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Correspondence to: Caroline C. Fernandes-Silva Laboratory of Phytochemistry, Department of
Botany, Institute of Biosciences, University of São Paulo, Rua do Matão 277 sala 154, CEP
This article has been accepted for publication and undergone full peer review but
has not been through the copyediting, typesetting, pagination and proofreading
process, which may lead to differences between this version and the Version of
Record. Please cite this article as doi: 10.1002/jsfa.7045
beeswax and plant material. Despite the wide spectrum of biological activity of propolis, to our
knowledge no studies have been carried out about phytotoxic properties of Brazillian propolis
and its constituents. The aims of this study were to analyze the chemical composition and to
evaluate the phytotoxic activity of the volatile fraction of a sample of Brazillian green propolis.
RESULTS: Main constituents are the phenylpropanoid 3-prenylcinnamic acid allyl ester
(26.3%) and the sesquiterpene spathulenol (23.4%). Several other sesquiterpenes and
The activity of solutions of the volatile fraction at 1.0, 0.5 and 0.1% was tested on lettuce seeds
and seedlings. The solution at 1% inhibited completely the seed germination and solutions at 0.1
and 0.5% reduced the germination rate index. The solution at 0.5% reduced the growth of the
CONCLUSIONS: The chemical composition of the volatile fraction of this Brazilian green
propolis is different from those previously described, and these results may contribute to a better
understanding about the chemical variations in propolis. The volatile fraction of Brazilian green
propolis influences both germination of seed lettuce and the growth of its seedlings, showing an
phytotoxic potential.
volatiles.
Considerable effort has been dedicated to studies about new allelochemicals with
potential use in agriculture for weed management.1 These compounds have advantages
over traditional herbicides, such as distinct modes of action and rapid biodegradability,
having thus lower environmental impact.1 Studies have shown that plant extracts often
have high phytotoxic activity. Several natural chemical compounds have long been
are valuable in the search for alternatives to the herbicides currently used in agriculture.
Due to the surge of organic agriculture in recent years, the importance of herbicides of
natural origin is gaining momentum, chiefly because synthetic compounds are not
permitted in this sort of plant farming.3 In particular, plant volatile oils, such as clove
oil, have been shown not only to possess insecticidal and antimicrobial activities, but
propolis, the most studied and exported type of propolis,5 possesses phytotoxic activity.
Its volatile oil inhibited the growth of Lolium multiflorum Lam. and Lactuca sativa L.6
beeswax and plant material.7 The resin provided by plants comprises a wide diversity of
for the several biological activities found in different types of propolis, such as
Recently, special attention has been given to its volatile fraction, and quite distinct
propolis.12 Ioshida et al. detected neither caryophyllene nor nerolidol in the volatiles
Although the chemical composition of propolis has been extensively studied, little
is known about the biological activity of its volatile fraction. A recent review reveals
volatile substances have been evaluated.10 Despite the wide spectrum of biological
activity of propolis, either from temperate or tropical countries, few studies have been
carried out about phytotoxic properties of propolis and its constituents. The first paper
about phytotoxic activity of propolis dates from 1968, reporting inhibition of the growth
of potato tubers.15 More recently, phytotoxic activity of ethanol and n-hexane extracts of
Portuguese propolis on in vitro culture of flax (Linum usitatissimum L.) was reported.16–
18
To our knowledge, there have been no studies about Brazilian propolis phytotoxic
activity.
fraction of propolis, and to enhance our understanding about their biological properties,
the present work aimed to analyze the volatile composition of a Brazilian green propolis
from the municipality of Viçosa (state of Minas Gerais) and to test its effect on lettuce
Propolis sample
from beehives located on the campus of the Federal University of Viçosa (state of
Clevenger extractor. The volatile fraction was diluted in n-hexane (1:10). One μL of the
ethereal solution was injected in a gas chromatograph 6850 (Agilent), equipped with a
capillary column DB-5HT (30 m x 0.32 mm, 0.25 μm), operating in the split mode and
impact at 70 eV. Temperatures of injector and detector were 300 °C and 250 °C,
respectively. Helium was used as carrier gas at 1.5 mL min-1. The column was
maintained at 50 ºC for 5 min and then heated to 100 °C at 6 °C min-1; the column
temperature was then ramped up to 250 °C at 3 °C min-1. The mass spectrometer was
programed to range from 40 to 700 mass units. Linear retention indices (LRI) were
Phytotoxic activity
The tests were conducted using hexane solutions of the volatile fraction at
concentrations 0.1, 0.5 and 1.0%. With the aid of an airbrush, 4 mL of each solution
was nebulized on filter papers inside Petri dishes. After total elimination of the solvent,
4 mL of water were added; 20 lettuce achenes were then placed on the filter papers. The
dishes were kept in a growth chamber for seven days at 25 ºC and 12 h photoperiod.
germination rate indices and lengths of hypocotyl-root axes and cotyledon leaves.
