample (0.15 mg/mL) and this mixture was allowed to stand for
5 min before the addition of 2 mL of 20% Na2CO3. The solution
was then allowed to stand for 10 min before reading at 730 nm
in a Shimadzu-UV-1201 (Shimadzu, Kyoto, Japan) spectrophotom-

Male Wistar rats weighing 270320 g and with age from 3 to

3.5 months from our own breeding colony were kept in cages of
3 or 4 animals each, with continuous access to food and water in
a room with controlled temperature (22 3 C) and on a 12-h
light/dark cycle with lights on at 7:00 a.m. The animals were maintained and in accordance to the guidelines of the Brazilian Association for Laboratory Animal Science (COBEA).
Rats were decapitated under mild ether anesthesia, and the
encephalic tissue was rapidly dissected and placed on ice. Tissues
were immediately homogenized in cold 10 mM TrisHCl, pH 7.5
(1/10, w/v). The homogenate was centrifuged for 10 min at
4000g to yield a pellet that was discarded and a low-speed supernatant (S1) was used for the TBARS assay (Puntel et al., 2007).
After centrifugation, an aliquot of 100 lL of S1 was incubated
for 1 h at 37 C with pro-oxidants agent (10 lM of Fe2SO4) in the
presence or absence of plant extracts and then used for TBARS
determination. The concentration range of each tested extract is
shown in Table 3. TBARS production was determined as described
by Ohkawa et al. (1979) and Puntel et al. (2007). Data were analyzed statistically by one-way ANOVA, followed by Duncans multiple range tests.
2.7. Isolation of the antioxidants avonol-derivatives compounds
The ethyl acetate fraction from the leaves (3 g) was subjected to
ash column chromatography on silica gel 60 (225 g) eluted with

6594

A.A. Boligon et al. / Bioresource Technology 100 (2009) 65926598

CH2Cl2/EtOH (7:30:1 v/v) to yield 47 fractions. These fractions

were combined to give two major fractions (I and II), based on similarities in their TLC proles. Fraction I (sub-fractions 130, 1.2 g),
was further puried by repeated chromatography column on silica
gel 60 (140 g) and eluted with CH2Cl2/EtOH (6:44:6 v/v), to yield
compounds 1 (17 mg) and 2 (29 mg). Fraction II (fractions 4547,
0.8 g), was further puried by repeated column chromatography
and eluted with CH2Cl2/EtOH (3:70:1 v/v), to yield compounds
3 (34 mg) and 4 (23 mg).
2.8. Quantication of avonol-derivatives compounds by HPLC
High performance liquid chromatography (HPLC-DAD) was performed with the HPLC system (Shimadzu, Kyoto, Japan), Prominence Auto-Sampler (SIL-20A), equipped with Shimadzu LC-20
AT reciprocating pumps connected to the degasser DGU 20A5 with
integrator CBM 20A, UVVIS detector DAD SPD-M20A and Software LC solution 1.22 SP1. Reverse phase chromatographic analyses were carried out under isocratic conditions using a C-18
column (4.6 mm  250 mm) packed with 5 lm diameter particles;
the mobile phase was methanolacetonitrilewater (40:15:45, v/v/
v) containing 1.0% acetic acid. The mobile phase was ltered
through a 0.45 lm membrane lter and then degassed by an ultrasonic bath prior to use. Stock solutions of quercetin and rutin standard references were prepared in the HPLC mobile phase at a
concentration range of 0.0180.280 mg/ml for quercetin and
0.01250.200 mg/ml for rutin. The ethyl acetate fraction from the
leaves was also dissolved in the mobile phase. All solutions and
samples were rst ltered through a 0.45 lm membrane lter
(Millipore). Quantication was carried out by integration of the
peaks using the external standard method. Isolated compounds
1, 2, 3 and 4 were quantied at 257 nm. The ow rate was
1.0 mL/min and the injection volume was 10 ll. The chromatographic peaks were conrmed by comparing their retention time
and Diode-Array-UV spectra with those of the reference standards
and by spiking the isolated compounds in the plant sample. All
chromatographic operations were carried out at ambient temperature and in triplicate.
2.9. Statistical analysis
Data from the TBARS assay were analyzed statistically by oneway analysis of variance (ANOVA), followed by Duncans multiple
range tests when appropriated using the statistical software SPSS
10.0 for Windows. TBARS graphics were constructed using the
Slide Write 4.032 Bit Edition program. Statistical p values were calculated to quantify levels of signicance for each treatment type. A
signicant p value (p < 0.05, p < 0.01, or p < 0.001 when appropriate) means that there exists signicant difference between the
two sets of data being analyzed. Correlation coefcient (r) to determine the relationship between two variables and the standard
deviations in the DPPH and total phenolics assays were calculated
from the data obtained from three separate experiments using MS
Excel for Windows. One-way ANOVA followed by Tukey test were
performed in the total phenolics and DPPH assays.

