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Revista Brasileira de Farmacognosia

Print version ISSN 0102-695XOn-line version ISSN 1981-528X

Rev. bras. farmacogn. vol.18  suppl.0 João Pessoa Dec. 2008 



In vitro anti-staphylococcal activity of Hyptis martiusii Benth against methicillin-resistant Staphylococcus aureus-MRSA strains


Atividade anti-estafilocócica in vitro de Hyptis martiusii Benth contra linhagens de Staphylococcus aureus resistentes à meticilina - MRSA



Henrique D. M. CoutinhoI, *; José G. M. CostaI; José P. Siqueira-JúniorII; Edeltrudes O. LimaIII

ILaboratório de Pesquisa em Produtos Naturais, Departamento de Ciências Físicas e Biológicas, Universidade Regional do Cariri, 63100-000 Crato-CE, Brazil
IILaboratório de Genética de Microrganismos, Departamento de Biologia Molecular, Universidade Federal da Paraíba, 58051-900 João Pessoa-PB, Brazil
IIILaboratório de Micologia, Departamento de Ciências Farmacêuticas, Universidade Federal da Paraíba, 58051-900, João Pessoa-PB, Brazil




This is the first report about the antibacterial activity of Hyptis martiusii Benth. In this study the ethanol extract of H. martiusii was tested for its antimicrobial activity against strains of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The growth of all bacterial strains tested was inhibited by the extract. The diameter of inhibition zones varied from 13 to 20 mm for the extract. The MIC and MBC values ranged from 128 to > 1024mg/mL and 256 to > 1024 mg/mL, respectively. It is therefore suggested that extracts from H. martiusii could be used as an anti-Staphylococcus agent. Compared with methicillin and gentamicin, the extract was more effective, being a promising antibacterial agent.

Keywords: Hyptis martiusii, Labiatae, anti-staphylococcal activity, antimicrobial activity.


Este é o primeiro relato de atividade antibacteriana de Hyptis martiusii Benth. Neste estudo, o extrato etanólico de H. martiusii foi avaliado para atividade antimicrobiana contra linhagens de Escherichia coli, Pseudomonas aeruginosa e Staphylococcus aureus. O crescimento de todas as bactérias testadas foi inibido pelo extrato. O diâmetro das zonas de inibição variaram de 13 - 20 mm. Os valores da CIM e CBM variaram de 128 a > 1024 mg/mL e 256 a > 1024 mg/mL, respectivamente. Devido a isso, podemos indicar que o extrato etanólico de H. martiusii pode ser usado como um agente anti-Staphylococcus. Quando comparado com outros antibióticos como meticilina e gentamicina, o extrato foi mais efetivo, demonstrando ser um promissor agente antibacteriano.

Unitermos: Hyptis martiusii, Labiatae, atividade anti-estafilocócica, atividade antimicrobiana.




Staphylococcus genus is widely spread in nature being part of the indigenous microbiota of skin and mucosa of animal and birds. Some Staphylococcus species are frequently recognized as etiological agents of many animal and human opportunistic infections (Nostro et al., 2004). S. aureus, S. epidermidis, S. saprophyticus and S. haemolyticus are the most important species as community and nosocomial human infection causing agents. In addition of causing different kinds of intoxications, S. aureus has been the most common etiological agent of festering infections that attack different tissues and/or organs (e.g. furuncle, carbuncle, abscess, myocarditis, endocarditis, pneumonia, meningitis, bacterial arthritis) (Verhoeff et al., 1999; Pereira et al., 2004). Capsule, peptidoglican, teicoic acids, adesins and synthesis of enzymes and extracelullar toxins are some virulence attributes present in/on S. aureus cell (Nostro et al., 2004).

Among the bacterial genera able to develop changes in their sensitivity to antimicrobials, Staphylococcus species have been recognized as having increasing and worrying antimicrobial resistance (Georgopapadakou, 2002; Nostro et al., 2004). For patients, the antimicrobial resistance increases the morbidity and mortality, whilst for healthcare institutions it means increasing costs (Dancer, 2001; Coutinho et al., 2005). Regarding the increasing clinical importance given to nosocomial and community bacterial infections and the progressive development of antimicrobial resistance, a great number of scientific researches emphasizing the antibacterial properties of plant products has been carried out (Hernández et al., 2003; Silva-Santos et al., 2004; Duarte et al., 2005; Gayoso et al., 2005; Michelin et al., 2005; Lima et al., 2006a,b; Santos et al., 2007; Serafin et al., 2007; Silva et al., 2007; Aguiar et al., 2008; Silva et al., 2008; Salvagnini et al., 2008; Simões et al., 2008). Filtrates, infusions, macerated, juices, extracts and cataplasms from plants with medicinal properties have been applied in the treatment of various diseases since antiquity (Annuk et al., 1999; Hernández et al., 2003).

