Antibacterial activity and interaction in vitro of extract from Piper montealegreanum Yuncker on antibiotics of clinical use

Wilma Wilma; Harley Harley; Luanne Luanne; Baldoíno Baldoíno; Maria Célia Chaves; Raïssa Raïssa

Antibacterial activity and interaction in vitro of extract from Piper montealegreanum Yuncker on antibiotics of clinical use

ARTÍCULO ORIGINAL

Antibacterial activity and interaction in vitro of extract from Piper montealegreanum Yuncker on antibiotics of clinical use

Actividad antibacteriana e interacción in vitro del extracto de Piper montealegreanum Yuncker con antibióticos de uso clínico

Atividade antibacteriana e interação in vitro do extrato de Piper montealegreanum Yuncker com antibióticos de uso clínico

Wilma Raianny Vieira da Rocha¹
Harley da Silva Alves^1*
Raïssa Mayer Ramalho Catão¹
Luanne Eugênia Nunes²
Baldoíno Sonildo da Nóbrega³
Maria Célia de Oliveira Chaves⁴

¹Universidade Estadual da Paraíba. Campina Grande, Paraíba. Brasil.
²Universidade Federal de Pernambuco. Recife, Pernambuco. Brasil.
³Instituto Federal de Ciência e Tecnologia da Paraíba. Cajazeiras, Paraíba. Brasil.
⁴Universidade Federal da Paraíba. João Pessoa, Paraíba. Brasil.

ABSTRACT

Introduction: Antibiotic and combination therapies are very commonly used nowadays to treat infectious diseases, and over the years new treatment strategies have been searched for and developed. Few chemical studies have been conducted about the biological activity of Piper montealegreanum Yuncker, a shrub native to northern Brazil.
Objective: Evaluate the antibacterial activity and in vitro interaction of crude ethanolic extract (CEE) of P. montealegreanum leaves against Escherichia coli ATCC 25922 with clinical antibiotics.
Method: Leaves from the plant were collected in Belém (Pará, Brazil) and identified at the Botany Department of the Federal University of Rio de Janeiro in Brazil. The plant material was dried, crushed in a mechanical mill and thoroughly extracted with ethanol (4 x 2.0 l). The solvent was removed by reduced pressure and a green residue was obtained. Determination was made of the antibacterial activity of the P. montealegreanum crude ethanolic extract (CEE) against E. coli using the disk diffusion method. Minimum inhibitory concentration (MIC) and interaction with clinical antibiotics such as ampicillin, ciprofloxacin, chloramphenicol, sulfamethoxasol and tetracycline, were determined by adding 20 μl of the 400 mg/ml^-1 crude ethanolic extract (CEE) solution to the antibiotic disks. The results of the interactions were subjected to the Wilcoxon test (p < 0.01).
Results: The CEE was not found to display any activity against E. coli ATCC 25922. However, interaction with the antibiotics was revealed by an increase in the diameter of the growth inhibition halo consistent with synergy interactions.
Conclusions: It was concluded that the crude ethanolic extract of P. montealegreanum was capable of modifying the behavior of antibiotics, hence the importance of the study. Further research should be conducted to identify and quantify plant substances and reveal the interaction mechanisms of CEE of P. montealegreanum with antibiotics.

Key words: Piperaceae; Escherichia coli; interactive effects.

RESUMEN

Introducción: En la actualidad es muy común el tratamiento antibiótico combinado y asociado para curar enfermedades infecciosas. De manera que, desde hace años, se buscan y desarrollan nuevas estrategias de tratamiento, pero todavía son escasas las investigaciones químicas que estudian la actividad biológica de la especie Piper montealegreanum Yuncker, arbusto autóctono del norte de Brasil.
Objetivo: Evaluar la actividad antibacteriana y la interacción in vitro del extracto crudo etanólico (ECE) de las hojas de P. montealegreanum con antibióticos de uso clínico contra Escherichia coli ATCC 25922.
Método: Las hojas de la planta se recolectaron en Belén (Pará, Brasil) y se identificaron en el Departamento de Botánica de la Universidad Federal de Rio de Janeiro (Rio de Janeiro, Brasil). Se secó el material botánico, se trituró en un molino mecánico y se extrajo completamente con etanol (4 x 2,0 L). El disolvente se eliminó con presión reducida y se obtuvo un residuo verde. Se determinó la actividad antibacteriana del extracto etanólico crudo (EEC) de P. montealegreanum contra E. coli utilizando el método de difusión de disco, su concentración inhibitoria mínima (CIM) y su interacción con antibióticos de uso clínico como ampicilina, ciprofloxacino, cloranfenicol, sulfametoxazol y tetraciclina añadiendo 20 μL de la solución del extracto etanólico crudo (EEC) 400 mg/mL^-1 a los discos de antibiótico. Los resultados de las interacciones fueron sometidos a la prueba de Wilcoxon ( p< 0,01).
Resultados: Se verificó que el extracto etanólico crudo no presentó actividad contra E. coli ATCC 25922. Sin embargo, se observaron interacciones con los antibióticos, las cuales quedaron demostradas por el aumento del diámetro del halo de inhibición del crecimiento en correspondencia con las interacciones de sinergismo.
Conclusiones: Se llegó a la conclusión de que el extracto etanólico crudo de P. montealegreanum fue capaz de modificar el comportamiento de los antibióticos, lo que destaca la importancia de esta investigación. Deben realizarse otros estudios para identificar y cuantificar las sustancias vegetales y descubrir los mecanismos de interacción del extracto crudo etanólico de P. montealegreanum con los antibióticos.