3. Results and discussion

3.1. Phenolic contents and free radical-scavenging activities (DPPH)
Antioxidant proles of stem bark and leaf fractions are shown in
Figs. 1 and 2, respectively. Total phenolic contents (TP) assayed by
the method of FolinCiocalteau expressed in milligrams equivalent
of gallic acid (GAE) per gram of each fraction and IC50 determinations by the DPPH method are given in Table 1. The DPPH assay

is used as a tool for the in vitro evaluation of extracts and fractions,

and its results can indicate the presence of phenolic and avonoid
compounds in plant extracts (Rice-Evans et al., 1996). DPPH solutions show a strong absorption band at 518 nm appearing as a deep
violet colour. In the presence of an active radical scavenger, the
absorption vanishes and the resulting decolourization is stoichiometric at a selected range with respect to the degree of reduction.
The remaining DPPH, measured after a certain time, corresponds
inversely to the radical-scavenging activity of the antioxidant,
i.e., the degree of decolouration indicates the free radical-scavenging efciency of the fractions (Kulisic et al., 2004).
Very good antioxidant activities were found for the butanolic
and ethyl acetate fractions from both stem bark and leaves, with
similar antioxidant proles. This can also be observed by comparing their IC50 (the amount of extract of the plant tested necessary
to decrease the concentration of initial DPPH absorbance by 50%)
in Table 1. Stem bark antioxidant activity was found to be slightly
higher than of the leaves and was also bigger than the well known
antioxidant ascorbic acid used as a reference. In fact, the IC50 of the
butanolic and ethyl acetate fractions were nearly 4-fold better than
the IC50 of ascorbic acid. Even in the leaves, the IC50 values of these
fractions were more than two times better than that of the ascorbic
acid. This fact could be explained on the basis of the similarity between compounds with high antioxidant activity extracted by
these organic solvents. Many other studies have demonstrated that
butanolic and ethyl acetate fractions are good sources of antioxidant compounds (Schubert et al., 2007; Tseng et al., 1997; Turkmen et al., 2006; Tung et al., 2009). On the other hand, the
dichloromethane fraction and the crude extract from stem bark
and leaves were inferior to ascorbic acid as antioxidants (Figs. 1
and 2 and Table 1). It is important to compare data obtained on
S. buxifolia with other plants under the same assay conditions:
the IC50 values of ethyl acetate and butanolic fractions from the
stimulating beverage popularly drunk every day in South Brazil,
Ilex paraguariensis (mat tea), were 13.26 and 27.22 lg/mL, respectively, while phenolic contents varied from 86.82 to 199.91 mg/g
(Schubert et al., 2007). If compared with green tea (Camellia sinensis), the results obtained with S. buxifolia were 2-fold higher than
those obtained for this plant, IC50 = 11.8 lg/mL (Almeida et al.,
2008). Considering isolated compounds, quercetin (IC50 = 3.73 lg/
mL) and rutin (IC50 = 12.44 lg/mL) (Galotta et al., 2008) demonstrate very good antioxidant activities. Quercitrin is a glycosylated
form of quercetin (quercetin-3-O-rhamnoside) which also has a
strong antioxidant activity with an IC50 value of 5.19 ug/mL (Artanti et al., 2006).
Butanolic and ethyl acetate fractions from the stem bark exhibit
97.45% and 96.29% of inhibition of DPPH at a concentration of
250 lg/ml, respectively. The antioxidant activity of these fractions
at lower concentrations (7.81 lg/mL) was also signicant with
89.60% of inhibition by the butanolic fraction and 90.23% by the
ethyl acetate fraction. The dichloromethane fraction was a much
weaker inhibitor at 7.81 lg/mL, with an inhibition of 34.14%, as
can be seen in Fig. 1. This behavior can be explained by the different composition of each fraction, since there are compounds that
react quickly with the DPPH and others that have a slower reaction
mechanism (Tsimogiannis and Oreopoulou, 2004).
Comparing the phenolic contents and IC50 values obtained for
stem bark, there were no statistically signicant differences between the butanolic and ethyl acetate fractions, but the same feature was not valid for the leaves (Table 1). The ethyl acetate
fraction from the leaves exhibited the higher phenolic contents obtained in this study, but its IC50 was similar to the butanolic fraction, in which phenolic levels were near 1.5 times lower. Several
authors have described a positive correlation between phenolic
content and antioxidant activity (Chandra and Mejia, 2004; Shyamala et al., 2005; Tung et al., 2007) using similar assay systems,

