Biochemical Systematics and Ecology 41 (2012) 119–125

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Biochemical Systematics and Ecology

journal homepage: www.elsevier.com/locate/biochemsyseco

Phytochemical and allelopathic studies of Terminalia catappa

L. (Combretaceae)
Tatiana de Gouveia Baratelli a, Anne Caroline Candido Gomes a, Ludger A. Wessjohann b,
Ricardo Machado Kuster a, *, Naomi Kato Simas a, *
a
Núcleo de Pesquisas de Produtos Naturais (NPPN), Centro de Ciências da Saúde (CCS), Bloco H, Universidade Federal do Rio de Janeiro, CEP: 21941-902,
Ilha do Fundão, Rio de Janeiro, RJ, Brazil
b
Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Weinberg 3, D-06120 Halle/Saale, Germany

a r t i c l e i n f o a b s t r a c t

Article history: The allelopathic potential of Terminalia catappa L. Combretaceae fruits and leaves on
Received 12 July 2011 Lactuca sativa L. (lettuce), Euphorbia heterophylla L. and Commelina benghalensis L. was
Accepted 9 December 2011 studied. Bioassays indicated the highest activity for dichloromethane and ethyl-acetate
Available online 16 January 2012
fractions of ethanolic extracts from fruits, and the mean effective concentration (EC50)
was determined. 2-Pentadecanone; vanillic, siringic, ferulic, p-coumaric, palmitic and
Keywords:
stearic acids were characterized in the dichloromethane fraction, and 3,4,40 -tri-O-methyl
Terminalia catappa L
ellagic acid and b-sitosterol-3-O-b-D-glucoside were isolated from it. No allelopathic effects
Allelopathy
Lactuca sativa L. were observed when the dichloromethane extracts of T. catappa fruit or leaf extracts were
Seasonal influence applied to the weeds E. heterophylla and C. benghalensis. Bioassays with seasonal sampling
revealed an influence on the allelochemical potential of T. catappa. Considering the
methodology adopted and the experimental results, the allelopathic activity of T. catappa
seems to be related to the interaction of different groups of substances, some of them
identified and characterized in this work.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Throughout the last decades, many research reports were published on compounds from plants with allelopathic activity
showing their inhibitory or stimulatory effects on various crop plants (Mizutani, 1999; Vyvyan, 2002; Macías et al., 2006).
Allelopathic compounds released by plants into the rhizosphere have effects on neighboring plants ranging from phyto-
toxicity to organogenesis induction. Allelochemicals release can occur by a variety of mechanisms such as volatile emission or
leaching from leaves, or exudation from roots (Inderjit and Weiner, 2001; Weir et al., 2004). Allelopathy phenomena among
plant species are responsible for natural selection in plant communities, e.g. protecting the donor plant against microor-
ganisms, viruses, insects and other pathogens or predators, or even inhibiting neighboring plant’s growth or stimulating the
growth of the seeds (Einhelling, 1996; Seigler, 1996; Dayan et al., 2000; Inderjit and Weiner, 2001).
Terminalia species are native from Africa and are now widely spread out in tropical and sub-tropical regions (Collins et al.,
1992). In Brazil, Terminalia catappa is an attractive, long-lived tree well suited to ornamental and amenity plantings. The
leaves and fruits drop results in organic matter under the trees. The allelochemical concentration in decomposing plant
residues is an important aspect in allelochemical interactions. Field observations indicated the reduction or even absence of

* Corresponding authors. Universidade Federal do Rio de Janeiro (UFRJ), Av. Carlos Chagas Filho, 373 – CCS, Bloco H, sala H0-017, Cidade Universitária, Rio
de Janeiro, RJ 21941-902, Brazil. Tel.: þ55 21 2562 6795; fax: þ55 21 2562 6512.
E-mail addresses: kuster@nppn.ufrj.br (R.M. Kuster), naomisimas@yahoo.com (N.K. Simas).

