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809
ISSN 0326-2383
KEY WORDS: Allelopathy, Antioxidants, Artemia salina, Epicatechin, Ochna serrulata.
* Author to whom correspondence should be addressed. E-mail: ines@qmc.ufsc.br
Latin American Journal of Pharmacy
(formerly Acta Farmacéutica Bonaerense)
Lat. Am. J. Pharm. 30 (4): 809-13 (2011)
Short communication
Received: August 17, 2010
Revised version: September 10, 2010
Accepted: September 15, 2010
Antioxidant, Allelopathic and Toxic Activity of Ochna serrulata
Guilherme COLLA, Mariana A. da SILVA, Gustavo S. QUEIR OZ,
Moacir G. PIZZOLATTI & Inês M.C. BRIGHENTE*
Laboratório de Química de Produtos Naturais, Departamento de Química,
Universidade Federal de Santa Catarina, Campus Universitário, 88040-900, Florianópolis, SC, Brasil
SUMMARY. Ochna serrulata (Ochnaceae) is an ornamental plant introduced into Brazil from Asia and
Africa. Species of the Ochna genus are rich in phenolic compounds, mainly flavonoids. The biological
screening of extracts and fractions showed that this plant exhibited a significant antioxidant activity, when
evaluated by the DPPH and reducing potential assays. Ochna serrulata also demonstrated slight toxic ac-
tivity against Artemia salina and a potential inhibitory allelopathic activity, when evaluated using the Lac-
tuca sativa seed germination test. The ethyl acetate fraction, the most active one, was partitioned on a silica
gel column to obtain epicatechin, which showed potential antioxidant activity.
INTRODUCTION
The biodiversity of the plant kingdom consti-
tutes a true therapeutic arsenal. Some ornamen-
tal plants are included within this context, con-
tributing to the discovery of new drugs, as in
the case of Papaver somniferum (papoula),
from which morphine is isolated and used as an
anesthetic and analgesic, and Digitalis purpurea
(foxglove), whose constituent known as digitalis
is used as a regulator in cardiopathy.
Ochna serrulata (Ochnaceae) is a native
plant from tropical regions of Africa and Asia
and introduced in Brazil as ornamental plant. It
possesses bright foliage short lateral branches
and yellow flowers with a pleasant scent and of
short duration that appear at the extremities of
the branches. The fruits are black with a red re-
ceptacle which has an arrangement resembling
Mickey Mouse’s face 1. The South African tribes
use the roots of O. serrulata in the form of a de-
coction for the treatment of osseous illnesses
and hemorrhoids 2.
The few available studies on the biological
activities of the different Ochna species reported
analgesic and antiinflammatory 3, anti-HIV 4, cy-
totoxic and antibacterial 5and antimalarial 6ac-
tivities. Moreover, studies with O. serrulata have
shown genotoxic properties 7.
Species of Ochnaceae are able to biosynthe-
size flavonoids and biflavonoids, the latter being
used as taxonomic markers in these plants 8.
Considering the scarcity of information re-
ported on O. serrulata, in this study the biologi-
cal screening of extracts and fractions of this
plant was carried out to ascertain its toxicity
against Artemia salina, and its potential antioxi-
dant and allelopathic activities. The partitioning
of the ethyl acetate fraction, the most active one
according to these assays, led to the isolation
and characterization of epicatechin which
showed potential antioxidant activity.
MATERIALS AND METHODS
The leaves and stems of Ochna serrulata
were collected at city of São José (Santa Catari-
na, Brazil). A voucher specimen was deposited
at the Botany department of the Federal Univer-
sity of Santa Catarina (Prof. Daniel de Barcellos
Falkenberg, number FLOR 15811).
The air-dried leaves (224.2 g) and stems
810
COLLA G., SILVA M.A. da, QUEIROZ G.S., PIZZOLATTI M.G. & BRIGHENTE I.M.C.
(161.8 g) were triturated and macerated sepa-
rately with 80 % ethanol at room temperature
for 15 days. The solvent was removed by rotary
evaporation (T < 55 ºC) and the extract (57.9 g)
was then suspended in methanol and refrigerat-
ed in order to decant the non soluble material.
This residue (20.9 %) was then filtered and the
solution was added to 30 % (v/v) ethanol and
partitioned with organic solvents of increasing
polarity. This procedure yielded three fractions,
hexane (0.2 %), ethyl acetate (27.2 %) and bu-
tanol (8.4 %), respectively.