6595

A.A. Boligon et al. / Bioresource Technology 100 (2009) 65926598

Antioxidant Activity(%

120
100
80
60
40
20
0
0

50

Crude extract

100

150

CH2Cl2

200

Ascorbic acid

250

AcOEt

300

BuOH

Fig. 1. Antioxidant activities of crude extract and dichloromethane, ethyl acetate and butanolic fractions from the stem bark of Scutia buxifolia.

Antioxidant Activity (%)

120
100
80
60
40
20
0
0

50

100

150

200

250

300

Concentration ( g/mL)

Crude extract

Ascorbic acid

CH2Cl2

AcOEt

n-BuOH

Fig. 2. Antioxidant activities of crude extract and dichloromethane, ethyl acetate, and butanolic fractions from the leaves of Scutia buxifolia.

Table 1
Total phenolics (TP) contents and antioxidant activities (IC50/DPPH) for the stem bark and leaves.
Parts of the plant/fractions

Butanolic
Ethyl acetate
Dichloromethane
Crude extract
Ascorbic acid

Stem bark

Leaves

TP + S.E. (mg/g)

IC50 S.E. (lg/mL)

TP + S.E. (mg/g)

IC50 S.E. (lg/mL)

323.47 2.62a
322.69 1.20a
166.88 0.66b
141.09 0.71c

4.35 1.30a
4.32 0.62a
28.76 0.46b
29.55 0.54b
15.98 1.30c

249.14 1.53a
383.34 2.31b
80.94 1.20c
72.09 0.50d

6.50 0.40a
6.12 0.83a
28.25 0.36b
30.54 1.14b
15.98 1.30c

TP: expressed as gallic acid equivalents (mg/g fraction + S.E.).

S.E.: standard error.
Averages followed by different letters in each column differ by Tukey test at p < 0.05.

but in the case of S. buxifolia, this kind of correlation could not be

well established. One explanation could be the presence of other
reducing compounds that probably interfere with the FolinCiocalteau assay and/or the presence of other non-phenolic compounds
with antioxidant effects. As an example, the total phenolic contents
in dichloromethane and crude extract of stem bark are almost
twice the values obtained for the same fractions from the leaves
(Table 1), but the IC50 of these four fractions were very similar,
ranging from 28.25 to 30.54 lg/mL.
3.2. In vitro Fe(II)-induced lipid peroxidation in rat brain
The antioxidant efciencies of the crude extract and the fractions from leaves and stem bark was tested against Fe(II), a known
pro-oxidant. We used encephalic tissue obtained from rats, since
brain is vulnerable to free radical damage in view of the fact of
its high consumption of oxygen and relatively low concentration

of antioxidant enzymes and free radicals scavengers. Fe(II)-induced

TBARS production in brain preparations was signicantly decreased by the stem bark of S. buxifolia. Ethyl acetate fraction
(p < 0.001) showed the highest activity, butanolic fraction
(p < 0.001) and the crude extract (p < 0.001) showed intermediate
activities and dichloromethane fraction (p < 0.01) the lowest activity (Fig. 3 and Table 2). Considering the results obtained with
leaves in the TBARS assay, the inhibitory potency was in the following order: ethyl acetate > butanolic > dichloromethane > crude
extract (Fig. 4 and Table 2). The inhibition of lipid peroxidation
by leaves from S. buxifolia showed a correlation with the phenolic
content of the leaves.
Several studies have focused in the use of natural therapeutic
antioxidant compounds that can afford protection in a variety of
in vitro and in vivo models of human pathologies, including neurotoxicity models (Bastianetto and Quirion, 2002; Patel et al., 2007;
Pereira et al., 2009; Wagner et al., 2006; Williams et al., 2004). Free

