Composition of the volatile fraction of a sample of Brazilian green

propolis and its phytotoxic activity

Caroline C. Fernandes-Silva,a* Carolina A. Lima,a Giuseppina Negri,b Maria L. F. Salatino,a

Antonio Salatino,a Marco A. S. Maywormc

a
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

05508-090, São Paulo SP, Brazil. E-mail: carolinecfs@usp.br

a
Laboratory of Phytochemistry, Department of Botany, Institute of Biosciences, University of

São Paulo, São Paulo Brazil.

b
CEBRID, Department of Preventive Medicine, Federal University of São Paulo, São Paulo,
SP, Brazil.
c
University of Santo Amaro, São Paulo, SP, Brazil

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Record. Please cite this article as doi: 10.1002/jsfa.7045

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 ABSTRACT

BACKGROUND: Propolis is a resinous material produced by honeybees, containing mainly

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

phenylpropanoids, in addition to linalool and α-terpineol (monoterpenes), were also detected.

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

hypocotyl-radicle axis and the development of the cotyledon leaf.

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.

Keywords: Allelopathy, Brazilian green propolis , phenylpropanoids, phytotoxicity, terpenoids,

volatiles.

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INTRODUCTION

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

pointed out as potential herbicides in management agricultural systems.2 Such studies

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

may be also valuable in weed management.4

Baccharis dracunculifolia D.C, the botanical source of the Brazilian green

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

Propolis is a resinous material produced by honeybees, containing mainly

beeswax and plant material.7 The resin provided by plants comprises a wide diversity of

secondary metabolites, including terpenoids and phenolic compounds and is responsible

for the several biological activities found in different types of propolis, such as

antimicrobial, antioxidant, antiviral and antitumoral.8

Main constituents from alcoholic extracts of Brazilian green propolis are

prenylated phenylpropanoids, such as artepillin C,7,8 as well as caffeoylquinic acids.9

Recently, special attention has been given to its volatile fraction, and quite distinct

chemical profiles have been reported.10 Kusumoto and co-workers detected

vinylchromenes, vinylprenylphenols, acetophenones, allylbenzene, dimetoxystyrene and

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the terpenoid spathulenol.11 On the other hand, De Albuquerque et al. reported

sesquiterpenes, such as caryophyllene and nerolidol, as major constituents of green

propolis.12 Ioshida et al. detected neither caryophyllene nor nerolidol in the volatiles

from Brazilian propolis; instead, other sesquiterpenes were reported, in addition to

monoterpenes.13 Oliveira and co-workers found a distinct composition, based mainly on

caryophyllene, acetophenone and β-farnesene, in addition to α-terpineol, δ-cadinene,

spathulenol, α-ylangene, γ-elemene and valencene.14

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

that only antibacterial, antimicrobial, antifungal and antioxidant properties of propolis

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.

In order to increase the knowledge on the chemical variation of the volatile

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

seed germination and seedling growth.

MATERIAL AND METHODS

Propolis sample

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 The propolis sample was collected in the period November 2008-March 2009,

from beehives located on the campus of the Federal University of Viçosa (state of

Minas Gerais, southeast Brazil).

Extraction of the volatile fraction and identification of the compounds

About 350 g of propolis was powdered and submitted to steam distillation in a

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

coupled to a mass spectrometer 5975C VL MSD (Agilent), operating with electron

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

determined as previously described by Viegas and Bassoli.19 Compounds were

identified by comparison of mass fragmentation patterns with mass spectra of NIST 08

Library and LRI with literature data.20,21

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.

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Phytotoxic effects were evaluated by determination of the germination rates,

germination rate indices and lengths of hypocotyl-root axes and cotyledon leaves.

Statistical analysis

Percent germination, growth germination rate and lengths of hypocotyl-radicle

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

“concentration” as independent variables, and Tukey’s test to evaluate the effect of

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

analyses were performed with GraphPad Prism version 6.0.

RESULTS AND DISCUSSION

Chemical characterization

The major constituents characterized in the volatile fraction were 3-

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-

terpineol (2), sesquiterpenes β-caryophyllene (5), β-copaene (6), cis-muuroladiene (7),

germacrene D (8) and δ-cadinene (9), as well as the phenylpropanoids

benzenepropanoic acid methyl ester (3) and benzenepropanoic acid ethyl ester (4) were

also detected (Fig. 1, Table 1).