Palabras claves: Piperaceae; Escherichia coli; efectos interactivos.

RESUMO

Introdução: Atualmente é muito comum o uso de antibioticoterapia combinada e associada no tratamento de doenças infecciosas. Dessa maneira, a busca por novas estratégias de tratamento vem surgindo ao longo dos anos. Piper montealegreanum Yuncker é um arbusto nativo do Norte do Brasil e estudos químicos envolvendo a avaliação da atividade biológica de esta espécie ainda são escassos.
Objetivo: Avaliar a atividade antibacteriana e a interação in vitro do extrato etanólico bruto (EEB) das folhas de P. montealegreanum contra Escherichia coli ATCC 25922 com antibióticos de uso clínico.
Metodologia: As folhas da planta foram coletadas em Belém (Pará, Brasil) e identificadas pelo Departamento de Botânica da Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil). O material botânico passou por secagem e foi triturado em moinho mecânico e extraído de forma exaustiva com etanol (4 x 2,0 L). O solvente foi eliminado sob pressão reduzida resultando em um resíduo verde. Determinou-se a atividade antibacteriana do extrato etanólico bruto (EEB) de P. montealegreanum contra E. coli utilizando o método de disco-difusão, bem como sua concentração inibitória mínima (CIM) e sua interação com antibióticos de uso clínico, como ampicilina, ciprofloxacina, cloranfenicol, sulfametoxazol e tetraciclina, adicionando 20 µL da solução de EEB 400 mg mL^-1 aos discos de antibiótico. Os resultados das interações foram submetidos ao teste de Wilcoxon (p < 0.01).
Resultados: Foi verificado que o EEB não apresentou atividade contra a E. coli ATCC 25922. Entretanto, foram observadas interações com antibióticos demonstradas pelo aumento do diâmetro do halo de inibição de crescimento, consistente com interações de sinergismo.
Conclusão: Concluiu-se que o EEB de P. montealegreanum foi capaz de modificar o comportamento dos antibióticos, destacando a importância deste estudo. Outros estudos devem ser realizados, a fim de identificar e quantificar as substâncias vegetais e descobrir os mecanismos de interação do EEB de P. montealegreanum com antibióticos.

Palavras-chave: Piperaceae; Escherichia coli; efeitos interativos.

INTRODUCTION

Treatment of infectious diseases is a problem that grows significantly. Taking into account the spread of bacterial resistance to drugs and the reduction of their effects,¹ there is uncertainty regarding bacteria resistant to multiple antibiotics. Thus, the need for new compounds with antimicrobial activity that may serve as alternative therapy against these microorganisms arises. According to Gonçalves et al.,² studying the application of new substances derived from the extraction of active ingredients from various plant species is important to find ways to inhibit or combat pathogens that are constantly resistant to the usual antibiotics.

Previous studies show the importance of determining the antimicrobial activity of various plant extracts, which can be seen as indications of new molecules with such activity or as an alternative treatment to infectious processes.^3-5

In developing countries, diseases are related to poor sanitation, malnutrition and poor access to medicines.⁶ In this context and due to ethnomedicinal use, herbal medicine is widely practiced. Among the most used medicinal plants by the population, few have their actions scientifically proven. Therefore, consolidated traditional use has been used as a guide for pharmacological research.⁷ Taking into account the biologically active properties of herbal products, approximately 80 % of the world's population use this practice as an alternative or in therapy combinations.⁸

The use of combined and associated antibiotic therapy is also a fact today. However, several authors reported studies on different effects caused by the combined use of natural products and conventional antimicrobials.^9-11

Piper montealegreanum Yuncker (Piperaceae) is a shrub native of northern Brazil,¹² and chemical studies involving the assessment of the biological activity of this species are still scarce. According to Pinto et al.,¹³ antibacterial activity of the ethyl acetate fraction obtained of the CEE was observed against strains ofBacillus subtilis, Escherichia coli and Pseudomonas aeruginosa.

