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Synergy effects of the antibiotics gentamicin and the essential oil of Croton zehntneri.

Abstract

The leaves of Croton zehntnevi Pax et Hoffm (Euphorbiaceae) were subjected to hydrodistillation, and the essential ail extracted was examined with respect to antibacterial and antibiotic modifying activity by gaseous contact. The gaseous component of the oil inhibited the bacterial growth of Staphylococcus aureus and Pseudomonas aeruginosa with a MID of 0.5 and < 1 mg/1 air, respectively. The activity of the antibiotic gentamicin was increased by 42,8% against P. aeruginosa after contact with the gaseous component, showing that this oil influences the activity of the antibiotic and may be used as an adjuvant in the antibiotic therapy of respiratory tract bacterial pathogens. [c] 2009 Elsevier GmbH. All rights reserved.

Keywords: Croton zehntneri; Essential oil; Modifying antibiotic activity; Minimal inhibitory dose; Staphylococcus aureus; Pseudomonas aeruginosa

Introduction

Staphylococcus aureus and Hemophilus influenzae are the most common bacteria isolated from the sputum, but in the second and third decade of life, Pseudomonas aeruginosa is the prevalent bacteria. In Germany, analysis of the sputum from patients with Cystic Fibrosis during a period of 12 months showed the presence of P. aeruginosa in 50% of these individuals and S. aureus in 63.3% arc the most prevalent pathogens (Coutinho et al., 2008; Valenza et al., 2008). Methicillin-resistant S. aureus (MRSA) has become a major nosocomial pathogen. In Europe, the spread ofMRSA varies widely among centers, ranging from 5 to 14% (Campana et al. 2004). Isolated from P. aeruginosa can be differentiated in terms of its morphotypes, including mucoid, not mucoid and those with biofilm, which vary their patterns of susceptibility to antibiotics. This differentiation causes several problems in the treatment, because is necessary identify the morphotype to choose the treatment strategy (Martin et al. 1993; Saiman et al. 1996).

Essential oils are natural, complex, multi-component systems composed mainly of terpenes in addition to some other non-terpene components (Edries 2007). Strong in vitro evidence indicates that essential oils can act as antibacterial agents against a wide spectrum of pathogenic bacterial strains including Listeria monocytogenes, L. innocua, Salmonella typhimurium, Escherichia coli O157:H7, Shigella dysenteria, Bacillus cereus, Staphylococcus aureus and Salmonella typhimurium (Burt, 2004; Nguefack et al., 2004; Schmidt et al., 2005). Croton zehntneri Pax et Hoffm is an aromatic-plant native to Northeastern Brazil, where it is popularly called "Canela de Cunha" and used in folk medicine principally as a sedative, as an appetite-stimulating antianorexigen, and for the relief of gastrointestinal disturbances (Albuquerque et al., 1995; Coelho-de-Souza et al., 1998). Despite this, little pharmacological investigation has been carried out on the effects of C. zehntneri. Published research on plant extracts has focused primarily on the central nervous system (Bernardi et al. 1991; Giorgi et al. 1991) and on muscle (Coelho-de-Souza et al. 1997; Leal-Cardoso and Fonteles 1999). The chemical composition is essentially characterized by a large percentage of oxygenated sesquiterpenes, being trans-Anethol (94.1%) the main one and trans-caryophyllene, myrcene, [alpha]-pinene, 1,8-cineole and estragole the minor compounds (Lima et al., 2006).

In this paper, we report the antibacterial and antibiotic modifying activity of the essential oil extracted from the leaves of Croton zehntneri Pax et Hoffm using the minimal inhibitory dose method and gaseous contact.

Material and methods

Plant

Leaves of Croton zehntneri Pax et Hoffm (Euphor-biaceae) were collected in Crato county, Ceara State, Brazil. The plant material was identified and a voucher specimen was deposited under the number 1619 at the Herbarium "Dardano de Andrade Lima" of Universidade Regional do Cariri - URCA.

Essential oil extraction

Fresh leaves (l.750 g) of C. zehntneri were collected in the Medicinal and Aromatic Plant Garden at the Pimenta Campus of the Regional University of Cariri (URCA), Crato, CE, Brazil, in March 2008. The leaves were triturated and extracted by hydrodistillation for two hours using a Clevenger-type apparatus (yeld 15.6 mg/g of leaves). The oil was collected, then dried using anhydrous sodium sulfate, and subsequently stored under low light conditions at < 10[degrees]C until analysis.

Strains

Pseudomonas aeruginosa (ATCC15442) and Staphylococcus aureus (ATCC12692) were used as the respective Gram-negative and Gram-positive standards. Both strains were stored at room temperature in heart infusion agar slants (HIA, Difco), and prior to assay, the cells were grown overnight at 37 [degrees]C in brain heart infusion (BHI, Difco).

Gaseous contact

Antibacterial activity of the essential oil from C. zehntneri was assayed using gaseous contact. An amount of 50 [micro]g of oil was dissolved in 50 [micro]l of DMSO (1:1). A twofold dilution series of this essential oil solution was prepared: 50, 25, 12.5, 6.25, 3.125 and 1.562 [micro]g of oil. Petri dishes with nutrient agar (Difco) were inoculated with [l0.sup.5] CFU/mL by the spread plate method. A volume of 100 [micro]l of each dilution was placed inside the upper part of Petri dish. The plates were incubated at 37 [degrees]C for 24 h. The minimal inhibitory dose (MID) was defined as the minimal inhibitory dose per unit space required to suppress the growth of microorganism in a closed system. The MID values were expressed as weight per unit volume (mg/L air), where the solution with 50 [micro]g equals 1 mg/L air (Inouye et al. 2001).