0305-1978/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bse.2011.12.008
120 T.deG. Baratelli et al. / Biochemical Systematics and Ecology 41 (2012) 119–125

vegetation around some trees of T. catappa. The delivery of allelochemicals into the rhizosphere can also occur through
leaching from leaves and other aerial plant parts, including breakdown-products of bark and fruit/leaf litter. This work aimed
at the assessment of the allelopathic potential of T. catappa’s leaves and fruits in laboratory bioassays, as well as at the
isolation and identification of the active principles. Seasonal influences of allelopathic activity of ethanol and aqueous extracts
were also investigated. This is the first investigation of allelopathic effects of T. catappa on Lactuca sativa on the weeds
Euphorbia heterophylla and Commelina benghalensis.

2. Materials and methods

2.1. Plant material

T. catappa’s fallen fruits and leaves (yellow and green) were collected in Ilha do Governador, Rio de Janeiro city, Brazil
during spring, summer, autumn and winter, between March 2004 and July 2005. All the material was identified by Dr Nilda
Ferreira from The Botanic Garden of Rio de Janeiro (herbarium voucher n RB401049). Leaves and fruits were dried in an oven
at 50  C for 5 days and then grinded using a mill. Lettuce (L. sativa ‘White Boston’), E. heterophylla and C. benghalensis were
used as bioassay species. Lettuce cypselae were purchased from Isla Pak, Rio Grande do Sul state, Brazil, the other seeds were
obtained from Agrocosmos, Sao Paulo, Brazil.

2.2. Germination and growth bioassays

Leaves and fruits of T. catappa were soaked in different solvents (ethanol, hexane, dichloromethane, ethylacetate and
water) and the extracts were tested for allelopathic activity on lettuce at different concentrations (ranging from 2200 to
2 ppm, n ¼ 550, for each extract). Two weeds (E. heterophylla and C. benghalensis) were also used as test species for allelo-
pathic growth assay with dichloromethane fractions obtained from fruits and green leaves of T. catappa at 336 ppm (EC50
previously calculated on lettuce). Twenty five cypselae of L. sativa, and 25 seeds of C. benghalensis and E. heterophylla were
sown in separate Petri dishes (9 cm diameter, 2 cm height) lined with Whatman N 1 filter paper discs (8 cm diameter). The
filter paper discs contained a defined concentration of the test compounds. After application of the extract, solvent was
allowed to evaporate within 24 h at room temperature and 5 ml of an aqueous solution (0.1% DMSO in distilled water) was
added to each Petri dish. When testing aqueous extracts, 1 ml of the test solution was supplemented with 4 ml of 0.1% DMSO
distilled water. Petri dishes with 5 ml of 0.1% DMSO in water were used as controls. Treatments and controls were assayed in
duplicate and replicated three times (n ¼ 150). Seedling measurements (length of hypocotyls and roots) were made on
millimetric paper.
Purified compounds were tested against lettuce at 336 ppm only. The experimental design was the same as described
above, but the Petri dishes used were smaller (3.5 cm diameter, 1 cm height), using 10 cypselae and 2.5 ml of 0.1% DMSO
distilled water. In this case, treatments and controls were carried out in triplicate and repeated three times (n ¼ 90).
All Petri dishes were incubated in the dark in a growth chamber, at 25  C for lettuce or at 30  C for C. benghalensis and E.
heterophylla. For the weed species growth assay 2.5 ml of aqueous solution (0.1% DMSO in water) were added on day 10 in
germination chamber.
Germination (root protrusion, Adegas et al., 2003) was evaluated after 24 h, only for L. sativa. Root and shoot lengths were
recorded after 5 d (L. sativa), 20 d (E. heterophylla) and 30 d (C. benghalensis). The same plants were used in germination and
growth tests.

2.2.1. Determination of EC50 values

Statistical analyses were made through variance analysis (ANOVA) by the t-test with level of significance of p > 0.05 using
the applicative GraphPad Instat 3.1 version. Mean effective concentration (EC50) was calculated by the software SPSS for
Windows.

