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O oleo essencial de Lippia gracilis Schauer, Verbenaceae, em ratos diabeticos

The essential oil from Lippia gracilis Schauer, Verbenaceae, in diabetic rats

Resumos

O óleo essencial das folhas de Lippia gracilis Schauer, Verbenaceae, foi examinado por CG e CG-MS. Quinze constituintes foram identificados, onde o carvacrol, p-cimeno e o γ-terpineno foram as substâncias majoritárias. No estudo in vitro, a solução a 5% do óleo de L. gracilis Schauer apresentou atividade antibacteriana para Staphylococcus aureus isolado de úlcera infectada de paciente diabético. O estudo avaliou a atividade antibacteriana da solução a 5% do óleo de L. gracilis Schauer em modelo experimental em diabetes utilizando ratos albinos Wistar machos com membro pélvico infectado com cepa de Staphylococcus aureus. No experimento foram utilizados 28 ratos Wistar distribuídos em quatro grupos (G1-branco, G2-controle negativo, G3-controle positivo, G4-teste) de sete ratos. Quando comparado o grupo G4 com G3, observou-se que a solução a 5% promoveu uma redução nas CFU/mL após 24h da administração do inóculo (S.aureus sem L. gracilis Schauer 10(8) ± 313 versus S.aureus com L. gracilis Schauer 13,28 ± 4,03). Os resultados foram expressos através do cálculo da média±EPM) e análise de variança (ANOVA). A diferença entre a concentração inibitória mínima no estudo in vitro foi determinada pelo teste Turkey (p<0.05). O teste Newman-Keuls com nível de significância (p<0.05) foi utilizado para o cálculo dos resultados obtidos no experimento in vivo. A solução a 5% do óleo essencial de Lippia gracilis Schauer apresentou boa atividade antibacteriana tanto no estudo in vivo como no in vitro.

Lippia gracilis Schauer; Verbenaceae; alecrim-de-tabuleiro; monoterpenos; carvacrol; p-cimeno; γ-terpineneo


The essential oil from Lippia gracilis Schauer (Verbenaceae) leaves was examined by GC and GC-MS. Fifteen constituents were identified. Carvacrol, p-cymene and γ-terpinene were found to be the major components. In the in vitro study, 5% solution of the Lippia gracilis Schauer oil presented antibacterial activity against Staphylococcus aureus isolated from diabetic patients with infected ulcers. The study evaluated the antibacterial activity of the 5% solution of the Lippia gracilis Schauer oil on the experimental model of diabetic adult male albino Wistar rats with leaft pelvic limb infected by Staphylococcus aureus strain. In this experiment, 28 diabetic Wistar rats were used, randomly distributed in four different groups of seven rats, (G1-white; G2-negative control; G3-positive control and G4-test). When comparing group G4 with G3, it was observed that the 5% solution presented a reduced CFU/mL level showing the antibacterial effect of the oil 24 hours after the administration of the inoculum (S .aureus without Lippia gracilis Schauer 108 ±313 versus S.aureus with Lippia gracilis Schauer 13.28±4.03). The results were expressed as mean±S.E.M. One-way analysis of the variance (ANOVA) was used. The differences between the minimum inhibitory concentration in vitro test were determined by the Tukey test (p<0.05). The Newman-Keuls test with level of significance (p<0.05) was used to measure the results in vivo. The findings have shown that 5% solution of the Lippia gracilis Schauer oil presented antibacterial activity in vitro and in vivo.

Lippia gracilis Schauer; Verbenaceae; alecrim-de-tabuleiro; monoterpenes; carvacrol; p-cymene; γ-terpinene


ARTIGO

O óleo essencial de Lippia gracilis Schauer, Verbenaceae, em ratos diabeticos

The essential oil from Lippia gracilis Schauer, Verbenaceae, in diabetic rats

Renato M. Neto* * E-mail address: renato@cb.ufrn.com.br, Tel. +55 84 99052977 ,I; Francisco J. A. MatosΦ Φ In memorian ,II; Vânia S. AndradeI; Maria C. N. de MeloI; Cibele B. M. CarvalhoIII; Sérgio B. GuimarãesIV; Otília D. L. PessoaII; Sônia L. SilvaV; Silvia F. R.SilvaV; Paulo R. L. VasconcelosIV

IDepartamento de Microbiologia e Parasitologia-Centro de Biociências –Universidade Federal do Rio Grande do Norte, Campus Universitário Lagoa Nova, s/n, 59072-970 Natal-RN, Brasil