Hyptis martiusii Benth ("cidreira-do-campo") is a small shrub belonging to family Labiatae used in Brazilian traditional medicine against intestinal and stomachic diseases (Agra et al., 2008), with few pharmacological reports. Antitumoral, cytotoxic and insecticidal activities were identified (Araújo et al., 2003; Costa-Lotufo et al., 2004; Costa et al., 2005; Araújo et al., 2006), but no antimicrobial activity has so far been reported according to a literature survey.

Chemical compounds as flavonoids (Isobe et al., 2006), triterpenes (Falcão et al., 2003), diterpenes (Ohsaki et al., 2005) and sesquiterpenes (Facey et al., 2005), with antimicrobial, insecticidal, analgesic, antiplasmodial activities and antidepressive effect (Okiemy-Andissa et al., 2004; Fragoso-Serrano et al., 2005; Chukwujekwu et al., 2005; Isobe et al., 2006; Silva et al., 2006; Bueno et al., 2006) were isolated on other plants from the genus Hyptis.

This study was carried out with the purpose of evaluating the antimicrobial effect of the ethanolic extract of H. martiusii to inhibit the growth and survival of S. aureus strains isolated from clinical samples.




Methicillin (SIGMA), Gentamicin (SIGMA). The solutions of antibiotics were prepared using the recommendations of Clinical and Laboratory Standards Institute - CLSI (NCCLS, 2003).


Escherichia coli (ATCC 8539 and ATCC10536), Pseudomonas aeruginosa (ATCC 25619 and 9027), Staphylococcus aureus (ATCC 6538 and 25923) were used as positive control. The clinical and methicillin-resistant Staphylococcus aureus (MRSA) were obtained from the Laboratório de Genética de Microrganismos - UFPB. All strains were stocked at room temperature in heart infusion agar slants (HIA, Difco) and, prior to assay, the cells were grown overnight at 37ºC in brain heart infusion (BHI, Difco).

Plant material

Leaves of Hyptis martiusii were collected in the city of Crato, State of Ceará, Brazil. The plant material was identified by Dra. Maria Arlene Pessoa da Silva and voucher specimen have been deposited with the number 464 at Herbarium "Dárdano de Andrade Lima" of Universidade Regional do Cariri - URCA.

Preparation of ethanol extract from Hyptis martiusii (EEHM)

200 g of aerial parts were oven-dried at room temperature and powdered. The powdered material was extracted by maceration using 1l of 95% ethanol as solvent at room temperature. The mixture was reserved for 72 h at room temperature. The extracts were then filtered and concentrated under vacuum in rotatory evaporator (Brasileiro et al., 2006). For the tests, the extract was diluted in DMSO and its highest concentration remaining after dilution into broth caused no inhibition of bacterial growth.

Antimicrobial activity test

Solid medium diffusion technique using agar wells was used for screening the antibacterial activity. For this, 1 ml of the bacterium suspension (approximately 105 cfu/ml) was uniformly spread on sterile agar Muller-Hinton Petri dishes. 50 µl of EEHM 10mg/mL were addicted within agar wells with 6 mm diameter (modified from Nair et al., 2005; Sahin et al., 2004). The system was incubated at 37 °C for 24 hours. It was considered as positive antibacterial activity when observed growth inhibition zone with diameter >10 mm diameter (Lima et al., 1993). MICs were determined in a microtitre assay (Javadpour et al., 1996) by inoculation of 100 µL of each strain suspended in BHI two - fold concentrated (final concentration 105 colony-forming units/mL) in a 96-well microtitre tray with two-fold serial dilutions by adding 100 µL of EEHM solution. The final concentrations of the EEHM was 512, 256, 128, 64, 32, 16 and 8 µg/mL. The MICs were recorded as the lowest concentration for growth inhibition. The Minimal bactericidal concentration (MBC) was determined inoculating samples from non - growth wells on plates with BHI agar. The MRSA strains 007 and 441 were assayed with methicillin and gentamicin with final concentrations of 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2 and 1 µg/mL. All plates were incubated aerobically for 24 h at 37 ºC. The MBCs were recorded as the lowest concentration without growth. All antimicrobial assays were performed twice and the results were expressed as average of the two repetitions.