6596

A.A. Boligon et al. / Bioresource Technology 100 (2009) 65926598

Fig. 3. Effects of different concentrations of crude extract, ethyl acetate dichloromethane, and butanolic fractions from the stem bark of Scutia buxifolia on Fe(II)
(10 lM)-induced TBARS production in brain homogenates. The samples were
incubated for 1 h with Fe(II) and the plant extracts or without (basal). Data show
means SEM values average from 3 to 4 independent experiments performed in
duplicate.

Fig. 4. Effects of different concentrations of crude extract (A), ethyl acetate (B),
dichloromethane (C), and butanolic (D) fractions from the leaves of Scutia buxifolia
on Fe(II) (10 lM)-induced TBARS production in brain homogenates. The samples
were incubated for 1 h with Fe(II) and the plant extracts or without (basal). Data are
means SEM values average from 3 to 4 independent experiments performed in
duplicate.

OH

Fe(II) can induce neurotoxicity (Bostanci and Bagirici, 2008) via

stimulation of the Fenton reaction (Fraga and Oteiza, 2002) and
its levels are increased in some degenerative diseases (Aisen
et al., 1999; Qian et al., 1997). Quercetin and its glycoside forms,
rutin and quercitrin where shown to have antioxidative action
in vitro and in vivo. The IC50 values of rutin, quercitrin and quercetin against the pro-oxidant Fe(II) were 25.8 lg/ml, 12.2 lg/ml, and
1.4 lg/ml, respectively (Pereira et al., 2009). They can act directly
by entering the redox reactions, and indirectly by chelation of
Fe(II).
3.3. Phytochemical characterization of the fractions and identication
of isolated compounds
Phytochemical screening from the leaves and stem bark fractions showed the presence of sterols, triterpenoids, alkaloids, avonoids, and tannins.
Successive chromatographic procedures with the ethyl acetate
fraction from the leaves led to the isolation of four quercetin-type
avonoids (Fig. 5), whose structures were identied based on analysis of data from 1H NMR and 13C NMR, and by comparison with
literature data. Compound 1 matched the literature data comparable to quercetin. Briey, the 1H NMR spectrum of compound 1
showed two peaks at 6.17 (1H, d, J = 2.0 Hz) and 6.37 ppm (1H,
d, J = 2.0 Hz) consistent with the meta protons H-6 and H-8 on
A-ring and an ABX system at d 7.72 (1H, d, J = 2.1 Hz, H-20 ), 7.62

OH
HO

OH

Fig. 5. Flavonol compounds isolated from Scutia buxifolia. 1 R = OH; 2 R = 3-O-Rha, 3

R = 3-O-Glc; 4 R = 3-OGlc(600 ? 100 )-Rha.

Table 3
Flavonols composition of Scutia buxifolia leaf ethyl acetate fraction.
Flavonol compound

Quercetin
Quercitrina
Isoquercitrina
Rutin

Quantities
mg/g of dry fraction

Percent of extract

27.1 0.03
183.2 0.24
6.6 0.04
48.1 0.18

2.71
18.3
0.66
4.81

Results are expressed as mean S.E. of three determinations.

a
Quantied as quercetin.

Table 2
Tested concentrations in the TBARS assay and IC50 (lg/mL) values for crude extract and fractions from the leaves and stem bark of Scutia buxifolia.
Plants

Extracts

Concentrations (lg dried plant/mL)

IC50 (lg/mL)

Leaves

Butanolic
Ethyl acetate
Dichloromethane
Crude extract
Butanolic
Ethyl acetate
Dichloromethane
Crude extract

0.28.5
0.28.5
0.28.5
0.283.0
0.28.5
0.28.5
0.830.0
0.28.5

4.96 1.56ab
2.93 2.17b
8.19 0.94a
40.46 2.51c
3.18 1.60a
0.66 0.12b
27.3 1.23c
5.15 0.77a

Stem bark

Averages followed by different letters differ by Tukey test at p < 0.05.