IIPrograma de Pós-graduação em Química Orgânica, Departamento de Química, Campus do Pici, Universidade Federal do Ceará, Av. Bezerra de Menezes s/n, 60021-970 Fortaleza-CE, Brasil

IIIPrograma de Pós-graduação em Microbiologia, Faculdade de Medicina, Universidade Federal do Ceará, Rua Prof. Costa Mendes, 1608, 60430-140 Fortaleza-CE, Brasil

IVPrograma de pós-graduação em Cirurgia, Faculdade de Medicina, Universidade Federal do Ceará, Rua Prof. Costa Mendes, 1608, 60430-140 Fortaleza-CE, Brasil

VCurso de Farmácia, Universidade de Fortaleza, Av.Washington Soares s/n, 60811-905 Fortaleza-CE, Brasil

RESUMO

O óleo essencial das folhas de Lippia gracilis Schauer, Verbenaceae, foi examinado por CG e CG-MS. Quinze constituintes foram identificados, onde o carvacrol, p-cimeno e o γ-terpineno foram as substâncias majoritárias. No estudo in vitro, a solução a 5% do óleo de L. gracilis Schauer apresentou atividade antibacteriana para Staphylococcus aureus isolado de úlcera infectada de paciente diabético. O estudo avaliou a atividade antibacteriana da solução a 5% do óleo de L. gracilis Schauer em modelo experimental em diabetes utilizando ratos albinos Wistar machos com membro pélvico infectado com cepa de Staphylococcus aureus. No experimento foram utilizados 28 ratos Wistar distribuídos em quatro grupos (G1-branco, G2-controle negativo, G3-controle positivo, G4-teste) de sete ratos. Quando comparado o grupo G4 com G3, observou-se que a solução a 5% promoveu uma redução nas CFU/mL após 24h da administração do inóculo (S.aureus sem L. gracilis Schauer 108 ± 313 versus S.aureus com L. gracilis Schauer 13,28 ± 4,03). Os resultados foram expressos através do cálculo da média±EPM) e análise de variança (ANOVA). A diferença entre a concentração inibitória mínima no estudo in vitro foi determinada pelo teste Turkey (p<0.05). O teste Newman-Keuls com nível de significância (p<0.05) foi utilizado para o cálculo dos resultados obtidos no experimento in vivo. A solução a 5% do óleo essencial de Lippia gracilis Schauer apresentou boa atividade antibacteriana tanto no estudo in vivo como no in vitro.

Unitermos:Lippia gracilis Schauer, Verbenaceae, alecrim-de-tabuleiro, monoterpenos, carvacrol, p-cimeno, γ-terpineneo.

ABSTRACT

The essential oil from Lippia gracilis Schauer (Verbenaceae) leaves was examined by GC and GC-MS. Fifteen constituents were identified. Carvacrol, p-cymene and γ-terpinene were found to be the major components. In the in vitro study, 5% solution of the Lippia gracilis Schauer oil presented antibacterial activity against Staphylococcus aureus isolated from diabetic patients with infected ulcers. The study evaluated the antibacterial activity of the 5% solution of the Lippia gracilis Schauer oil on the experimental model of diabetic adult male albino Wistar rats with leaft pelvic limb infected by Staphylococcus aureus strain. In this experiment, 28 diabetic Wistar rats were used, randomly distributed in four different groups of seven rats, (G1-white; G2-negative control; G3-positive control and G4-test). When comparing group G4 with G3, it was observed that the 5% solution presented a reduced CFU/mL level showing the antibacterial effect of the oil 24 hours after the administration of the inoculum (S .aureus without Lippia gracilis Schauer 108 ±313 versus S.aureus with Lippia gracilis Schauer 13.28±4.03). The results were expressed as mean±S.E.M. One-way analysis of the variance (ANOVA) was used. The differences between the minimum inhibitory concentration in vitro test were determined by the Tukey test (p<0.05). The Newman-Keuls test with level of significance (p<0.05) was used to measure the results in vivo. The findings have shown that 5% solution of the Lippia gracilis Schauer oil presented antibacterial activity in vitro and in vivo.

Keywords: Lippia gracilis Schauer, Verbenaceae, alecrim-de-tabuleiro, monoterpenes, carvacrol, p-cymene, γ-terpinene.