2.3. Extraction and isolation of compounds

Dry-grinded leaves and fruits were separately soaked in ethanol until extraction was complete. The extract was
concentrated in vacuum and partitioned against hexane, dichloromethane and ethyl-acetate. The original crude, and hexane,
dichloromethane and ethylacetate extracts, and the aqueous residue were bioassayed using lettuce as a test plant. Sephadex
LH-20 column chromatography (45 mm diameter  30 cm height) of the dichloromethane fraction (2.6 g) from T. catappa’s
fruits resulted in eight fractions. Each of them was bioassayed on lettuce and among these fractions F2, F3, F4 and F5 were the
most active ones. Fraction F2 was subjected to GC–MS analysis. Two different precipitates (both insoluble in methanol) were
obtained from fractions F2 and F3, compounds 1 (17 mg) and 2 (49 mg), respectively. Only compound 2 was active at
336 ppm. These compounds were further identified as 3,4,40 -tri-O-methyl ellagic acid (1) and b-sitosterol-3-O-b-D-glucoside
(2). F4 (0.6 g) was subjected to MPLC in an SR 10/50 column (10 mm diameter  50 cm height), using Merck’s Silica gel 60 (70–
230 Mesh-ASTM) as stationary phase. Another portion of F4 (0.8 g) was subjected to two sequential silica gel column
chromatography separations (15 mm diameter  10 cm height) producing subfractions which were bioassayed. The most
active (sub-)fractions were then co-injected with p-coumaric, siringic, ferulic, salicylic and ellagic acid into an HPLC with
 T.deG. Baratelli et al. / Biochemical Systematics and Ecology 41 (2012) 119–125 121

diode-array detection to check for the occurrence of these compounds. HPLC analyses were carried out using a SHIMADZU
CBM-10A chromatograph consisting of an LC-10AD pump and an SPD-M10A UV detector. A Lichrosorb-RP-18 column (25 cm
height  5 mm diameter) was used. Chromatographic conditions were done as described in Wen et al. (2005).

H3C CH3

O CH3
CH3 CH3
O O
CH3 H3C

O OH
H3C OH O
CH3 O
O O OH
HO
O HO

2.4. Structural identification

Compounds 1 and 2 were determined by nuclear magnetic resonance spectroscopy (NMR). Mass spectroscopic techniques
were used only for the identification of compound 1. NMR spectra were run using a Bruker AM-200 spectrometer operating at
500 MHz for 1H and 200 MHz for 13C. Spectra were recorded using DMSO-d6 as solvent and internal reference. MS spectra
were recorded on a LC–MS System from Applied Biosystems API-150EX (ESI – 70 kV) and using a direct inlet system. GC–MS
spectra were carried out on Agilent Network 6890 under electron impact (70 eV) conditions, using a quadrupole ion detector
model 5973, Wiley 7N database, Agilent capilar column DB-5MS (30 m  0.25 mm), helium carrier gas at 0.5 ml/min,
temperature programmed from 250  C to 290  C (5  C/min) and 1 ml injection.

3. Results and discussion

3.1. EC50 determination

Bioassays were carried out in a concentration series, ranging from 2200 to 2 ppm, n ¼ 550 for each, using lettuce as
a species test, in order to calculate the EC50 (mean effective concentration) values, which are presented in Table 1.
It is important to point out that other works described allelopathic effects in higher concentrations, ranging from 5000 to
330,000 ppm (Chon et al., 2002, 2003; Mazzafera, 2003; Gatti et al., 2004; Periotto et al., 2004; Nasir et al., 2005; Souza Filho
et al., 2005a,b). The mean effective concentration obtained for T. catappa extracts was lower (336–1504 ppm), which rein-
forces its potential as a species with allelopathic effect.

3.2. Bioassay-directed chemical fractionation (T. catappa fruit extracts)

From all extracts and fractions tested at 1000 ppm, only those obtained with dichloromethane as solvent show an
inhibitory effect on lettuce germination (77% inhibition relative to the control).

Table 1
EC50 values for different extracts from T. catappa, calculated by the software SPSS for Windows.