A.A. Boligon et al. / Bioresource Technology 100 (2009) 65926598

6597

Fig. 6. High performance liquid chromatography phenolic prole of ethyl acetate fraction from the leaves: 1 quercetin, 2 quercitrin, 3 isoquercitrin and 4 rutin.
Chromatographic conditions described in Section 2.

(1H, dd, J = 8.4, 2.1 Hz, H-60 ) and 6.87 (1H, d, J = 8.4 Hz, H-50 ) corresponding to the catechol protons on B-ring. The 13C NMR spectrum indicated the presence of 15 carbon atoms, the signal at: d
177.3 was attributed to a carbonyl carbon placed at C-4, the other
signals were compatible with those of quercetin (Fossen et al.,
1998; Liu et al., 2008; Slimestad et al., 1995; Slimestad, 2003).
Compound 2 presented the same aglycone signal patterns of compound 1 but the presence of a methyl doublet at d 0.95 (J = 6.0 Hz)
together with the small coupling constant of the anomeric proton
doublet at d 5.34 (J = 1.6 Hz) was indicative of a rhamnopyranose
moiety (Ma et al., 2005). The data were in agreement with the literature data reported for quercetin 3-O-rhamnoside (quercitrin)
(Lawrence et al., 2003; Ma et al., 2005). Compound 3 exhibited a
similar NMR spectra to that of quercetin, but, the signal at d 5.23
(1H, d, J = 7.2 Hz) followed by other characteristic additional signals indicate the presence of a sugar moiety in 3. The hexose
was determined to be a glucopyranosyl unit bound to the 3 position of the aglycone by comparison of proton and carbon shift
NMR values with the literature data (Fossen et al., 1998; Liu
et al., 2008; Slimestad et al., 1995; Slimestad, 2003). Compound
3 was quercetin-3-O-b-D-glucopyranoside (isoquercitrin); the b
conguration of the glucopyranoside unit was based on the observation of a large 1H1H coupling constant of the anomeric proton
(J = 7.2 Hz) (Slimestad et al., 1995). The aglycone signals of compound 4 corresponded well with the shifts for quercetin (1), the
only signicant difference being an upeld shift of 1.6 ppm for
the C-3. This shift is analogous to that reported when the 3-hydroxy group is glycosylated in a avonol glycoside. The two anomeric
proton signals at d 5.75 (d, J = 8.0 Hz) and at d 5.25 (d, J = 2.0 Hz)
were assignable to H-1 of a b-glucosyl proton and to the H-1 of
a-rhamnosyl proton, respectively. In the I3C NMR of 4, the C-6 signal (d 68.5) of glucose showed a downeld shift of 7.3 ppm in comparison with the corresponding C-6 signal (d 61.2) of quercetin 3glucoside (3), indicating a 16 linkage between the C-3-glucose
and the rhamnose (Rastrelli et al., 1995). The identity of this compound was further conrmed by cochromatography with authentic rutin standard and literature data comparisons (Lawrence et al.,
2003; Rastrelli et al., 1995).