The phytochemical investigations of Piperaceae species showed the presence of mevalonic acid (monoterpenes and sesquiterpenes) and acetic/shikimic acid (flavonoids) metabolites, in addition to shikimic acid pathways (lignoids, arylpropanoids and amides).¹⁴ The most frequently isolated metabolites are amides, aristolactams, lignoids and phenylpropanoids. Flavonoid isolation is often observed, represented by flavones, dihydroflavonols, chalcones and dihydrochalcones.^14,15

In face of Piper genus potential for antimicrobial and antifungal activity, besides other biological activities,^16-21 the aim of this study was to assess the antibacterial activity of the crude ethanolic extract (CEE) obtained of the leaves from P. montealegreanum Yuncker against the standard strain of E. coli ATCC 25922 and assess the effects of interactions between antibiotics of conventional use and CEE.

METHODS

Plant material

Leaves from P. montealegreanum Yuncker were collected in Belém (Pará State, Brazil; latitude 14° 10' 00" S, longitude 53° 05' 00" W). The botanical material was identified by Dra. Elsie F. Guimarães, at the Botanical Department of UFRJ, and a specimen voucher was deposited at Emilio Goeldi Museum, Belém, under serial number MSP-010. Subsequently, it was sent to IPEFARM - Federal University of Paraiba for phytochemical study.

The botanical material was dried at 40 °C for 72 h and powdered in a mechanical mill providing 1.3 kg of powder, exhaustively extracted with ethanol (4 x 2.0 L). The solvent removed under reduced pressure furnished a green residue (115.0 g).

Preparation for analysis

The crude ethanolic extract (CEE) was solubilized in 1.0 mL of absolute alcohol (99.8 %) and was subsequently subjected to an ultrasonic bath (Ultrassonic 1440 A) for 15 min.

Microorganism and bacterial inoculum preparation

In order to conduct this study, the Escherichia coli ATCC 25922 standard strain was used, which was suspended in BHI enrichment broth (DIFCO^®). After incubation at 37 °C/24 h, it was seeded by streaking technique on Mueller Hinton agar and incubated again at 37 °C/24 h, enabling the microorganism to be in the exponential growth phase. After the incubation period, bacterial suspension (standard inoculum) was prepared in 0,85 % saline solution, with turbidity corresponding to the 0,5 McFarland standards, equivalent to 1.5 x 10 ⁸UFC mL^-1.²²

Antibacterial activity and determination of the Minimum Inhibitory Concentration (MIC)

In order to determine the MIC, CEE serial dilutions were conducted in absolute alcohol, starting from the initial concentration of 400 mg/mL ^-1, corresponding to the extraction yield. Five solutions were prepared at the following concentrations (w/v): 200 mg/mL^-1; 100 mg/mL^-1; 50 mg/mL^-1; 25 mg/mL^-1 and 12.5 mg/mL^-1. Antibacterial activity and MIC determinations were carried out through disk diffusion²² with adaptations, using sterile disks (6 mm) soaked with 20 µL CEE at different concentrations. Tests were conducted in triplicate and values were expressed by the arithmetic mean of growth inhibition halos, measured by a halometer. Solutions were considered active when they had growth inhibition halos ≥8 mm.²³ Cecon^® gentamicin disks (5 µg/mL^-1) and sterile disks impregnated with absolute alcohol were respectively used as positive (activity presence) and negative (no activity) controls.

Antibiogram and association tests with the CEE from P. montealegreanum

E. coli ATCC 25922 strain sensitivity profile to antibiotics (antibiogram), as well as the interactive effect of the association between CEE and the tested antibiotics were carried out simultaneously against representatives of five5 antibiotic classes of Cecon^®. Ampicillin 10 µg (AMP), ciprofloxacin 5 µg (CIP), chloramphenicol 30 µg (CLO), sulfamethoxazole 25 µg (SUT) and tetracycline 30 µg (TET) antibiotics were tested, both isolated and after addition of 20 µL of the CEE from P. montealegreanum at a concentration of 400 mg/mL^-1. Disks were added to the previously seeded and incubated Mueller Hinton agar plates at 37 °C/24 h, and growth inhibition halos diameter was measured. The assays were performed in triplicate.

Statistical analysis

Statistical analysis was carried out through the Shapiro-Wilk test, using the Assistat 7.7 beta software. For the comparison of results obtained in the association tests between each antibiotic and CEE from P. montealegreanum was used Wilcoxon non-parametric t test. Differences were considered significant at p< 0.01.

