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Acta Scientiarum 

 

http://www.uem.br/acta 
ISSN printed: 1679-9283 
ISSN on-line: 1807-863X 
Doi: 10.4025/actascibiolsci.v39i3.34801 

 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 373-380, July-Sept., 2017 

Antioxidative enzymes of Cucumis sativus seeds are modulated by 
Leucaena leucocephala extracts 

Vandjore de Mattos Ribeiro

1*

, Ariane Spiassi

2

, Thaís Regina Marcon

2

, Gislaine Piccolo de Lima

2

Jaqueline Malagutti Corsato

3

 and Andréa Maria Teixeira Fortes

1

 

1

Programa de Pós-Graduação em Conservação e Manejo de Recursos Naturais, Universidade Estadual do Oeste do Paraná, Rua Universitária 

2069, Jardim Universitário, 85819-110, Cascavel, Paraná, Brazil. 

2

Centro de Ciências Biológicas e da Saúde, Universidade Estadual do Oeste do 

Paraná, Cascavel, Paraná, Brazil. 

3

Programa de Pós-Graduação em Engenharia Agrícola, Universidade Estadual do Oeste do Paraná, Cascavel, 

Paraná, Brazil. *Author for correspondence. E-mail: vandm_@hotmail.com  

ABSTRACT. This study aimed to analyze oxidative stress produced in cucumber seeds and seedlings 
when exposed to aqueous extract from dried leaves of leucaena, as well as its effect on the germination 
behavior, early growth and the antioxidant enzymes activity. It was evaluated the percentage, the speed 
index, the average time, the frequency and germination synchronization, the root length and shoot, as well 
as the catalase and peroxidase enzymes activity. There was no significant inhibitory effect of the leaf 
extracts on the germination percentage. However, there was delay in the seeds germination, as the extract 
proportion increased. A stimulatory effect of the extract compared to the shoot length was observed, 
however the root growth was significantly reduced. The catalase activities had a peak at 24 hours after 
soaking the seeds, however, the activities were reduced in seedlings. The peroxidase activity was low in the 
seeds and increased in the seedlings at 168 hours after immersion. The results suggest that there was 
oxidative stress due to allelochemicals present in the leaves extracts from leucaena, verified by germination 
and initial growth changes, causing alterations in the plants rootlets.

 

Keywords: allelopathy, reactive oxygen species, antioxidant enzymes. 

Enzimas antioxidativas de sementes de Cucumis sativus são moduladas por extratos de 
Leucaena leucocephala 

RESUMO. Este trabalho objetivou analisar o estresse oxidativo produzido em sementes e plântulas de 
pepino quando submetidas a extrato aquoso de folhas secas de leucena bem como, seu efeito sobre o 
comportamento germinativo, crescimento inicial e atividade de enzimas antioxidantes. Foram avaliadas a 
porcentagem, índice de velocidade, tempo médio, frequência e sincronização da germinação, comprimento 
de raiz e parte aérea e atividade das enzimas catalase e peroxidase. Não houve efeito inibitório significativo 
dos extratos sobre a porcentagem de germinação. No entanto, houve atraso na germinação das sementes, à 
medida que se aumentou a proporção do extrato. Foi observado efeito estimulatório do extrato em relação 
ao comprimento da parte aérea, porém o crescimento da raiz foi reduzido significativamente. As atividades 
da catalase tiveram pico às 24 horas de embebição das sementes, tendo sido reduzido nas plântulas. No 
entanto, a atividade da peroxidase foi baixa nas sementes e teve aumento nas plântulas, às 168 horas após a 
embebição. Os resultados sugerem que houve estresse oxidativo devido aos aleloquímicos presentes nos 
extratos foliares de leucena, verificado pelas alterações na germinação e de crescimento inicial, o que causou 
alterações nas radículas das plântulas. 

Palavras-chave: alelopatia, espécies reativas de oxigênio, enzimas antioxidantes. 