3.4. HPLC analysis

The HPLC prole of one of the active fractions was also acquired,
as well as the quantication of rutin and quercetin by HPLC-DAD
based on reference rutin and quercetin standards. Quercitrin and
isoquercitrin were also quantied but they were expressed separately as quercetin content (Table 3). The major component was
found to be quercitrin, followed by rutin, quercetin and isoquercitrin. Quercitrin is a glycosylated form of quercetin (quercetin-3-Orhamnoside) which also has a strong antioxidant activity with IC50
value of 5.19 lg/mL (Artanti et al., 2006). HPLC analysis of the ethyl
acetate fraction from the leaves is shown in Fig. 6.
4. Conclusions
The fractions and crude extract from the leaves and stem bark of
S. buxifolia tested here are effective inhibitors of TBARS production
and also presented DPPH scavenger activity. These effects can be
related mainly to their phenolic and avonoids contents. Quercetin, quercitrin, isoquercitrin and rutin were isolated from the S.
buxifolia for the rst time. Quercitrin was the major avonol present in the plant. These results indicated that the plant S. buxifolia
has many chemical compounds able to scavenge free radicals.
Therefore, the plant has promising compounds to be tested as potential antioxidant drugs for the treatment of diseases resulting
from oxidative stress. The benecial effects of intake of antioxidant
substances have been shown in several experimental and epidemiological studies. In fact, an association between ingestion of diets
rich in fruits, vegetables, red wine with a decline in degenerative
diseases has been demonstrated in the literature (Hollman and Katan, 1999; Virgili and Marino, 2008). More studies are needed to
determine whether this medicinal plant could be used industrially
as an important biosource of antioxidants.
Acknowledgements
The authors would like to thank V. Batista (Tarum, Dom Pedrito, Brazil) for plant collection, R.B. Zacchia (Botanical Department

6598

A.A. Boligon et al. / Bioresource Technology 100 (2009) 65926598

of Federal University of Santa Maria) for providing the identication of S. buxifolia and Maria Anglica da Silveira Lima (Chemistry
Department of Federal University of Santa Maria) for providing the
NMR Spectra.

References
Aisen, P., Wessling-Resnick, M., Leibold, E.A., 1999. Iron metabolism. Curr. Opin.
Chem. Biol. 3, 200206.
Almeida, I.F., Valento, P., Andrade, P.B., Seabra, R.M., Pereira, T.M., Amaral, M.H.,
Costa, P.C., Bahia, M.F., 2008. In vivo irritation potential of a Castanea sativa
(Chestnut) leaf extract, a putative natural antioxidant for tropical application.
Basic Clin. Pharmacol. Toxicol. 103, 461467.
Amico, V., Chillemi, R., Mangiaco, S., Spatafora, C., Tringali, C., 2008. Polyphenol
enriched fractions from Sicilian grape pomace. HPLC-DAD analysis and
antioxidant activity. Bioresour. Technol. 99, 59605966.
Artanti, N., Marifa, Y., Hana, M., 2006. Isolation and identication of active
antioxidant compound from Star fruit (Averrhoa carambola) Mistletoe
(Dendrophthoe pentandra L.) Miq. ethanol extract. J. Appl. Sci. 68, 16591663.
} u
} u
} rk, M., Ku
} ltu
} r, S., Oks