Statistical analysis
axis and cotyledon leaf were expressed as means and standard deviation. Two-way
ANOVA was used to compare control and tested solutions, using “days” and
time. The differences between the germination rate index (GRI), the length of the
hypocotyl-root axis (HRA) and the length of cotyledon leaves among the tested
solutions were evaluated by one-way Anova, with Tukey’s post-test. All statistical
Chemical characterization
prenylcinnamic acid allyl ester (12 - 26.3%), spathulenol (10 - 23.4%) and 7-phenyl-5-
oxo-heptanol (11 – 13.3%) (Figure 1, Table 1). The monoterpenes linalool (1) and α-
benzenepropanoic acid methyl ester (3) and benzenepropanoic acid ethyl ester (4) were
variability holds also for the propolis volatile fraction, even among samples of a same
propolis type, e.g. Brazilian green propolis. Several compounds reported by Kusumoto
propolis.12,14 Some compounds detected in the present work have been reported by other
authors in the volatile fraction of Brazilian propolis. Examples are the monoterpenes
linalool (1) and α-terpineol (2) and the sesquiterpenes β-caryophyllene (5), δ-cadinene
(9) and spathulenol (10).20 The present work reports for the first time the sesquiterpenes
cis-muuroladiene (7) and germacrene D (8) in Brazilian green propolis, as well as the
allyl ester of 3-prenyl cinnamic acid (12) (Fig. 1; Table 1). The latter compound was
fraction, it has a considerably high retention time (Figure 1) and is characterized by LRI
2016.
Phytotoxic activity
of the lettuce seeds (Table 2). Solutions at 0.1% and 0.5% did not affect the final
germination rate. However, the germination rate index (GRI) was substantially affected,
because germination rate on days 1 and 4 (0.5% solution) and on day 1 (0.1% solution)
decreased substantially (Table 2). Another phytotoxic effect exerted by the solution at
0.5% was the inhibition of the growth of the hypocotyl-radicle axes and the cotyledon
seed lettuce and the growth of its seedlings (Table 2). Among the constituents of the
analyzed volatile fraction, some have already been reported as possessing phytotoxicity.
For example, spathulenol (10; Fig. 1, Table 1), is the main constituent of the essential
oil of Eucalyptus camaldulensis Dehnh; such oil completely inhibits the germination of
oleracea L.25 Linalool (1; Fig. 1, Table 1) is by far the main component of both leaf
(81%) and flower (95%) of Lippia adoensis Hochst ex. Walp.26 At 400 ppm, the
essential oil of leaves and flowers of L. adoensis, as well as linalool, inhibit the growth
of the root of lettuce seedlings to nearly 60% relative the control. Among the
α-terpineol (2), were the most effective growth inhibitors of seedlings of radish and
Brazilian green propolis (Fig. 1, Table 1). Even at low concentrations (e.g. 3 mg L-1),
this sesquiterpene was shown to inhibit both germination rate and seedling growth of
Brassica napus L. and Raphanus sativus L.28 β-Caryophyllene was reported also as a
constituent of the volatile oil of Ageratum conyzoides L., which is highly inhibitory to
seedlings of crops in south China.29 It was shown, however, that the volatile oil is more
inhibitory than its isolated constituents, a reason why it has been suggested that there is
29
a phytotoxic synergism among allelochemicals of volatile oils. Two of the most
abundant constituents of the fraction studied in the present work are 3-prennylcinnamic
acid allyl ester (12) and benzenepropanoic acid methyl ester (3; Figure 1, Table 1). To
our knowledge, nothing has been published about their phytotoxic effect.
The present results suggest that at least part of the previously reported
the plant.
This is the first report regarding the phytotoxic activity not only of Brazilian
propolis but also of the volatiles of overall propolis types. Given the wide diversity of
the chemical composition of propolis,5,8 the present work demonstrates that much lies
potential herbicides.
The chemical composition of the volatile fraction of the studied Brazilian green
propolis is different from those previously described. Due to the wide variation found in
chemical composition of propolis these results were expected and may contribute to a
better understanding of the chemical variation of the different types of propolis. Also,
the volatile fraction of Brazilian green propolis influences both germination of seed
lettuce and the growth of its seedlings, showing that the studied sample possess an
allelopathic potential.
AKNOWLEDGEMENTS
Estado de São Paulo) for financial support and Professor Dejair Message for the
provision of the propolis sample. AS and MLFS are research fellows of CNPq
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the chemistry of plant products. Nat Prod Rep 28: 925–36 (2011).
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20 Maróstica Junior RM, Daugsch A, Moraes CS, Queiroga CL, Pastore GM, Park
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and a novel compound from chloroform extracts. J Apic Res 42: 1–3 (2003).
26 Elakovich SD. The Essential oil of Lippia adonensis leaves and flowers. J Nat
constituents of volatile oil from Ageratum conyzoides. J Chem Ecol 25: 2347–
2356 (1999).
municipality of Viçosa (state of Minas Gerais, southeast Brazil). RT: retention time; LRI: linear retention
index.
11 1619 nd*** 13.3 206, 188 (60), 133 (100), 77 (10) 7-phenyl-5-oxo-
heptanol 4
germination rate index; HRA: hypocotyl-radicle axis; Cot. Leaf: cotyledon leaf.
Control 95.0 ± 4.1a 100 ± 0.0a 100 ± 0.0a 26.9 ± 0.8a 71.3 ± 1.0a 9.2 ± 0.2a
0.1% 0.0 ± 0.0a 91.7 ± 4.7b 98.3 ± 2.4b 7.4 ± 0.2b 61.5 ± 3.8a 7.7 ± 0.3b
0.5% 0.0 ± 0.0a 20.0 ± 4.7b 95.0 ± 7.1c 3.7 ± 5.7c 31.6 ± 5.7b 5.9 ± 0.6c
1.0% 0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0d 0.0 ± 0.0c 0.0 ± 0.0d
* Results with the same letters in comparisons among lines are not significantly different (p < 0.05).
** Results with the same letters in comparisons among columns are not significantly different (p < 0.05
propolis from the municipality of Viçosa (state of Minas Gerais, southeast Brazil).