Introduction 

Invasive species can have profound effects on 

ecosystems (Kimbro et al., 2009), through changes 
in the structure and habitat quality (Espinola & Julio 
Junior, 2007), making changes in the diversity and 
relative abundance of native species and altering the 
succession dynamics of communities. 

In this way, some species are able to change the 

environment of others via chemicals released mostly 

into the soil, a phenomenon known as allelopathy 

(Rice, 1984). According to Rizvi et al. (1992) some 

invasive plants release allelochemicals in the 

environment, being in the aqueous phase of the soil 

or substrate, or by volatilized gaseous substances in 

the air surrounding soil plants. The secondary 

metabolites, after being produced and released, can 

cause direct and indirect effects on other plants. 

Direct effects are characterized by alterations on 

plant metabolism and growth, affecting membranes 

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374 

Ribeiro et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 373-380, July-Sept., 2017 

and its permeability, hormone concentrations, and 
enzyme activity (Sunmonu & Van Staden, 2014; 

Cheng & Cheng, 2015). Gholami, Faravani, and 

Kashki (2011) report that inhibitory effects on root 

and shoot elongation can contribute to reduction in 

cell division, due to damage of cell membrane 

caused by allelochemicals. The retarded germination 

and root and shoot length might be influenced by 

damage of root and shoot cells due to interference of 

absorption of nutrients and other growth processes 

caused by allelochemicals found in seed and leaf 

extracts (Elisante, Tarimo, & Ndakidemi, 2013). 

Many of these processes being the allelochemicals 

action results, which can act on the cell degradation 

processes signaling, through the production and 

accumulation of reactive oxygen species (ROS), 

resulting in oxidative stress (Aumonde et al., 2013). 

To cope with oxidative stress conditions that are 

often imposed by the environment, plants have 
developed ROS removal systems, performed by the 
enzymatic antioxidant system, highlighting the 
peroxidase (POD) and catalase (CAT) (Gill & 
Tuteja, 2010). CAT is an enzyme that promotes 
H

2

O

2

 decomposition into H

2

O and O

2

. It is mainly 

found in peroxisomes and can also be present in 
mitochondria and cytoplasm. The Peroxidases 
(POD) are enzymes belonging to the class of 
oxidoreductases, and its role is to catalyze the 
hydrogen peroxide oxidation (H

2

O

2

) or organic 

peroxides (Sharma, Jha, Dubey, & Pessarakli, 2012). 
The peroxidases have been correlated in plants 
resistant to diseases, ethylene biosynthesis, 
lignification and suberization, and protection against 
H

2

O

2

 and other oxidants (Gomes, Smedbol, 

Carneiro, Garcia, & Juneau, 2014).  

Therefore, this study aimed to evaluate the 

allelopathic potential of L.  leucocephala aqueous 
extract, through the antioxidative enzymes analysis, 
along with the germination and early growth of the 
Cucumis sativus (cucumber), due to its characteristic 
bioindicator species, which presents fast and 
uniform germination, besides expressing 
phytotoxicity results even in low concentrations.  

Material and methods 

The study was conducted at the Plant Physiology 

Laboratory at the Universidade Estadual do Oeste do 
Paraná
, Cascavel Campus - PR, from June to 
September 2014. 

The seeds used in the experiment were from C. 

Sativus, long green variety, lot 30129, commercially 
acquired. 

The L. leucocephala extract was obtained from 

leaves, at a senescence stage, observed due to loss of 

green color in leaves results from chlorophyll 
degradation, from a permanent preservation area, 

located between coordinates 25°40’23.03” and 

54°39’46.50”. The leaves were dried at 40° C in air 

circulating oven until obtaining its constant weight 

to avoid the loss of volatile compounds. After drying 

out, they were crushed in a Willey knife mill and 

packaged in labeled glass vials, protected from 

humidity, at ambient temperature and stored for 

three months until their usage. 

For the matrix extract obtention, it as prepared a 

solution containing 100 g of L. leucocephala leaves 

powder for 1 L of distilled water solution, which 

rested for 24 hours and was filtered through a fine 

mesh strainer tissue, resulting in a matrix extract at 

10% (w/v).  