INTRODUÇÃO

Diabetes Mellitus (DM) is one of the world's most serious health problems (Motta et al., 2003, Vischer al., 2009). In Brazil, there are about 5 million diabetics; 7.6% of Brazilians between the ages of 30 and 65 suffer from diabetes. The four important factors that make diabetic subjects more prone to complications are: susceptibility to infections, hyperglycemia,vascular disease and nerve damage (Levin, 1995). Diabetic foot infection represents one of the most serious chronic complications of DM (Goldstein et al., 1996). In this country, as in other developing countries, there is a widespread and uncontrolled use of antibiotics, and patients often do not take a full course treatment because they are unable to afford it. Thus, it is important to find inexpensive antimicrobial agents (Guzmán-Blanco et al., 2000). Essential oils are rich sources of biologically active compounds (Lemos et al., 1990, Aguiar & Jaciana, 2008) Recently, great interest has been shown in the antimicrobial properties of extracts from aromatic plants, particularly essential oils (Matos et al., 2004). Many oils and extracts from different plants have been investigated because of their antimicrobial properties against bacteria and yeasts (Salgueiro et al., 2003). Essential oils have been found to be antibacterial, antifungal and therapeutic in cancer treatment and can present other pharmacological properties. Thus, the use of natural antimicrobial compounds seems to be important not only in the preservation of food, but also for the control of human and plant diseases of microbial origin (Rasooli & Irmostafa, 2002, Barbosa et al., 2005). The genus Lippia has been credited with a long list of pharmacological properties. Lippia spp oils present high antimicrobial effects on various microorganisms (Bassole al., 2002). The variability of the oils in the Lippia genus has been the subject of several studies reviewed by Matos (Matos et al., 2004). The aims of this work were to study the chemical composition and antimicrobial activity of the 5% solution of the Lippia gracilis Schauer oil in diabetic adult male Wistar rats subjected to Staphylococcus aureus pelvic limb infection in vivo and in vitro experiments.

MATERIAL AND METHODS

Plant material and essential oil extraction

Lippia gracilis Schauer fresh leaves were collected in August 2005 at the Medicinal Plant Nursery - Natural Products Laboratory of the Federal University of Ceará, Brazil. The plant was identified by botanists of the Biology Department of the Federal University of Ceará, where a voucher specimen was deposited at Herbarium Prisco Bezerra under number 23427. The leaves were placed in a glass container and the essential oil (EO) was extracted by steam distillation in a Clevenger type apparatus. The extraction was performed for 2 h in 500 mL of water. The oils were stored in glass bottles in a freezer until they were used.

Analysis of Lippia gracilis EO

The EO was analyzed using a Hewlett-Packard 5971 GC/MS instrument under the following conditions column: dimethyl-polysiloxane DB-1 fused silica capillary column (30m x 0.25 mm); carrier gas: He (1 ml/min); injector temperature: 250 ºC; detector temperature: 200 ºC; column temperature: 35-180 ºC at 4 ºC/min then 180-25 ºC at 10 ºC/min; mass spectrum: electronic impact 70eV. The constituents were identified by computer-based library search, retention indices and visual interpretation of the mass spectra (Alencar et al., 1984; Adams et al., 1989).

Animals

Twenty-eight adult male Wistar rats (Rattus norvegicus - albinos), aged 60 days and weighing 180±25 g, bred in the Central Animal House of the Federal University of Ceará, were used. The animals were housed in polycarbonate cages in a room with a 12 h light/dark cycle, temperature of 22±2 ºC, and humidity of 45-64% during the whole experimental period. They were fed with a balanced commercial diet (Nuvital, Curitiba, PR, Brazil) and water ad libitum. All the animals were carefully monitored and maintained in accordance with ethical recommendations of the Brazilian Veterinary Medicine Council (CMV) and the Brazilian College of Animal Experiments (COBEA). The Ethics Committee of The Federal University of Ceará approved the protocols employed (Reg. Nº. 46/04 dated 11 October 2005).

Induction of experimental diabetes mellitus

After an overnight fasting, the rats were induced by intraperitoneal injection (150 mg/kg) of alloxan monohydrate (Acros Organics Research Laboratories, Inc., New Jersey, USA) dissolved in 0.1 M sodium citrate buffer (pH 3.0) (Zarrow et al., 1964). After two weeks, rats with marked hyperglycemia (fasting serum glucose > 250 mg/dL) were selected and used for study. The animals had free access to food and water after the alloxan injection.

Determination of the serum glucose concentration

Blood samples from the tail vein of the anesthetized (Ketamine cloridrate 90 mg/kg/im) rat were collected and (100 µL) centrifuged, and the serum was used to determine the glycemia by the glucose oxidase method (Calore et al., 2007).