Oxacillin (OXA); Penicillin (PEN); inductive Erythromycin (EMI); Kanamycin (KAN); Streptomycin (SM); Gentamicin (GEN); Amikacin (AMI); Tobramycin (TOB); Chloramphenicol (CHL); Tetracyclin-minocyclin (TCM); Neomycin (NEO); Paramomycin (PARA); Butirosin (BUT); Sisomicin (SIS); Netilmicin (NET); Constitutive Erythromycin (ERIC); Tetracyclin (TC); Ampicillin (AMP); Amoxicillin (AMOX); Cefalotin (CF); Cefadroxil (CFR); Cefalexin (CFL); Clindamycin (CN); Ciprofloxacin (CIP); Gatifloxacin (GAT); Ampicillin-Sulbactan (AMPS); Rifampicin (RIF); Novobiocin (NOV).



In the last years there has been a great scientific interest in chemical and pharmacological investigations regarding the biological properties of medicinal plants (Almeida et al., 2001; Silva et al., 2003; Rocha et al., 2005; Barbosa-Filho et al., 2005; Barbosa-Filho et al., 2006a,b,c; Saúde-Guimarães & Faria, 2007; Barbosa-Filho et al., 2007; Biavatti et al., 2007; Oliveira et al., 2007; Barbosa-Filho et al., 2008). It is known that medicinal plants have been source of many drugs applied in clinical procedures (e.g morphine, emetine, rutine). The use of extracts as antimicrobial agents presents a low risk of rising microbial resistance to their action because are complex mixtures, making more difficult the microbial adaptability (Daferera et al., 2003).

Table 1 shows the inhibitory activity of EEHM against clinical isolates of S. aureus. EEHM showed effectiveness in inhibiting the all strains with inhibition zones between 13-20 mm (average: 16,5 ±1.8). Six strains showed inhibition zones with diameter >18 mm. Smallest inhibition zones (13 mm) were found on the MRSA strain 358, while the largest one (20 mm) were found on the MRSA strains 365, 10C and 19L. MIC and MBC values were 128 - 512 µg/mL and 256 > 1024 µg/mL for the S. aureus strains, respectively. The effect was not observed on the P aeruginosa and E. coli strains. As far as we know, it is the first report of the antimicrobial activity of H. martiusii.



Table 2 shows the anti-staphylococcal efficacy of EEHM when compared with the aminoglycoside gentamicin and the β-lactam methicillin. The EEHM was 2 - 4 times more effective to inhibit the S. aureus growth than these drugs. Regarding the MIC values found for all assayed S. aureus strains, the classification criteria above cited confirms the strong anti-staphylococcal property of EEHM.



Plants remaining to the genus Hyptis are used in the folk medicine by populations around the world as an antimicrobial remedy (Goun et al., 2003; Wiart et al., 2004; Kala, 2005). These antimicrobial properties have been assayed and proven in ethanol extracts and essential oils of H. ovalifolia (Hasimoto and Souza et al., 2002; Souza et al., 2003), in methylene chloride extract from H. brevipes (Goun et al., 2003). Furthermore, flavones from H. suaveolans (Isobe et al., 2006) and pectinolides from H. pectinata (Fragoso-Serrano et al., 2005) showed antibacterial activity against Helicobacter pylori, E. coli and S. aureus respectively.

The results obtained in this study showed the strong anti-staphylococcal property of the ethanol extract of H. martiusii, noted by small MIC value and effectiveness in inhibiting the microbial growth of S. aureus. These data are promising and could encourage further researches on phytochemical, toxicological and pharmacological aspects of H. martiusii by-products in order to support their possible rational use in the antimicrobial therapy, particularly, in anti-S. aureus therapy.



This work was supported by the following Brazilian agencies CNPQ and FAPESQ/PB.



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Received 10 October 2008
Accepted 16 November 2008



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