The ethyl acetate fraction (14.9 g) was then
run on a silica gel-based chromatographic col-
umn and eluted using mixtures of hexane, ethyl
acetate and methanol at different ratios. Fraction
7-11 (hexane/AcOEt 1:9) was partitioned in suc-
cessive flash chromatography runs yielding epi-
catechin (81.7 mg). The physic and spectroscop-
ic data obtained (IR, 1H and 13C NMR) were in
agreement with data reported in the literature
for epicatechin 9.
Biological activity
The phenolic and flavonoids contents were
determined using the Folin-Ciocalteau and AlCl3
(2 %) reagents, respectively. Antioxidant activity
was evaluated using 2,2-diphenyl-1-picryl-hy-
drazyl (DPPH) and reducing potential assays as
previously described 10. Toxic activity against
larvae of Artemia salina was also evaluated 11.
The allelopathic activity was determined
through the Lactuca sativa (lettuce) seed germi-
nation test. The vegetal extracts were dissolved
in ethanol in different concentrations (0.25 to
2.00%) and later impregnated (1 mL) into What-
man Nº 1 filter paper. After the solvent evapora-
tion, the filter papers were placed into 6-cm
Petri dishes, 1.5 mL of tween 80 (100 µg/mL)
added and the samples were left in stand for 24
h. Each filter paper then received 25 L. sativa
seeds. Previously, the lettuce seeds were surface
sterilized in a 1:10 (v/v) dilution of commercial
hypochlorite bleach for 10 min and rinsed sev-
eral times with distilled water. Tests were car-
ried out in triplicate. A negative control was
used following the same procedure but in the
absence of the plant extract. The dishes were
incubated in a growth chamber at 25 ± 1 °C
with a 12 h light/12 h dark cycle. The filter pa-
per was wetted with water distilled daily, germi-
nation recorded daily and the test was moni-
tored until the 5th day for standardization. Seeds
were considered germinated when the protru-
sion of the radicle was evident. After the 5th day
the radicle and hypocotyl growth were mea-
sured and the results compared with the nega-
tive control to evaluate the seedling develop-
ment. The seed germination speed (SGS) was
evaluated through Eq. [1]:
SGS = G1/N1 + G2/N2 + ...Gn/Nn [1]
where G1, G2, Gn are the number of normal
seedlings recorded in the first, second and last
counting sessions and N1, N2, Nn correspond to
the number of days after sowing for the first,
second and last counts. These results were com-
pared with the negative control. Data were con-
sidered significant at the 0.05 level using analy-
sis of variance (ANOVA) and the Fisher test.
RESULTS AND DISCUSSION
The Ochna serrulata crude extracts of leaves
and stems, and the fractions obtained from the
liquid-liquid partitioning of the leaf extracts,
were evaluated for toxic activity against Artemia
salina, antioxidant activity and allelopathic ac-
tivity.
The antioxidant assay using the free radical
DPPH demonstrated an excellent antioxidant ac-
tivity for all vegetal extracts and fractions. Ex-
cept the hexane fraction, all the extracts and
fractions had EC50 values comparable with
known antioxidant compounds such as BHT
(EC50 = 17.3 µg/mL) and ascorbic acid (EC50 =
9.2 µg/mL), as seen in Table 1.
The capacity to reduce metallic ions or inter-
mediates of the lipid peroxidation process
through electron donation is characterized as
the reducing potential of a sample 12. This assay
showed similar activity to the DPPH test, being
the ethyl acetate fraction the most active one
followed by the butanol and hexane fractions.
The two antioxidant assays used on the frac-
tions of O. serrulata leaf extract showed a posi-
tive correlation with the phenolic content
(DPPH assay, r2= 0.7292; reducing potential
test, r2= 0.7296), but not with the flavonoid
one. The greater antioxidant activity observed
for the most polar fractions of O. serrulata
leaves may be attributed to the phenolics con-
centration. The leaf extract had greater concen-
trations of phenolics and flavonoids and, conse-
quently, higher antioxidant activity than the
stem extract (the flavonoids provide greater pro-
tection against the radiation emitted by the sun
to the leaves than to the stems).
The chromatographic partition of the ethyl
acetate fraction yielded epicatechin. This com-
pound has been previously isolated from other
811
Latin American Journal of Pharmacy - 30 (4) - 2011
Ochna species 13-16 and showed high antioxi-
dant capacity when evaluated by the tests ap-
plied in this study (Table 1).
