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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.
60 
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,
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)
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. 
BIBLIOGRAPHIC REFERENCES 
BARROS, I.M.C. Contribuição ao estudo 
químico e biológico de Hancornia speciosa 
GOMES (Apocynaceae). 2008. 194 f. 
Dissertação de Mestrado – Universidade de 
Brasília, Brasília. 
BRAVIM, L.S. Avaliação da atividade 
antinociceptiva e antiinflamatória do óleo 
essencial de Hyptis crenata (Pohl) ex Benth. 
2008. 74 f. Dissertação de Mestrado – Instituto de 
Ciências da Saúde, Universidade Federal do Pará. 
Belém. 
CARVALHO, A.C.B. et al. Aspectos da legislação 
no controle de medicamentos fitoterápicos. T & C 
Amazônia. Ano V, n. 11, 2007. 
CLINICAL AND LABORATORY 
STANDARDS INSTITUTE. Performance 
standards for antimicrobial disk susceptibility 
tests: approved standard, 20th edition: 
Pennsylvania. document M02-A10, 2009. ISBN 1- 
56238-688-3. 
FALCÃO, D.Q. Estudo químico e farmacológico 
de quatro espécies de Hyptis do Estado do Rio 
Grande do Sul. 2003. 178 f. Dissertação de 
Mestrado – Centro de Ciências da Saúde, 
Universidade Federal do Rio de Janeiro, Rio de 
Janeiro. 
HUSSAIN, A.I. Characterization and biological 
activities of essential oils of some species of 
Lamiaceae. 2009. 257 f. Thesis – Department of 
Chemistry and Biochemistry, Faculty of Sciences, 
University of Agriculture. Faisalabad. Pakistan. 
MEYER, B.N. et al. Brine shrimp: a convenient 
general bioassay for active plant constituents. 
Journal of Medicinal Plants Research. v. 45, p. 
31-34, 1982. 
NASCIMENTO, J.E. et al. Estudo fitoquímico e 
bioensaio toxicológico frente a larvas de Artemia 
salina Leach. de três espécies medicinais do 
gênero Phyllanthus (Phyllanthaceae). Revista de 
Ciências Farmacêuticas Básica e Aplicada. v. 
29, n.2, p. 145-150, 2008. 
VIOLANTE, I.M.P. Avaliação do potencial 
antimicrobiano e citotóxico de espécies vegetais 
do Cerrado da Região Centro-Oeste. 2008. 89f. 
Dissertação (Mestrado em Saúde e
63 
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).

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Artigo bioterra v15_n1_07

  • 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.
  • 2. 60 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. BIBLIOGRAPHIC REFERENCES BARROS, I.M.C. Contribuição ao estudo químico e biológico de Hancornia speciosa GOMES (Apocynaceae). 2008. 194 f. Dissertação de Mestrado – Universidade de Brasília, Brasília. BRAVIM, L.S. Avaliação da atividade antinociceptiva e antiinflamatória do óleo essencial de Hyptis crenata (Pohl) ex Benth. 2008. 74 f. Dissertação de Mestrado – Instituto de Ciências da Saúde, Universidade Federal do Pará. Belém. CARVALHO, A.C.B. et al. Aspectos da legislação no controle de medicamentos fitoterápicos. T & C Amazônia. Ano V, n. 11, 2007. CLINICAL AND LABORATORY STANDARDS INSTITUTE. Performance standards for antimicrobial disk susceptibility tests: approved standard, 20th edition: Pennsylvania. document M02-A10, 2009. ISBN 1- 56238-688-3. FALCÃO, D.Q. Estudo químico e farmacológico de quatro espécies de Hyptis do Estado do Rio Grande do Sul. 2003. 178 f. Dissertação de Mestrado – Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro. HUSSAIN, A.I. Characterization and biological activities of essential oils of some species of Lamiaceae. 2009. 257 f. Thesis – Department of Chemistry and Biochemistry, Faculty of Sciences, University of Agriculture. Faisalabad. Pakistan. MEYER, B.N. et al. Brine shrimp: a convenient general bioassay for active plant constituents. Journal of Medicinal Plants Research. v. 45, p. 31-34, 1982. NASCIMENTO, J.E. et al. Estudo fitoquímico e bioensaio toxicológico frente a larvas de Artemia salina Leach. de três espécies medicinais do gênero Phyllanthus (Phyllanthaceae). Revista de Ciências Farmacêuticas Básica e Aplicada. v. 29, n.2, p. 145-150, 2008. VIOLANTE, I.M.P. Avaliação do potencial antimicrobiano e citotóxico de espécies vegetais do Cerrado da Região Centro-Oeste. 2008. 89f. Dissertação (Mestrado em Saúde e
  • 5. 63 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).