From this extract, 2.5, 5.0 and 7.5% proportions 

(w/v) of the extract in distilled water were obtained. 

In the control treatment only distilled water was 

used. In order to assess this extract’s effect on 

germination, the early growth and antioxidative 

enzymes of C. sativus seeds and seedlings, the 

following tests were performed: 

Germination test:

 For the germination tests four 

repetitions of 20 seeds were performed. The seeds 

were placed in petri dishes with three sheets of 

wetted filter paper with distilled water (control) or 

by the extract different proportions in the 

proportion of 2.5 times the paper weight, and then 

put in a growth chamber at 25° C and 12 hours 

photoperiod (Brasil, 2009). Germination was 
recorded daily, for seven days, being taken into 

consideration the germinated seeds with 2 mm of 

primary root (Hadas, 1976). To analyze the 

germination, the following parameters were taken: 

germination percentage (GP%), germination speed 

index (GSI), as per Maguire (1962), average 

germination time (AGT) and frequency and 

synchronization of germination, both as per 

Labouriau (1983). At the end of the germination 

test, after seven days, a measure of the germinated 

seedlings primary root and shoot was made using a 

ruler. The seedlings measurement results were 

expressed in centimeters.  

For the germination tests, the experimental 

design was completely randomized (CRD), with five 

treatments and four replicates with 20 seeds per 

repetition. The means were compared statistically by 

Tukey test. 

Enzymatic extraction: Five collection points were 

chosen: seeds at 0, 2, 12 and 24 hours after soaking 

period and seedlings at 168 hours after the soaking 

period. 100 mg of vegetal material were 

homogenized in potassium phosphate buffer, at 0.1 

mol L

-1

 and pH 6.8. The homogenate was 

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Antioxidative enzymes modulated by Leucaena leucocephala 375 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 373-380, July-Sept., 2017 

centrifuged at 12,000 rpm for 20 minutes at 4° C. 
The total protein determination was carried out 

according to Bradford (1976) for the enzyme 

specific activity calculation purpose. 

Peroxidase (POD): The peroxidase activity was 

measured according with the described by Teisseire 

& Guy (2000), with the addition of crude enzyme 

extract, 20 mmol L

-1

 of pyrogallol (1,2,3-

benzenetriol), 50 mmol L

-1

 of potassium phosphate 

buffer, pH 6.5, and hydrogen peroxide (H

2

O

2

) 5 

mmol L

-1

. The purpurogallin formation was 

measured by a UV-visible spectrophotometer at 430 

nm and its molar extinction coefficient (2.5 mmol L

-

1

 cm

-1

) was used to calculate the specific enzyme 

activity.  

Catalase (CAT): The catalase activity was 

calculated according to Peixoto, Cambraia, Sant’ana, 

Mosquim, and Moreira (1999), with the addition of 

50  μL of enzyme extract, 0.05 mol L

-1

 of sodium 

phosphate buffer pH 7.0, and H

2

O

2

 12.5 mmol L

-1

The absorbance reading was performed at 240 nm. 
The enzyme activity was calculated using the molar 

extinction coefficient

 of H

2

O

2

 (39.4 mmol L

-1

 cm

-1

). 

The enzymatic activities data presented were the 

average values of assays in duplicate, in which the 

enzymes behavior were observed. The enzymatic 

activities were expressed in specific activity (POD - 
μmol of purpurogallin min

-1

 mg

-1

 of protein; CAT- 

nmol H

2

O

2

 min

-1 

mg

-1

 protein).  

For the analysis of antioxidative enzymes, there 

was no adjustment for parametric statistics, in this 

way, the data were analyzed by descriptive statistics.  

Results and discussion 

Effect of Leucaena leucocephala extract on seeds 

germination and early seedling development in C. sativus 

Table 1 shows the results for the C. sativus seeds 

germination. There was no significant difference in 

the seeds germination percentage, however it was 

found that, with respect to GSI and AGT variables, 

all proportions of L. leucocephala extract differed from 

the control, with delay and greater time for this 

process occurence as the extracts proportions 

increased, corroborating with Ferreira & Aquila 

(2000), which state that the allelopathic effect is 

generally not observed on the germination final 

percentage, but on the germination speed or another 

parameter such as the average time of germination. 