Sample EC50 (ppm) on L. sativa roots EC50 (ppm) on L. sativa hypocotyls

Ethanol extract 1448 –a
(green leaves)
March 2004

Ethanol extract 1504 –a

(fruits)
March 2004

Ethylacetate fraction 383 943

(fruits)
March 2004

Dichloromethane fraction 336 797

(fruits)
March 2004
a
EC50 not established, insufficient interval.
122 T.deG. Baratelli et al. / Biochemical Systematics and Ecology 41 (2012) 119–125

At 1000 ppm, the ethanol, dichloromethane and ethylacetate extracts show inhibition effects against lettuce root growth
(55%, 86% and 73% inhibition, respectively). Effects on radicle elongation were greater than those observed on shoot growth
(41% and 49% inhibition for dichloromethane and ethylacetate fractions, respectively, at 1000 ppm).
The dichloromethane and ethylacetate fractions are the most active ones, but the latter exhibits no effect on germination,
even at 1600 ppm. Therefore, further fractionation was pursued with the dichloromethane sample.
Successive bioassay-directed chromatographies were carried out. The eight fractions obtained were tested at 336 ppm
(EC50 previously established for the dichloromethane sample). GC–MS analysis of fraction F2 revealed the major presence of
fatty acids such as palmitic and stearic acid, and 2-pentadecanone. Experimental data are consistent with previous studies
that indicated fatty acids as potential allelochemicals (Suzuki et al., 1998; Kim and Kil, 2001; Bhowmik and Inderjit, 2003;
Inderjit and Duke, 2003; Chiang et al., 2004; Nakai et al., 2005). Fraction F4 showed growth inhibition of 66% while the
others inhibited 20–34%.

3.3. Bioassay of purified compounds

Compounds 1 and 2 were bioassayed at 336 ppm. Only 2 inhibited lettuce growth. Spectroscopic data of compounds 1 and
2 (MS, 1H and 13C NMR) were in accordance with data previously reported in the literature (Cheng et al., 1998; Gauvin et al.,
1998; Lingbo et al., 2005; Chung et al., 2005).
Chung et al. (2005) isolated b-sitosterol-3-O-b-D-glucoside (among other compounds) from Oryza sativa L. and carried out
allelopathic tests against duckweed (Lemna paucicostata) and this substance showed about 13–20% inhibitory activity based
on chlorophyll reduction, while b-sitosterol did not show any herbicidal activity. These results support the allelopathic effect
attributed to b-sitosterol-3-O-b-D-glucoside isolated from T. catappa against lettuce.

3.4. Identification of phenolic acids

Fraction F4-B4 was co-injected in HPLC with 5 standards of phenolic acids: p-coumaric, siringic, ferulic, salicylic and ellagic
acid. Comparison of the UV spectra, retention times and peak enhancements revealed the presence of p-coumaric, siringic
and ferulic acid in F4-B4. HPLC-DAD analysis of fraction 49–55 pointed out the presence of vanillic and p-coumaric acid. The
role of phenolic acids in different ecosystems has been widely studied with special focus on their potential phytotoxicity.
Extensive research has been done in the field of allelopathy, and, consequently, a great number of allelochemicals have been
reported. The essential role that secondary metabolites such as phenolic compounds play in complex interactions among
living organisms in the natural environment has been unraveled gradually. In the last three decades, the potential of phenolic
acids as allelochemicals has been widely described in the literature, not only in laboratory bioassays but also in field studies
(Dietz and Winterhalter, 1996; Chung et al., 2002; Inderjit et al., 2002; Chon et al., 2003; Iqbal et al., 2003; Beninger et al.,
2004; Djurdjevic et al., 2004; Sánchez-Moreiras et al., 2004; Kim et al., 2005; Souza Filho et al., 2005b; Blum and Gerig, 2005).