RESULTS

The CEE of the leaves from P. montealegreanum, as well as the dilutions used in the in vitro antimicrobial activity tests failed to inhibit the growth E. coli ATCC 25922 strain (table 1).

It was observed that, although not showing antibacterial activity evidence by the method used in this study, CEE showed interactive capacity with some conventional antibiotics. It modified the growth inhibition halos diameter of antibiotics tested alone and associated with CEE, which characterizes the main finding of this study (Table 2). There was no growth inhibition halo size interference in the association between the CEE from P. montealegreanum and the absolute alcohol used in extract dilution.

From the data obtained, it was found that the strongest synergism evidence in the association was between CEE and the chloramphenicol disk, in which the interaction increased the inhibition halos media from 21.33 mm (chloramphenicol) to 28.33 mm (chloramphenicol + CEE), followed by increases of 20.33 mm (tetracycline) to 25.67 mm (tetracycline + CEE), 17.0 mm (ampicillin) to 20,33 mm (ampicillin + CEE), 30.33 mm (ciprofloxacin) to 33.33 (ciprofloxacin + CEE) and 24.67 mm (sulfamethoxazole) to 26.67 mm (sulfamethoxazole + CEE). Different increase percentages 32.82 %, 26.27 %, 19.59 %, 9.89 % and 8.11 % were observed, respectively.

Non-parametric Wilcoxon t test was used as statistical test to compare the performance of each subject (or pairs of subjects) and to check if there were significant differences between their results in the two distinct situations, through P value analysis. In this study, samples with p< 0.01 were considered significant. Thus, there were significant differences between the compared situations regarding isolated antibiotic use compared to the antibiotic-extract interaction.

The synergistic effect of the association between antibiotics and the CEE from P. montealegreanum, based on the media diameter of inhibition halos of antibiotics tested in isolation, was significantly lower when compared to the combinations (Figure A, B, C, D and E).

The synergism found between the CEE from P. montealegreanum and antibiotics tested was evidenced by inhibition halos diameter increase, confirmed by their medians increase against E. coli ATCC 25922 (Figure).

DISCUSSION

The fact that CEE did not show antibacterial activity by disk diffusion does not determine its inactivity. For security, in vitro antimicrobial activity tests should be conducted with at least two different methodologies,²³ due to many interfering factors that may influence results, including test product solubility and diffusivity. Furthermore, it should be considered that, according to Yokota and Fujii,²⁴ Gram-negative bacteria have structural particularities that hinder antibiotics penetration, such as the lipopolysaccharides outer layer that determines surface properties, such as permeability and susceptibility to antibiotics.

According to Von Baum and Marre,²⁵ E. coli strains have been identified as cause of urinary tract infections, neonatal meningitis, nosocomial septicemia and enteritis. In addition, E. coli strains are commonly resistant to at least two antibiotic classes, restricting the available therapeutic options. This fact highlights the use of combined antibiotic therapy, either with traditional antibiotics or in the search for new substances.²⁶

This study showed that all interactions when compared with the inhibition halo formed by the action of the antibiotic tested alone can be considered as synergistic effect, as inhibition halos diameter increase of ≥ 2 mm was observed.¹⁰

According to Alves,¹⁵ the CEE from P. montealegreanum has flavonoids in its composition, which had antibacterial activity against Bacillus subtilis,Escherichia coli and Pseudomonas aeruginosa strains.¹³Such compounds may be involved in the activity between the CEE and antibiotics used in this study, and flavonoids antibacterial activity has been already proved by several author.^27-29 Moreover, different species of the family Piperaceae demonstrated antimicrobial activity against clinically important species.³⁰ In addition, it has already been established that flavonoids bind to the microorganism cell wall, resulting in rupture and possible cell lysis.^31-35

The results show that the interference of the CEE from P. montealegreanum in the action of antibiotics used in the clinical practice serves as a warning to people that use this drug class along with plant products, such as teas or herbs. This type of association may cause problems in the treatment of patients. In this context, additional studies to determine the mechanism of action of CEE from P. montealegreanum and to verify in which point the interaction with antibiotics occurs, besides determining if this interaction is dose-dependent.

ACKNOWLEDGMENTS

The authors acknowledge the financial support of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for this work.

Interest conflicts

The authors declare that they do not have any potential conflict of interest in this article.

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Recibido: 14/9/2016.
Aprobado: 4/5/2018.

Harley da Silva Alves. Universidade Estadual da Paraíba. Campina Grande, Paraíba, Brasil. Correo electrónico harley.alves@hotmail.com