Antibacterial assay

Bacterial strain

Staphylococcus aureus strain isolated from diabetic patients exhibiting infected ulcers was used in the study.

Antibacterial activity (in vitro)

The Minimal Inhibitory Concentration (MIC) was estimated by the broth dilution method (CLSI, 2008). The microorganism (Staphylococcus aureus) was grown overnight at 37 ºC in 10 mL of Mueller Hinton Broth (v/v) (Bioxon). The culture was adjusted with sterile DMSO (dymethylsulfoxide; Sigma) solution 2% (v/v). The density of the suspension of the respective microorganism was adjusted to 0.5 Mc Farland turbidity standards. The suspension was then inoculated onto the agar plate (15 x 100 mm) containing Mueller Hinton Agar (Bioxon). Concentrations of 10 to 1.25 % (v/v) of each essential oil were prepared. Discs of filter paper (Whatman no 5) of 5 mm diameter were impregnated with 5 µL of the essential oil placed on the agar surface. Discs impregnated with DMSO solution 2% (v/v) was used as negative control, and oxacillin (DIFCO) (1 µg) was used as positive control. The plate was incubated overnight at 37 oC and the diameter of any resulting zones of inhibition (mm) of growth was measured. Each experiment in vitro was performed three times.

Antibacterial activity (in vivo)

Twenty-eight adult male Wistar (Rattus norvegicus-albinus) diabetic rats were used in the experiment, distributed in four groups of seven rats. The rats were inoculated intracutaneously (left pelvic limb) with different solutions. Group 1 (negative control-white): the rats were inoculated with 0.1 mL of 2% DMSO solution (v/v). Group 2 (negative control-oil): the rats were inoculated with 0.1 mL of the 5% Lippia gracilis Schauer solution (v/v) preparation with 2% DMSO solution (v/v). Group 3 (positive control): the rats were inoculated with 0.1 ml of 2% DMSO solution (v/v) containing 105 CFU(colony-Forming Units)/mL of Staphylococcus aureus. Group 4 (test): the rats were inoculated with 0.05 mL of 2% DMSO solution (v/v) containing 105 CFU/mL of Staphylococcus aureus; after 24 h, they were inoculated with 0.05 mL of Lippia gracilis Schauer solution (v/v) preparation with 2% DMSO solution (v/v). Upon 48 h inicial experiment the rats were sacrificed by decapitation, using Ketamine Cloridate anesthesia (90 mg/kg/i.m) (Calore et al., 2007).

Bacterial culture

Pelvic limbs from 28 rats were used for bacterial analysis. After disinfecting them with 70% alcohol, left pelvic limb samples corresponding to the injection sites were deposited in 20 x 150 mm sterilized plastic tubes, homogenized, and suspended in 2.0 mL of BHI (Brian Heart Infusion) (DIFCO). Aliquots of 0.1 mL of the suspension from each of three dilutions (10-1, 10-2 e 10-3) were added to the center of the 15x100mm Petri containing agar 5 % sheep blood (v/v) and incubated at 37 ºC for 24 h. The number of Colony-Forming Units (CFU/mL) per pelvic limb surface on the plates was determined by the method described by Serguei et al., (2009), and bacterial colonies were tested for coagulase and catalase activity. Quality control was done by the Staphylococcus aureus ATCC (American Type Culture Collection) 29213. The antimicrobial agent tested was oxacillin (CLSI, 2008).

Statistical analysis

The results were expressed as mean±S.E.M. One-way analysis of variance (ANOVA) was used. The differences between the minimum inhibitory concentrations in vitro test were determined by Tukey test (p<0.05). A Newman-Keuls test (mean±S.E.M) with level of significance (p<0.05) was conducted to analyze the results.

RESULTS

Chemical analysis

The chemical analysis of Lippia gracilis Schauer EO is displayed in Table 1 according to the order of elution from a non-polar column. The main constituent as previously reported was carvacrol. The percentage yield in this oil was 50.13%.

Antibacterial activity (in vitro)

The results show that the concentration of Lippia gracilis Schauer EO displays a moderate antibacterial activity against Staphylococcus aureus. L. gracilis Schauer EO inhibited the growth of the bacteria producing a zone diameter of 26±0.88 and 25±0.57 mm in the oil concentrations of 10 and 5% (v/v) respectively.

Antibacterial activity (in vivo)

The concentration of Lippia gracilis Schauer EO 5% (v/v), was injected in the left pelvic limb, and after 24 h, the solution containing 105CFU/mL of Staphylococcus aureus was administered. A moderate antibacterial activity was displayed. When comparing Group 4 with 3, it was observed that the 5% solution presented an antibacterial effect, and reduced the number of CFU/mL.

