This study evaluated the toxicity and antimicrobial activity of the aqueous extract of leaves of Hyptis crenata Pohl ex Benth. The toxicity test on Artemia salina found an LC50 value of 1028.30 μg/mL, considered non-toxic. The antimicrobial assay showed inhibition halos only against gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, indicating antibacterial activity. These results suggest H. crenata as a potential source of bioactive metabolites, and further research is needed to identify its active principles through monitored studies.

1. REVISTA DE BIOLOGIA E CIÊNCIAS DA TERRA ISSN 1519-5228
59
Volume 15 - Número 1 - 1º Semestre 2015
TOXICITY TEST AND ANTIMICROBIAL ACTIVITY OF AQUEOUS EXTRACT OF
LEAVES OF Hyptis crenata Pohl ex. Benth
Líbio José Tapajós Mota1; Erick Dias da Silva2; Sheylla Susan Moreira da Silva de Almeida3
RESUMO
A espécie Hyptis crenata, conhecida como “salva-do-marajó”, é usada por sua atividade antiinflamatória e
repelente de insetos. O objetivo deste estudo foi realizar o teste de toxicidade em relação ao microcrustáceo
Artemia saina e avaliar o perfil da atividade antimicrobiana do extrato aquoso da espécie Hyptis crenata Pohl ex
Benth. A espécie foi coletada em Itaubal - AP, identificada e armazenada no Herbário da Universidade Federal
do Amapá, com número de identificação 703. No ensaio toxicológico, foram utilizadas larvas de A. salina Leach,
utilizando a metodologia de Meyer et al., (1982) e Nascimento et al., (2008) com modificações, determinando o
valor de LC50 com o software BioEstat®. O ensaio microbiológico utilizou duas espécies bacterianas gram-positivas:
Enterococcus faecalis e Staphylococcus auereus, e duas gram-negativas: Escherichia coli e P.
aeruginosa em concentrações de 1, 10, 25, 50 e 100 mg/mL de H. crenata, utilizando-se a metodologia do CLSI
(2009). O extrato bruto aquoso teve um valor de LC50 = 1028,30 mg/mL, considerado atóxico nas concentrações
testadas, ao passo que o extrato da planta em relação a A. salina foi considerado não tóxico quando LC50 > 1000
mg/mL. O ensaio microbiológico do extrato aquoso das folhas de H. crenata apresentou halos de inibição apenas
em espécies de bactérias gram-negativas, Escherichia coli e P. aeruginosa, indicando atividade antibacteriana.
Estes resultados indicam a espécie como uma potencial fonte de metabólitos com atividade biológica e mais
pesquisas são necessárias para aprofundar a identificação de seus princípios ativos por meio de estudos
biomonitorados.
Palavras-chave: Toxicidade, A. salina, salva-do-marajó, produtos naturais.
TESTE DE TOXICIDADE E ATIVIDADE ANTIMICROBIANA DE EXTRATO AQUOSO DE
FOLHAS DE Hyptis crenata Pohl ex. Benth
ABSTRACT
The species Hyptis crenata, known as "salva-do-marajó", is used as anti-inflammatory and insect repellent. The
objective of this study was to conduct the toxicity test in relation to the microcrustacean Artemia salina Leach
and evaluate the antimicrobial activity profile of the aqueous extract of the species Hyptis crenata Pohl ex Benth.
The species was collected in Itaubal – Ap, exsiccated and stored at the Herbarium of the Federal University of
Amapá, identification number 703. In the toxicological assay, it was used larvae of A. salina Leach, using the
methodology of Meyer et al. (1982) and Nascimento et al. (2008) with modifications, by determining the value
of LC50 using the software BioEstat®. The microbiological assay had two gram-positive bacterial strains:
Enterococcus faecalis and Staphylococcus aureus and two gram-negative: Escherichia coli and Pseudomonas
aeruginosa, at concentrations of 1, 10, 25, 50 and 100 mg/mL of H. crenata extract, using CLSI methodology
(2009). The aqueous crude extract had a value of LC50=1028.30 mg/mL, considered nontoxic at tested
concentrations, because for plant extract in relation to A. salina it is considered nontoxic when LC50>1000mg/mL.
The microbiological assay of the aqueous extract of the leaves of H. crenata presented inhibition halos only in
gram-negative strains, E. coli and P. aeruginosa, indicating antibacterial activity. These results turn the species
in a potential source of metabolites with biological activity, and it is required further research to deepen the
identification of its active principles through monitorated studies.
Keywords: Toxicity, A. salina, salva-do-marajó, natural products.

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INTRODUCTION
Several species of Amazonian plants have
aroused the interest of scientific studies in many
areas, among them, those with medicinal interest
and therapeutic properties.