} z, S., 2007. Screening of antioxidant activity of
Barla, A., Ozt
three Euphorbia species from Turkey. Fitoterapia 78, 423425.
Bastianetto, S., Quirion, S., 2002. Natural extracts as possible protective agents of
brain aging. Neurobiol. Aging 23, 891897.
Behera, B.C., Verma, N., Sonone, A., Makhija, U., 2008. Antioxidant and antibacterial
properties of some cultured lichens. Bioresour. Technol. 99, 776784.
Bostanci, M.O., Bagirici, F., 2008. Neuroprotective effect of aminoguanidine on ironinduced neurotoxicity. Brain Res. Bull. 76, 5762.
Chandra, S., Mejia, E.G., 2004. Polyphenolic compounds, antioxidant capacity, and
quinone reductase activity of an aqueous extract of Ardisia compressa in
comparison to mate (Ilex paraguariensis) and green (Camellia sinensis) teas. J.
Agric. Food Chem. 52, 35833589.
Chang, S.K., Sung, P.M., 2008. Antioxidant activities of ethanol extracts from seeds in
fresh Bokbunja (Rubus coreanus Miq.) and wine processing wast. Bioresour.
Technol. 99, 45034509.
Choi, C.W., Kim, S.C., Hwang, S.S., Choi, B.K., Ahn, H.J., Lee, M.Y., Park, S.H., Kim, S.K.,
2002. Antioxidant activity and free radical scavenging capacity between Korean
medicinal plants and avonoids by assay-guided comparison. Plant Sci. 63,
11611168.
Chua, M.T., Tung, Y.T., Chang, S.T., 2008. Antioxidant activities of ethanolic extracts
from the twigs of Cinnamomum osmophloeum. Bioresour. Technol. 99, 1918
1925.
Fossen, T., Pedersen, A.T., Andersen, O.M., 1998. Flavonoids from red onion (Allium
cepa). Phytochemistry 47, 281285.
Fraga, C.G., Oteiza, P.I., 2002. Iron toxicity and antioxidant nutrients. Toxicology 80,
2332.
Galotta, A.L.Q.A., Boaventura, M.A.D., Lima, L.A.R.S., 2008. Antioxidant and cytotoxic
activities of Aa (Euterpe precatoria Mart). Quim. Nova 31, 14271430.
Hollman, P.C.H., Katan, M.B., 1999. Dietary avonoids: intake, health effects and
bioavailability. Food Chem. Toxicol. 37, 937942.
Kulisic, T., Radonic, A., Katanilic, V., Milos, M., 2004. Use of different methods for
testing antioxidative activity of oregano essential oil. Food Chem. 85, 633640.
Lawrence, O.A.M., Ugi, I., Lemmne, P., Hermann, R., 2003. Flavonol glycosides of
Warburgia ugandensis leaves. Phytochemistry 64, 891896.
Liu, X., Cui, C., Zhao, M., Wang, J., Luo, W., Yang, B., Jiang, Y., 2008. Identication of
phenolics in the fruit of emblica (Phyllanthus emblica L.) and their antioxidant
activities. Food Chem. 109, 909915.
Ma, X., Tian, W., Wu, L., Cao, X., Ito, Y., 2005. Isolation of quercetin-3-O-Lrhamnoside from Acer truncatum Bunge by high-speed counter-current
chromatography. J. Chromatogr. A 1070, 211214.
Maksimovic, Z., Malencic, F.F.L., Kovacevic, N., 2005. Polyphenol contents and
antioxidant activity of Maydis stigma extracts. Bioresour. Technol. 96, 873877.
Morel, F.A., Maldaner, G., Ilha, V., Missau, F., Silva, F.U., Dalcol, I., 2005. Cyclopeptide
alkaloids from Scutia buxifolia Reiss and their antimicrobial activity.
Phytochemistry 66, 25712576.
Nascimento, M.A., Silva, A.K., Fan, L.C.B., Quignard, E.L.J., Lopes, J.A., Almeida, M.G.,
2006. Turnera ulmifolia L. (Turneraceae): preliminary study of its antioxidant
activity. Bioresour. Technol. 97, 13871391.