Bioassays with leaf extracts of exotic species such 

as  Emilia sonchifolia (L.) DC. (Oliveira Belinelo, 

Almeida, Aguilar, & Vieira Filho, 2011) Lolium 

multiflorum Lame and Brachiaria brizantha cv. 

Marandu (Castagnara et al., 2012) also found that 

extracts delayed the C. sativus seeds germination. 

Table 1. Percentage, germination speed index (GSI) and average 
time of germination (AGT) of Cucumis sativus L. seeds submitted 
to aqueous extract of Leucaena leucocephala (Lam.) De Wit. 
Cascavel - PR, 2015. 

Treatment  

% (w/v) 

Germination 

percentage (%) 

Germination  

Speed Index 

Average 

Germination Time 

(days) 

88 

13.87 a 

1.58 b 

2.5 

86 

10.49 b 

1.93 ab 

79 

7.68 bc 

2.26 a 

7.5 

85 

7.86 bc 

2.40 a 

10 

78 

6.95 c 

2.45 a 

CV (%) 

9.76 

12.81 

11.06 

Numbers followed by the same letter do not differ significantly as determined by 
Tukey test (p<0.05) 

The germination rate of the C. sativus seeds 

results are illustrated in Figure 1. The graphs show 

different behavior between control and other L. 

leucocephala extract proportions. It can be seen that in 

the control (0% w/v) the germination peak occurred 

on day 1 after the experiment installation, showing a 

better performance in terms of germination speed 

and increased process synchronization (U). 

As for the C. sativus seeds under L. leucocephala 

extract different proportions showed a peak 

germination 48 hours after the experiment 

installation (Day 2), with a lower germination 

synchronization followed by higher AGT and lower 

GSI (Table 1, Figure 1).  

It is worth mentioning that the frequency and 

synchronization are inversely related parameters, the 

higher the synchronization value (U), the lower the 

frequency, resulting in a more uniform germination 

at a certain point of time (Bufalo et al., 2012). 

Therefore, despite the C. sativus seeds 

germination final percentage not being influenced 

when subjected to L. leucocephala extract different 

proportions, it is possible to observe in a more 

detailed and joint analysis of other variables, as seen 

in Table 1 (GSI , AGT, frequency and 

synchronization) (Figure 1), that in general, the 

main effects found were delays in C. sativus seeds 

germination in contact with the extracts, 

demonstrated by the frequency curves, an increase 

in the germination average time and a reduction of 

the germination speed index. 

Therefore, the plant extract under study can 

interfere with other species germination process by 

the allelochemicals presence.  

Regarding the seedlings length, the results 

indicated that there was L. leucocephala extract effect 

on the C. sativus growth, as the applied extract 

proportion increased (Table 2). Regarding the 

primary root length, all the extract proportions 

differed from the control, showing a negative effect 

for this variable. However, the seedlings shoots had 

growth increase at 2.5; 5 and 7.5% proportions 

(w/v), however at 10% (w/v) there was no significant 

difference compared to the control. 

 

 

 

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376 

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Maringá, v. 39, n. 3, p. 373-380, July-Sept., 2017 

 

 

Figure 1. Relative frequency of germination of Cucumis sativus L. seeds in seed development. a. extract 0% (w/v); b. extract 2.5% (w/v); c. 

Extract 5% (w/v); d. Extract 7.5% (w/v); e. Extract 10% (w/v). 

Table 2. Primary root length and shoot of Cucumis sativus L. 

seedlings subjected to aqueous extracts of Leucaena leucocephala 
(Lam.) De Wit. Cascavel - PR, 2015. 