According to the World Health
Organization (WHO), about 80% of the population
of developing countries still uses traditional
practices in primary health care; of this total, 85%
uses medicinal plants. Brazil follows this global
trend, by encouraging the use of complementary
practices in health care programs (Carvalho et al.,
2007; Barros, 2008).
The species Hyptis crenata (Pohl) ex Benth
is from the family Lamiaceae and Lamiales order
according to taxonomic classification proposed by
Dahlgren. This family consists of herbs, shrubs or
trees (Falcão, 2003) and comprises over 252
genera and 7.000 species (Hussain, 2009).
It is an aromatic and medicinal plant
(Bravim, 2008) and is known as "salva-do-marajó",
"salsa-do-campo" or "hortelã-do-campo"
and it is used by coastal communities as spices for
flavoring food and in medicine as anti-inflammatory
(Rebelo et al., 2009).
This research aimed to perform
toxicological assay in relation to the
microcrustacean A. salina, to determine the
toxicity of bioactive compounds of the aqueous
extract of leaves of Hyptis crenata Pohl ex Benth
through lethal concentration (LC50), using a
marine organism. This study also included the
investigation of the antimicrobial profile of the
aqueous extract of the leaves of H. crenata, using
two strains of bacterium gram-negative and two
strains of gram-positive.
MATERIAL AND METHODS
Vegetal Material
The species Hyptis crenata Pohl ex Bent
was collected in Itaubal – AP. The identification of
vegetal material was made by Dr. Wegliane
Campelo da Silva Aparício, at the Herbarium of
Federal University of Amapá (HUFAP).
Preparation, drying of the vegetal material and
obtaining the aqueous extract
The leaves of H. crenata were dried in an
oven at 45°C to eliminate water and inhibition of
proliferation of micro-organisms. The vegetal
material was milled in a mechanical mill and it was
subjected to hot aqueous extraction under reflux at
45°C, for 45 minutes, in 700 mL of distilled water.
The aqueous extract of leaves of H. crenata
(EBAFHC) was obtained by simple filtration and
dried at room temperature.
Toxicity testing in vitro in relation to Artemia
salina Leach.
The EBAFHC was tested for toxicity in
relation to A. salina Leach according to the
methodology described by Meyer et al. (1982) and
Nascimento et al. (2008) with some modifications.
The solution was prepared at a
concentration of 2 mg/mL. For EBAFHC, it was
dissolved 100 mg in 50 mL of Tween 80 and
solution of synthetic sea salt.
The test consisted of placing eggs of A.
salina in a container with water and synthetic sea
salt at a concentration of 30 g/L with constant
illumination and good oxygen saturation.
After 24 hours the larvae were replaced in
suitable container for 24 hours without lighting.
After this period, the larvae in nauplii phase were
collected with the aid of a Pasteur pipette and
counted macroscopically.
Ten larvae were added to the test tubes
containing the samples diluted at concentrations of
1, 10, 100, 250, 500, 1000 and 1250 μg/mL. After
24 hours of incubation, they were examined
against a light background with the aid of a
magnifying glass being counted and recorded the
number of survivors in each test tube. The test was
performed in triplicate and the results are
expressed in μg/mL, as the concentration required
killing 50% (LC50) of larvae, obtained by Probit
analysis.
Antimicrobial Activity
This assay relied on the use of EBAFHC, it
was used four bacterial strains, two gram-positive
(Enterococcus faecalis – ATCC 29212 and
Staphylococcus aureus – ATCC 25923) and two
gram-negative (Escherichia coli – ATCC 25922
and Pseudomonas aeruginosa – ATCC 10145), it
was held by obeying the rules and procedures of
the Clinical and Laboratory Standards Institute -
CLSI (2009). The test for the evaluation of
antibacterial activity was performed in duplicate.
The stock solution EBAFHC was prepared
at a concentration of 250 mg/mL, using DMSO as
solvent. Dilution was carried out for the
concentrations 1 mg/mL, 10 mg/mL, 25 mg/mL,

3. 61
50 mg/mL and 100 mg/mL, using as a positive
control the antibiotic tetracycline (dose?) for
bacterium gram-positive and gentamicin (dose?)
for bacterium gram-negative. Distilled water was
used as negative control.
In four Falcon test tubes, bacterial strains
were reactivated in 5 mL of brain and heart broth
infusion (BHI) at 37.5°C in an oven for 24 hours.
From this material the suspensions were prepared
in 0.9% saline solution until the final concentration
was, approximately, 1.5 x 108 cells/mL, using a
McFarland scale 0.5.
Petri plates were prepared with Müeller
Hinton agar (MH), in which they were seeded,
with the aid of the Drigalski´s handle, 600 μl of
each bacterial suspension prepared previously. On
the agar surface of each plate there were arranged
6 mm diameter sterilized discs, soaked in 20 μL of
the solutions in the respective concentrations
already mentioned, in addition to positive and
negative controls.