Antibiotic modifying activity

The antibiotic modifying activity of the gaseous component was determined using the same method, but only the solution with a total of 50 [micro]g of oil was used. In these plates, antibiotics disks with gentamicin and tetracycline were used to determine changes in the inhibition zone diameter of P. aerugionsa ATCC15442. Plates without the essential oil and with DMSO alone were used as control.

Results and discussion

In the Table 1, P. aeruginosa was shown to be less susceptible to the essential oil of C. zehntneri, in accordance with previous studies. In the Table 2, the antibiotic activity of gentamicin against P. aeruginosa was enhanced in the presence of the essential oil.

Synergy research aims to find the scientific reasons to the superiority of many herbal drugs extracts as compared with single constituents thereof (Wagner and Ulrich-Merzenich 2009). Essential oils may interact with and affect the plasma membrane, interfering with respiratory chain activity and energy production (Nicolson et al. 1999). The weak antibacterial activity against Gram-negative bacteria was ascribed to the presence of an outer membrane with hydrophilic polysaccharide chains serving as barriers to hydrophobic active compounds in the essential oil (Mann et al. 2000; Tassou and Nychas 1995).
Table 1. Antibacterial activity of the volatile compounds of essential
oil of Croton zehntneri by gaseous contact.

Strain               MID (mg/L
                     air)

                     1          0.5  0.25  0.125  0.0625  0.03125

S. aureus ATCC12692  -          -    +     +      +       +

P. aeruginosa        +          +    +     +      +       +
ATCC15442

+ growth observed; - no growth observed; MID minimal inhibitory dose.
Table 2. Modification of the antibiotic activity of the volatile
compounds of Croton zehntneri essential oil by gaseous contact.

Treatments   P. aeruginosa ATCC 15442
             (mm[+ or -]SD %)

             Gentamicin     Enhancement  Tetracyclin      Enhancement
                            (%)                           (%)

Not          14[+ or -]1.6  -            32[+ or -]1.3    -
treatment

DMSO         14[+ or -]1.3  -            32,5[+ or -]1.6  -

EOCZ (MID 1  20[+ or -]1.6  42.8         32[+ or -]1.6    0
mg/L air)

EOCZ - Essential oil of Croton zehntneri.
SD - Standard deviation.


The mechanisms by which essential oils can inhibit microorganisms involve different modes of action, and in part may be due to their hydrophobicity. As a result, they get partitioned into the lipid bilayer of the cell membrane, Saffecting the respiratory chain and the energy production (Nicolson et al. 1999), rendering it more permeable to the uptake of antibiotics and leading to leakage of vital cell contents (Burt, 2004; Juven et al. 1994). Several components of essential oils as thymol and carvacrol may act as membrane permeabilizers, enhancing the intake of antibiotics (Helander et al. 1998). Impairment of bacterial enzyme systems may also be a potential mechanism of action (Wendakoon and Sakaguchi 1995). These mechanisms of action may be derived by the combination of direct vapor absorption on microrganisms with indirect effect of the vapour absorption through the medium (Moleyar and Narasinham 1986).

This strategy are named "herbal shotgun" or "Synergistic multi-target effects" and refers to the use of herbals and drugs in a multitargeted approach, due the fact of mono or multi-extract combinations affect not only a single target, but several ones, cooperating in an agonistic-synergistic way. This approach are not exclusive for extract combinations, but combinations between single natural products or extracts with chemosynthetics or antibiotics are possible too (Hemaiswarya et al. 2008; Wagner and Ulrich-Merzenich 2009).

Individual compounds of essential oils from Melaleuca leucodendron and Ocimum gratissimum presented synergism with several antibiotics by direct contact (Jedlickova et al., 1992), but this is the first report of synergism between gaseous compounds of essential oils and antibiotics as gentamicin.

Several reports indicate different antibiotic combinations assyed in vitro and applied in the clinics, but combinations of natural products and synthetic drugs are not reported. The results obtained in this investigation suggest that the volatile compounds of essential oil of C. zehntneri may suppress the growth of bacterial pathogens of respiratory infections and could be a source of metabolites with antibacterial modifying activity to be used as adjuvants to antibiotic therapy against these pathogens. So research should be stimulated to identify more natural compounds with synergistic behavior.

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Fabiola F.G. Rodrigues (a), Jose G.M. Costa (a), (b) Henrique D.M. Coutinho (b)

(a) Faculdade Leao Sampaio, Juazeiro do Norte, CE, Brazil

(b) Laboratorio de Pesquisa em Produtos Naturals, Centra de Ciencias Biologicas e da Saude, Universidade Regional do Cariri, Crato, CE, Brazil

* Correspondence to: Universidade Regional do Cariri - URCA; Centro de Ciencias Biologicas e da Saude - CCBS; Laboratorio de Pesquisa em Produtos Naturais - LPPN. CEP:63105-900. Crato, CE - Brasil. Rua Cel. Antonio Luis 1161. Pimenta, 63105-000. Tel: + 55(88)31021212; fax: +55(88)31021291.

E-mail addresses: fabiolatere gmail.com (F.F.G. Rodrigues). galberto@urai.br (J.G.M. Costa), hdmcoutinho@gmail.com (H.D.M. Coutinho).

0944-7113/$-see front matter [c] 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.04.004
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Title Annotation:SHORT COMMUNICATION
Author:Rodrigues, Fabiola F.G.; Costa, Jose G.M.; Coutinho, Henrique D.M.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3BRAZ
Date:Nov 1, 2009
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