Treatment % (w/v)  Root length (cm) 

Seedlings shoots length (cm) 

10.11 a 

5.76 b 

2.5 

8.56 b 

7.18 a 

6.79 c 

7.33 a 

7.5 

5.73 c 

7.43 a 

10 

1.84 d 

5.54 b 

CV(%) 7.26  8.54 

Numbers followed by the same letter do not differ significantly as determined by 
Tukey test (p<0.05) 

It is observed that the plant extracts usually affect 

more specifically the root than the shoot growth, as 

observed in this experiment, mainly due to the roots 

direct and prolonged contact with the extract (Tuan 

Noorfatiehah, Nashriyah, Hasbullah, Raja Danial, & 

Muhamad Azhar, 2011; Aliyu & Mustapha, 2014).  

This  root  length  decrease  may  be  due  to  the 

interaction between allelochemicals and plant 
hormones, for studies show that allelopathic 
compounds tend to inhibit the gibberellins action as 
well as the acetic indole acid function (AIA), thereby 
preventing the cycle stages and cell elongation (Rice, 
1984; De Klerk, Guan, Huisman, & Marinova, 
2011). 

According to the L. leucocephala phytochemical 

profile, there are in its composition compounds as 
mimosine (b-[N-(3-hydroxy-4-oxopyridyl)] - a-
aminopropionic acid), and gallic acids, protocatechuic, 
p-hydroxybenzoic, p-hydroxyphenylacetic, vanillic, 
ferulic, caffeic and p-coumaric acids (Chou & Kuo, 
1986; Aderogba, McGaw, Bezabih, & Abegaz, 2010; 
Hassan,    Tawfik,    &    Abou-Setta,   2014).   These  

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Antioxidative enzymes modulated by Leucaena leucocephala 377 

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phenolic compounds can both reduce as increase the 

indole acetic acid concentration (IAA) in plant 

tissues, which is a plant hormone of the auxin 

group, having the cell elongation as its primary 

function (Rahman, 2013). The L. leucocephala 

allelopathic effects have been attributed to the 

phenolic compounds presence, such as those 

mentioned above. The coumaric acid and the 

hydroxybenzoic acid potentiate the IAA oxidase 

system, responsible for its inactivation, causing the 

roots inhibition (Souza Filho & Alves, 2002).  

According to Cothren and Oosterhuis (2010), 

the root growth is inhibited by auxin in proportions 

promoting the stems and coleoptile elongation, 
which could explain the shoot growth stimulation in 
contrast with the root inhibition, as demonstrated in 
Table 2.  

The extracts effect on the enzyme activity 

To determine the oxidative stress presence in the 

C. sativus seeds and seedlings submitted to L. 
leucocephala
 extract, it was analyzed the antioxidant 
enzymes activity involved in ROS detoxification and 
balance, during the (2, 12, 24 and 168 hours) 
immersion periods, as seen in Figure 2. 

 

 

Figure 2. Peroxidase activity (POD) and catalase (CAT) in seeds (2, 12 and 24 hours) and seedlings (168 hours) of Cucumis sativus L. 
submitted to treatments with Leucaena leucocephala (Lam.) De Wit extract at different soaking periods 

 

 

 

 

 

 

 

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378 

Ribeiro et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 373-380, July-Sept., 2017 

 

It was found that the CAT activity increased after 

24 hours in all the extract proportions, as well as in 
the control, but decreased after 168 hours soaking, 
that is, this enzyme activity was low in C. sativus 
seedlings and high in the seeds after 24 hours 
immersion in all the tested proportions. This 
catalase high activity, also present in the control (0% 
extract), leads us to suppose that the oxidative 
damage arose in response to the stress caused in the 
natural process of the seeds soaking, that way the 
catalase activity increase occurred in order to start 
the process of damage repair, this repair process 
being efficient, for the seeds showed high rates of 
germination percentage, the germination average 
speed and their germination occurring in a short 
time (Table 1). 

The CAT is the enzyme that has a high potential 

in the H

2

O

2

 dismutation process into H

2

O and O

2

being essential for the ROS detoxification during 
the toxic radicals production conditions (Garg & 
Manchanda, 2009). According to Figure 3, the 
catalase activity was similar in all the extract 
proportions in the germination first two hours.  