Wide variation in chemical composition is characteristic of propolis.22 Such

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

et al. in the volatile fraction of Brazilian propolis, such as vinylchromenes and

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vinylprenylphenols, have never been detected by other authors.11 Chiefly sesquiterpenes

have been reported as major compounds of the volatile fraction of Brazilian

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

reported as a new component of Brazilian green propolis,23 and it was later

characterized as a marker of this type of propolis.24 Although abundant in the volatile

fraction, it has a considerably high retention time (Figure 1) and is characterized by LRI

2016.

Phytotoxic activity

The volatile fraction at 1% concentration inhibited completely the germination

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

leaves (Table 2).

The volatile fraction of Brazilian green propolis influences both germination of

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

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the seeds and the growth of seedlings of Amaranthus hybridus L. and Portulaca

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

constituents of essential oils of four studied Teucrium species, monoterpenes, including

α-terpineol (2), were the most effective growth inhibitors of seedlings of radish and

garden cress.27 β-Caryophyllene (5) is a minor constituent of the volatile fraction of

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

allelopathic properties of B. dracunculifolia6 are accounted for volatile compounds of

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

ahead to be studied about volatile compounds from propolis as environmentally friendly

potential herbicides.

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 CONCLUSIONS

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

The authors would like to thank FAPESP (Fundação de Amparo à Pesquisa do

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

(Conselho Nacional do Desenvolvimento Científico e Tecnológico).

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Table 1. Main compounds of the volatile fraction of a sample of Brazilian green propolis from the

municipality of Viçosa (state of Minas Gerais, southeast Brazil). RT: retention time; LRI: linear retention

index.

Peak LRI obt. LRI lit. Relative m/z Proposed compound

Amount (%)

1 1097 1095 2.1 154(5), 121(20), 93(80), Linalool 2

80(30), 71(100)

2 1180 1186 1.3 154(10), 121(60), 93(75), 81 (40), α-Terpineol 2

59(100)

3 1267 1264 9.3 164(30), 133(10), 104(100), 91 Benzenepropanoic

(60), 77(20) acid methyl ester 3

4 1339 1349 5.7 178(30), 133(10), 104(100), 91(60), Benzenepropanoic

77(20) acid ethyl ester 3

5 1405 1408 2.6 204, 133(90), 105(60), 90 (100), β-Caryophyllene 2

69(80)

6 1423 1432 3.2 204, 161(90), 105(80), 93(80), β-Copaene 2

91(100)

7 1448 1450 1.6 204, 161(100), 119(60), 105 (60), cis-Muuroladiene 2

91(60)

8 1500 1484 1.5 204, 161(100), 105(40), 119 (40), Germacrene D 2

93(30), 91(40)

9 1512 1513 4.3 204(50), 189(20), 161(100), δ-Cadinene 2

134(60), 11(70), 105 (85), 91 (80)

10 1557 1577 23.4 220, 205(60), 119(80), 105 (70), Spathulenol 2

91(90), 93(100)

11 1619 nd*** 13.3 206, 188 (60), 133 (100), 77 (10) 7-phenyl-5-oxo-
heptanol 4

12 2016 nd*** 26.3 256(80), 201(90), 185(100), 3-prenylcinnamic

145(90), 115(40) acid allyl ester 5
*LRI obt. = Linear Retention Index obtained on the present work; ** LRI lit. = Linear Retention Index previously described in
literature; ***nd = not described previously; characterization based on: 1 Adams (2009); 2: NIST 08 library and Adams (2009); 3
NIST 08 library and Maróstica Júnior (2008); 4 suggested structure obtained through fragmentation pattern; 5 isolated and fully
characterized standard (Negri et al., 2003)

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Table 2. Phytotoxic activity of the volatile fraction of a Brazilian green propolis from Viçosa, MG. GRI:

germination rate index; HRA: hypocotyl-radicle axis; Cot. Leaf: cotyledon leaf.

Germination (%)* Length (mm)

Sample Day 1 Day 4 Day 7 GRI (%) ** HRA** Cot. 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

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Figure 1. Chromatogram, obtained by CG-MS, of the volatile fraction of a sample of Brazilian green

propolis from the municipality of Viçosa (state of Minas Gerais, southeast Brazil).

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