Ohkawa, H., Ohishi, H., Yagi, K., 1979. Assay for lipid peroxide in animal tissues
thiobarbituric acid reaction. Anal. Biochem. 95, 351358.
Papetti, A., Daglia, M., Aceti, C., Quaglia, M., Gregotti, C., Gazzani, G., 2006. Isolation
of an in vitro and ex vivo antiradical melanoidin from roasted barley. J. Agric.
Food Chem. 54, 12091216.
Patel, R., Garg, R., Erand, S., Maru, G.B., 2007. Chemopreventive herbal anti-oxidant:
current status and future perspectives. J. Clin. Bochem. Nutr. 40, 8291.
Pereira, R.P., Fachinetto, R., Prestes, A.L., Puntel, R.L., Silva, G.N.S., Heinzmann, B.M.,
Boschetti, T.K., Athayde, M.L., Burger, M.E., Morel, A.F., Morsch, V.M., Rocha,
J.B.T., 2009. Antioxidant Effects of Different Extracts from Melissa ofcinalis,
Matricaria recutita and Cymbopogon citratus. Neurochem Res 34, 973983.
Puntel, R.L., Roos, D.H., Grotto, D., Garcia, S.C., Nogueira, C.W., Rocha, J.B., 2007.
Antioxidant properties of Krebs cycle intermediates against malonate prooxidant activity in vitro: a comparative study using the colorimetric method
and HPLC analysis to determine malondialdehyde in rat brain homogenates.
Life Sci. 81, 5162.
Qian, Z.M., Wang, Q., Pu, Y., 1997. Brain iron and neurological disorders. Chin. Med.
J. 110, 455458.
Rastrelli, L., Saturnino, P., Schettino, O., Dini, A., 1995. Studies on the constituents of
Chenopodium pallidicaule (Canihua) seeds. Isolation and characterization of two
new avonol glycosides. J. Agric. Food Chem. 43, 20202024.
Rice-Evans, C.A., Miller, N.J., Paganga, G., 1996. Structureantioxidant activity
relationships of avonoids and phenolic acids. Free Radic. Biol. Med. 20, 933
956.
Schubert, A., Pereira, D.F., Zanin, F.F., Alves, S.H., Beck, R.C.R., Athayde, M.L., 2007.
Comparison of antioxidant activities and total polyphenolic and
methylxanthine contents between the unripe fruit and leaves of Ilex
paraguariensis A. St. Hil. Pharmazie 62, 876880.
Shyamala, B.N., Gupta, S., Lakshmi, A., Prakash, J., 2005. Leafy vegetable extracts
antioxidant activity and effect on storage stability of heated Oils. Innovative
Food Sci. Emerging Technol. 6, 239245.
Slimestad, R., Andersen, O.M., Francis, G.W., Marston, A., Hostettman, K., 1995.
Syringetin 3-O-(600 -acetyl)-(-glucopyranoside and other avonols from needle
of Norway spruce, Picea abies. Phytochemistry 40, 15371542.
Slimestad, R., 2003. Flavonoids in buds and young needles of Picea, Pinus and Abies.
Biochem. Syst. Ecol. 31, 12471255.
Tseng, T.H., Kao, E.S., Chu, C.Y., Chou, F.P., Lin, H.W., Wang, C.J., 1997. Protective
effects of dried ower extracts of Hibiscus sabdariffa L. against oxidative stress in
rat primary hepatocytes. Food Chem. Toxicol. 35, 11591164.
Tsimogiannis, D.I., Oreopoulou, V., 2004. Free radical scavenging and antioxidant
activity of 5,7,30 40 -hydroxy-substituted avonoids. Innovative Food Sci.
Emerging Technol. 5, 523528.
Tsimogiannis, D.I., Oreopoulou, V., 2006. The contribution of avonoids C-ring on
the DPPH free radical scavenger efciency. A kinetic approach for the 30 ,40 hydroxy substituted members. Food Sci. Emerging Technol. 7, 140146.
Tung, Y.T., Wu, J.H., Huang, C.Y., Kuo, Y.H., Chang, S.T., 2009. Antioxidant activities
and phytochemical characteristics of extracts from Acacia confuse bark.
Bioresour. Technol. 100, 509514.
Tung, Y.T., Wu, J.H., Kuo, Y.H., Chang, S.T., 2007. Antioxidant activities of natural
phenolic compounds from Acacia confuse bark. Bioresour. Technol. 98, 1120
1123.
Turkmen, N., Sari, F., Velioglu, Y.S., 2006. Effects of extraction solvents on
concentration and antioxidant activity of black and black mate tea
polyphenols determined by ferrous tartrate and FolinCiocalteu methods.
Food Chem. 99, 835841.
Virgili, F., Marino, M., 2008. Regulation of cellular signals from nutritional
molecules: a specic role for phytochemicals, beyond antioxidant activity.
Free Radic. Biol. Med. 45, 12051216.
Wagner, C., Fachinetto, R., Dalla-Corte, C.L., Brito, V.B., Severo, D., Dias, G.O.G., Morel,
A.F., Nogueira, C.W., Rocha, J.B.T., 2006. Quercitrin, a glycoside form of
quercetin, prevents lipid peroxidation in vitro. Brain Res. 1107, 192198.
Wagner, H., Bladt, S., Zgainski, E.M., 1984. TLC screening. In: Plant Drug Analysis a
Thin Layer Chromatography Atlas. Springer-Verlag, Berlin, Heidelberg.
Wasicky, R., Wasicky, M., Joachimovits, R., 1964. Erstuntersuchungen na Coronilha
Scutia buxifolia Reissek. Planta Med. 12, 1325.
Williams, R.J., Spencer, J.P.E., Rice-Evans, C., 2004. Flavonoids: antioxidants or
signaling molecules? Free Radic. Biol. Med. 36, 838849.
World Health Organization, 1998. Regulatory situation of herbal medicines: a
worldwide review. Geneva, p. 45.