After 12 hours of soaking, small changes in the 

seeds activity were observed, on the extract different 
proportions. However, at 24 hours a high CAT 
activity was seen in 0, 2.5 and 5% proportions (w/v) 
compared to 7.5 and 10% proportions (w/v), for 
which the enzyme activity decreased. This CAT 
activity reduction did not reflect in the germination 
percentage decrease of the seeds submitted to 7.5 

and 10% extract (w/v), however may have led to 
increased time and low germination rate index 
(Table 1). 

According to Umair, Ali, Tareen, Ali, and 

Tareen (2012) the damage repair caused by the lipid 
peroxidation occurs during the Phase I of the water 
procurement by the seeds, mainly through the 
antioxidant enzymes production. It is at this 
germination stage that higher activities of enzymes, 
promoting the reactive oxygen species elimination, 
are observed, such as CAT and POD. According to 
Gurgel Jr., Torres, Oliveira, and Nunes (2009) the 
C. sativus  seeds  have  moisture  gain  mainly  after  12 
and 26 hours of immersion, which may explain the 
fact that cucumber seeds soaked in water also 
showed increase of CAT 24 hours after immersion 
(Figure 2). 

After 168 hours of immersion, an increased 

activity of this enzyme at 0% (w/v) extract ratio was 
observed, with a decline at 2.5 and 5% proportions 
(w/v), increasing again at 7.5 to 10% ratios (w/v), 
reaching then values similar to the control.  

The POD activity was similar in all ratios during 

the first 24 hours of germination (comprising values 
between 0.14 and 0.30 umol min mg protein), 
however, at 168 hours after immersion at 5% extract 
proportion (w/v), an increase in its activity occured, 
as shown in Figures 2 and 3. Siddique & Ismail 
(2013) also observed a POD activity increase in rice 
seedlings, when submitted to Fimbristylis miliacea 
extract. 

 

 

 

Figure 3. Activity of peroxidase (POD) and catalase (CAT) in seeds and seedlings of Cucumis sativus L. submitted to different proportions 
of Leucaena leucocephala (Lam.) de Wit extract at different times of soak. 

 

 

 

 

 

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Antioxidative enzymes modulated by Leucaena leucocephala 379 

Acta Scientiarum. Biological Sciences 

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This enzyme activity was more pronounced in 

seedlings (exposed to 168 hours immersion) (Figure 

3), its activity peak being at 5% proportion (w/v) of 

the extract, in contrast with the catalase low activity, 

significantly reducing its activity at 7.5 to 10% 

proportions (w/v) of the extract. Apel & Hirt (2004) 

suggest that when changes in the antioxidant 

enzymes balance happen, there is a compensatory 

mechanism occurrence in the tissues. For example, 

when the catalase activity is reduced, other enzymes 

are generated in large quantities in a compensatory 

effect, in order to guarantee the oxidative damage 

repair. However, this was not observed in the C. 

sativus roots development. It is possible that the low 

POD activity in the extract higher proportions was 

not sufficient to repair the oxidative damage, causing 

the seedlings stress, due to possible membrane 

damage, as observed in the roots length average low 

values (Table 2). 

Therefore, plants subjected to stressful 

conditions increase the antioxidant enzymes 

activities in order to prevent damage. Consequently, 

the energy of the seedling allelochemicals mediated 

stress is directed to the antioxidants biosynthesis, 

enabling this way to face adverse environmental 

conditions. This energy shift may be responsible for 

reducing the growth of the plants that are 

conditioned to this stress (Sunaina & Singh, 2014). 

Conclusion 

The  Leucaena leucocephala extract has allelopathic 

potential.  

There was a delay in the seeds cucumber 

germination and a decrease in the radicle length of 

seedlings due to the oxidative stress facing the 

allelochemicals present in the extract. 

An oxidative stress was induced by the 

allelochemicals present in the extract. 

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Received on January 10, 2017. 

Accepted on May 17, 2017. 

 

 

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