The plates were incubated at 37.5°C for 24
hours. The diameter formed by the halo of
inhibition promoted by the extract and controls is
used as a parameter of inhibition power of each
substance against the tested microorganisms. The
reading of the results consisted in measuring the
diameter of the inhibition halos, and the results are
expressed in terms of the diameter, in millimeters
(mm) of zone of inhibition of bacterial growth. The
formation of halos equal or above 8 mm indicates
bacterial activity.
RESULTS AND DISCUSSION
This research allowed us to assess the
toxicological profile of EBAFHC, performing
toxicity test in Artemia salina by finding the values
of LC50 of 1028.30 μg/mL and considered
nontoxic, because it is above 1000 μg/mL,
according to Meyer et al. (1982). Graphic 1
expresses the relation between mortality and
extract concentrations for dilution.
Y = 13.7010 + 0.0353X
R2 = 0.9638
Coef. correlation = 0.9848
It is observed that by the adjusted
coefficient of determination (R2) of 96.38%, the
rate of survival or mortality is explained by the
concentration, and other factors should act as
predictors to increase it.
This result could also be compared with
earlier studies conducted by Violante (2008),
whose values of LC50 for crude extracts and
organic phases of H. crenata were above 1000
μg/mL.
The evaluation of antimicrobial profile of
EBAFHC, there was formation of inhibition halos
only in strains gram-negative of P. aeruginosa and
E. coli, in which the antibiotic gentamycin was
used as a positive control. The inhibition halo
formed of the positive control in E. coli was
measured giving a mean value of 14.5 mm
diameter, considered sensitive. For P. aeruginosa
the mean value was 16 mm in diameter, and it is
compatible with the inhibition of halos of aqueous
extract at tested concentrations, and also sensitive
degree, as shown in Table 1 for bacterial
sensitivity standards.
TABLE 1 – Interpretive standards of zone diameter (halo)
inhibition (CLSI, 2009)
ANTIBIOTIC BACTERIAL
SPECIES
HALO DIAMETER (mm)
Resistant Intermediate Sensitive
Gentamicin E. coli (gram-negative)
≤ 12 13 – 14 ≥ 15
Gentamicin P. aeruginosa
(gram-negative)
≤ 12 13 – 14 ≥ 15
Tetracycline S. aureus (gram-positive)
≤ 14 15 – 18 ≥ 19
Tetracycline E. fecalis ( gram-positive)
≤ 14 15 – 18 ≥ 19
The inhibition halos of the aqueous extract
were lower than the standard antibiotic used in the
test for E. coli. for P. aeruginosa the
measurements of the inhibition halos of the
aqueous extract were measured around the positive
GRAPHIC 1 - Mortality rate of larvae of Artemia salina
Leach exposed to concentrations of EBAFHC: CL50 =
1028.30 μg/mL.
Mortality (%)
EBAFHC Concentrations (μg/mL)

4. 62
control, 16 mm diameter, only at the concentration
of 100 mg/mL, in which the value of measured
inhibition halo coincides with the control positive,
as shown in Table 2.
TABLE 2 – Measurement of halos in the tested bacterial
strains in the extract EBAFHC. The results are given as mean
(mm) and standard deviations.
EBAFHC
CONCENTRATION (mg/mL) E. coli P aeruginosa
100 10.05 ±
0.05
16.00 ± 2.82
50 12.50 ±
0.50
14.00 ± 0.70
25 10.05 ±
0.05
14.25 ± 2.25
10 11.00 ±
1.15
-
1 8.50 ± 0.60 14.25 ± 2.25
Positive Control 14.50 ±
0.70
16.00 ± 0.00
Negative Control - -
CONCLUSION
The toxicity test performed in this study
confirms the nontoxic profile in crude aqueous
crude extract of the leaves of Hyptis crenata Pohl
ex Benth at tested concentrations and in conditions
of the experiment, allowing its use as vegetal
compound potentially pharmacological.
The antibacterial activity test of EBAFHC
was positive only for two strains gram-negative
and did not present antimicrobial profile in both
tested strains gram-positive. According to the
results of this study, it can be concluded that the
species is promising in the search for chemical
constituents with biological potential, but not
antibacterial agente, and further research should be
carried out as regards obtaining the main active
principles.
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Desenvolvimento da Região Centro Oeste) -
Universidade Federal de Mato Grosso do Sul,
Campo Grande.
________________________________________
1-Programa de Pós-Graduação em Ciências da
Saúde, Universidade Federal do Amapá
(UNIFAP); Docente do Colegiado de Química da
Universidade do Estado do Amapá (UEAP) –
Macapá (AP).
2-Acadêmico do Curso de Ciências Farmacêuticas
da Universidade Federal do Amapá (UNIFAP) –
Macapá (AP).
3-Docente do Programa do Pós-Graduação em
Ciências da Saúde da Universidade Federal do
Amapá – Macapá (AP).