DISCUSSION

Diabetic foot infections are generally polymicrobial. S. aureus are among the most frequently isolated microorganisms from the lesion (Motta et al., 2005).The essential oil of Lippia gracilis Schauer is mostly constituted by monoterpenes and sesquiterpenes. The major components are: carvacrol (50.13%), p-cymene (10.73%) and β-caryophyllene (5.96%) (Table 1). The 5% solution of the L. gracilis oil presented antibacterial activity in vitro and in vivo against the tested strain of Staphylococcus aureus isolated from diabetic patients exhibiting infected ulcers (Tables 2 and 3). The antimicrobial activity of different essential oils is well documented, particularly those which contain relatively high concentrations of the antibacterial phenols. Lippia sp oils are potential candidates for emulsion formulation as a topical product (Oladimeji et al., 2000; Oliveira et al.,2007). While essential oils are noted for their antimicrobial activities (Hammer et al., 1999; Smith-Palmer et al., 1998), as demonstrated by Lippia oil in this study, such activities vary, depending on their composition. The composition of Lippia oil has already been reported (Oladimeji et al., 2001). Among its components, some that have been confirmed to have antimicrobial activities are linalol, geraniol, (Onawunmi et al., 1984), thymol (Mwangi et al., 1994), α-pinene and terpineol (Weston et al., 1997). Such antimicrobial activities could form the basis to apply the oil in food preservation and in natural therapy (Albuquerque et al., 2007). The antimicrobial activity of carvacrol is well established (Kunle et al., 2003) and this study has shown that it is a major component of Lippia gracilis Schauer. One of the important cell types that are affected by diabetes is polymorphonuclear neutrophils (PMN). These PMN granulocytes represent the host's defense barrier against bacterial agents (Mandrup et al., 1987). In diabetic patients, these cells show functional alterations in chemotaxis, adhesion, phagocytosis and intracellular killing of the microorganism. PMN dysfunction has been attributed to an increased glucose concentration in PMN, which could lead to protein glycosylation (Marhoffer et al., 1992), depletion of NADPH (Wilson et al., 1987) and increased concentration of advanced glycosylation and products in macrophages, which in turn induce the release of cytokines such as interleukin-1 and tumor necrosis factor (Radoff et al., 1997). Diabetes has also been known to affect cell and tissue metabolism in different ways and numerous research studies have demonstrated these effects in other chronic diseases, despite the fact that the precise mechanism of such alterations remains unclear. One of the studies, conducted by Lebovici et al., 1996), showed that a crucial risk factor for the evolution of an infectious process in the foot is the length of time the person has been a diabetic. The antibacterial activity shown by the essential oil from Lippia gracilis Schauer may be attributed to the presence of some components such as carvacrol, terpineol acetate, cymene, tymol, pinene, cineole and linalool, which are already known to exhibit antibacterial activity (Knobloch et al., 1985; Juven et al., 1994; Harborne & Williams, 1995; Kim et al., 1995; Cimanga et al., 2002; Albuquerque et al., 2007; Aguiar&Jaciana et al., 2008). In conclusion, the 5% solution of the Lippia gracilis Schauer oil significantly reduced the colony count when compared with the control group, revealing its potential antibacterial qualities.

The results were expressed as mean+S.E.M. a5µL of concentration EO. Tukey test; (p<0.05) when compared to control.

Values are means+S.E.M. of the number of colony forming units. In parenthesis is the number of animals per group; a p<0.001 when compared with 1, as compared to controls (ANOVA and test t-Student-Newman-Kewls as the post hoc test); b p<0.001 when compared with 2, as compared to controls (ANOVA and test t-Student-Newman-Kewls as the post hoc test); c p<0.001 when compared with 3, as compared to controls (ANOVA and test t-Student-Newman-Kewls as the post hoc test).

ACKNOWLEDGEMENTS

The authors wish to thank CAPES for supporting this work and Terezinha de Jesus dos Santos Rodrigues for technical assistance.

Received 15 October 2008; Accepted 7 February 2009

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  • *
    E-mail address:
    renato@cb.ufrn.com.br, Tel. +55 84 99052977
  • Φ
    In memorian
  • Datas de Publicação

    • Publicação nesta coleção
      11 Jun 2010
    • Data do Fascículo
      Maio 2010

    Histórico

    • Recebido
      15 Out 2008
    • Aceito
      07 Fev 2009
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