59

REVISTA DE BIOLOGIA E CIÊNCIAS DA TERRA ISSN 1519-5228

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 E XTRATO 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).