3.5. Weed bioassays

No allelopathic effect was observed for the dichloromethane extract from the leaves and fruits of T. catappa at 336 ppm
when evaluated on hypocotyl and radicle elongation of two soy weeds, E. heterophylla and C. benghalensis.
It’s important to note that the herbicidal activity of sorgoleone, for instance, is strongest on small-seeded weeds (Nimbal
et al., 1996; Rimando et al., 1998; Souza et al., 1999; Almeida et al., 2001; Dayan, 2006). Large seeded weeds tend to be less
sensitive to allelochemicals such as sorgoleone, because these plants may avoid the phytotoxic effect by having lower
absorption and translocation or faster metabolic degradation of the allelochemicals (Dayan, 2006).
As the weed species bioassayed in this study have larger seeds than lettuce, and considering Dayan’s observation, it could
be inferred that the weeds selected tend to be more resistant than lettuce to allelochemicals. The lack of effect observed could
also be related to the fact that the EC50 established for lettuce may be smaller than the EC50 for the weeds. The sensitivity of
target species may also vary considerably with plant developmental stages or under particular environment conditions.

3.6. Seasonal analyses

Plants can change their allelopathic potential according to their exposure to environmental conditions and are affected by
different factors such as water stress, light incidence, latitude, nutrients supply, temperature, pollution and microorganisms
(Kato-Noguchi, 1999). Seasonal analyses are relevant because they allow correlating the period of the year and the production
of metabolites with biological activity (Nogueira, 1998).
Green leaves of T. catappa were collected in spring (October, 2004), summer (January, 2005), autumn (April, 2005) and
winter (July, 2005). Ripe fruits were collected during the fructification period of the species, in summer (January, 2005),
autumn (April, 2005) and winter (July, 2005). Yellow leaves were collected in winter (July, 2004).
Ethanol extraction is supposed to maximize the extraction of allelochemicals, while aqueous extraction simulates natural
conditions that trees are subjected to, as precipitation passes through the plant litter into the ground. Also the canopy
participates in leaching chemicals into the environment. After this consideration, aqueous and ethanol extracts from seasonal
 T.deG. Baratelli et al. / Biochemical Systematics and Ecology 41 (2012) 119–125 123

sampling were used in order to assess some influence on the allelochemical effect. Bioassays were carried out at 1448 and
1504 ppm (EC50 previously established for the leaf and fruit ethanolic extract, respectively).
Major allelopathic activity was evidenced in winter (July, 2005) for the fruit ethanol extract (30% inhibition on germination
and 65% inhibition on lettuce root growth). A hypothesis for this allelopathic activity crest in winter (end of fructification
period) is that there may be a redirection of secondary metabolites from senescent leaves to other parts of the plant such as
fruits, seeds and stems. Nutrients that were invested in the leaf may be reallocated to younger leaves and to growing seeds, or
may be stored for the next growing season (Quirino et al., 2000; Lim et al., 2003; Yoshida, 2003; Guiboileau et al., 2010).
For the leaf ethanol extract, the highest activity was observed in southern summer (January, 2005) with 80% lettuce root
growth inhibition. The allelopathic effect decreased in autumn (60% root growth inhibition) and was restored in winter (Fig. 1).
Aqueous extracts exhibited lower levels of allelopathic inhibition in relation to ethanol ones. Aqueous extracts from fruits
and leaves showed higher inhibitory effects in autumn (April, 2005).
As some substances may alter the pH of the environment and interfere with the allleopathic effect (Ferreira and Aquila,
2000; Macías et al., 2000), the pH of aqueous samples was verified. A variation from pH 3–5 was observed, but it was not
possible to establish a direct relation between pH and allelopathic effect (Fig. 2).
Dichloromethane extracts from ripe fruits (March, 2004), green (March, 2004) and yellow leaves (July, 2004) were also
bioassayed at 200 ppm. It was observed that the yellow leaves dichloromethane fraction inhibited lettuce root growth by 9%
while green leaves and fruit extracts reach inhibition levels of 25–31%. This indicates that the concentration of allelochemicals
in yellow leaves is lower than in green leaves. Senescence contributes to the plant survival and its developmental program, by
allowing nutrient recycling and reallocation all along plant life, and this may include allelochemicals not required outside the
growth period (Quirino et al., 2000; Lim et al., 2003; Yoshida, 2003; Guiboileau et al., 2010). Also, in winter with its lower
temperatures there is a slowdown in vegetal metabolism. Both effects may explain the different allelopathic potency
observed for green and yellow leaves. Bioassay results pointed out that fallen leaves and riped fruits are involved in allelo-
pathic phenomena of T. catappa, while yellow leaves do not.
There was a significant reduction of the EC50 value when comparing ethanol extract, dichloromethane and ethylacetate
fruit fractions. However, the allelopathic effect was scattered over several fractions, indicating that more than one compound
is responsible for the effect. Although a wide range of chemicals has been identified from several plants, it is not yet
demonstrated beyond doubt that isolated chemicals are actually responsible for allelopathic activity under field conditions
(Bhowmik and Inderjit, 2003). Allelopathic activity in the field often is thought to be a joint action of several allelochemicals
rather than to one unique allelochemical (Einhelling, 1996; Inderjit et al., 2002; Bhowmik and Inderjit, 2003; Inderjit and
Duke, 2003).
In conclusion, allelopathic bioassays indicate that the dichloromethane and ethyl-acetate fractions of T. catappa’s fruits
were the most active ones. Their EC50 were established at 336 and 383 ppm, respectively, against lettuce radicles. Only
dichloromethane fractions inhibit L. sativa germination. Lettuce roots showed to be more sensitive to allelochemicals than the
shoots. The allelopathic activity of T. catappa might be related to the interaction of different groups of substances such as fatty
acids (stearic and palmitic acids), 2-pentadecanone, phenolic acids (vanillic, siringic, ferulic and p-coumaric acids) and b-
sitosterol-3-O-b-D-glucoside identified and characterized in this work. This study points out, that the allelochemicals are
released into the environment through green leaves and accumulated fallen fruits of T. catappa. Allelopathy might play a role
in the successful invasion of T. catappa into non-native environments, but further field studies are required to evaluate the
significance of the allelochemicals on neighborhood species that co-occur in nature with this species.