The lethality assay using the microcrustacean
Artemia salina is widely used as a screening
test to verify the toxic and, consequently, bio-
logical activity of vegetal extracts and their iso-
lated compounds 17. The vegetal extracts are
considered active in this bioassay when the LD50
values give up to 1000 ppm. Table 1 shows the
results of this bioassay, where it can be ob-
served that the ethyl acetate fraction was the
Phenolic Flavonoids DPPH EC50 Reducing Potential Artemia salina
mg GA/g* mg QE/g* µg/mL mg AA/g* LD50 µg/mL
(CI 95%)
Stem extract 219.9± 1.1 20.1±0.1 15.3 261.7±5.2 933.2
(897.4-970.5)
Leaf extract 290.9±2.4 105.8±0.5 13.2 617.8±4.4 998.4
(907.8-1096.7)
Hexane fraction 234.4±3,3 48.6±1.0 32.6 506.4±1.1 868.0
(755.3-997.5)
Ethyl acetate fraction 339.±2,5 123.0±2.1 10.7 981,4±22,4 457.1
(385.8-542.0)
Butanol fraction 290.9±2.0 18.8±0,5 13.6 597.2±1.3 >1000
Epicatechin - - 1.8 810.4±4.4 691.8
(837.5-571.5)
Table 1. Antioxidant assay and toxicity against Artemia salina of vegetal extracts of Ochna serrulata. * mg of
gallic acid (GA), quercetin (QE) or ascorbic acid (AA)/g of vegetal material; EC50 = effective concentration 50%;
LD50 = lethal dose 50 %.
Ochna serrulata
Figure 1. Influence of vegetal extracts of Ochna serrulata on seed germination speed (SVS) of Lactuca sativa.
Averages followed by the same letters do not differ significantly according to the Fisher test (5%).
most active. The epicatechin value was LD50 =
691.8 ppm, showing that this compound was
not the component mainly responsible for the
toxic activity of this plant.
Finally, allelopathy is a phenomenon in-
volved in the plant defense system, producing a
diversity of secondary metabolites and releasing
them to the external environmental, thus exert-
ing an effect on other plants 18. The observation
of seed germination and seedling development
is a useful bioassay to study the allelopathic ac-
tivity. Figure 1 shows the influence of extracts
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COLLA G., SILVA M.A. da, QUEIROZ G.S., PIZZOLATTI M.G. & BRIGHENTE I.M.C.
and fractions of O. serrulata on the seed germi-
nation and seedling development of lettuce.
Overall, the results showed that the inhibition of
seed germination increased when the concentra-
tion of the extract or fraction increased, with the
exception of the butanolic fraction for which
concentrations of 0.50 and 1.0 g% stimulated
seed germination. The leaf extract of O. serrula-
ta presented a greater effect on the seed germi-
nation than the stem extract. After fractioning of
the leaf extract the hexane fraction showed
higher activity. The seedling development was
evaluated by calculating the percentage of radi-
cle and hypocotyl growth (Fig. 2). In most of
the vegetal samples the radicle and hypocotyl
development was affected, with greater inhibi-
tion being observed when the extract concentra-
tion increased, with some exceptions where
stimulation of the seedling growth occurred
with an increase in the concentration.
Epicatechin, a recognized allelochemical 9,19,
showed an inhibition of 33.6 % in the seed ger-
mination at a concentration of 1 %, inhibiting
the development of the radicle (72.1 %) more
than that the hypocotyl (33.1 %) at this concen-
tration.
Considering the inhibitory effect on the let-
tuce seed germination and seedling develop-
ment of the O. serrulata extracts and fractions,
this plant must possess, besides epicatechin,
other compounds with potential herbicidal ac-
tivity.
CONCLUSIONS
The hydroalcoholic extracts of O. serrulata
leaves and stems clearly showed antioxidant
and allelopathic activity, however, it demonstrat-
ed only slight toxic activity against Artemia sali-
na. The liquid-liquid partitioning of the leaf ex-
tract showed that the ethyl acetate fraction was
the most active in terms of toxicity toward A.
salina and antioxidant activity. In relation to al-
lelopathic activity the hexane fraction was the
most active. The epicatechin, isolated from the
ethyl acetate fraction, showed antioxidant, al-
lelophatic, and toxicity against A. salina.
Acknowledgement. The authors are grateful to CNPq
and CAPES for financial support.
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after 5 days. Averages followed by the same letters do not differ significantly according to the Fisher test (5%).
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