90
% Effects on radicle lenghts of L. sativa (%

c
80
b b,c
70 d
inhibition from control)

60
a a a a a
50

40

30

20

10

0
mar/04 abr/04 mai/04 jun/04 jul/04 ago/04 set/04 out/04 nov/04 dez/04 jan/05 fev/05 mar/05 abr/05 mai/05 jun/05 jul/05
Months of the year

Fig. 1. Influence on allelochemical effect of ethanolic extracts from seasonal sampling of green leaves at 1448 ppm (full line with squares), and of ripe fruits at
1504 ppm (dotted line with triangles), of T. catappa against L. sativa’s radicles. Data were the means of an application to 150 plants (two independent replicates
measured as technical triplicates, each with 25 plants). Letters indicate results from statistical comparisons, data from tests sharing the same letter do not differ
significantly (p > 0.05, t-test).
124 T.deG. Baratelli et al. / Biochemical Systematics and Ecology 41 (2012) 119–125

% Effects on radicle lenghts of L. sativa (%

60
a a a d
50 4,98

inhibition from control)

b b b
40 5,05
5,04
4,92 3,75
d
30
c
c
20 3,05
4,12

10

0
mar/04 abr/04 mai/04 jun/04 jul/04 ago/04 set/04 out/04 nov/04 dez/04 jan/05 fev/05 mar/05 abr/05 mai/05 jun/05 jul/05
Months of the year

Fig. 2. Influence on allelochemical effect of aqueous extracts from seasonal sampling of green leaves at 1448 ppm (full line with squares), and ripe fruits at
1504 ppm (dotted line with triangles) of T. catappa on L. sativa’s radicles, and their respective pH values. Data were the means of 150 plants (two independent
replicates in triplicate, each with 25 plants). Letters indicate results from statistical comparisons, data from tests sharing the same letter do not differ significantly
(p > 0.05, t-test).

Acknowledgments

The authors thank CAPES (Conselho de Administração de Pessoal de Ensino Superior) for a master scholarship for T. de G.
Baratelli; NPPN-UFRJ (Natural Product Chemistry Pos-Graduating Program of Federal University o Rio de Janeiro) and CNPq
for financial support.

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