**4. Materials and methods**

#### **4.1 Collection of botanical material**

Fresh leaves of *M. suaveolens* were collected in the municipality of Quixelô located in the state of Ceará (Brazil) under coordinates −6°14′22.40″S, −39°16′14.29″W in March 2015 (**Figure 4**). The collection area is characterized as being part of the Caatinga, a seasonally dry tropical forest. The leaves were dried in an oven at 30°C. The plant material was identified and a voucher specimen was deposited in the Herbarium Caririense Dárdano of Andrade-Lima – HCDAL under #12.104.

## **4.2 Extraction of essential oil**

After drying, the leaves were packed in a volumetric flask containing 4 L of distilled water and subjected to constant boiling for 2 hours. Then the essential oil was collected and stored in an amber bottle under constant refrigeration until the conduction of the chemical analyzes and microbiological tests [8].

#### **Figure 3.**

*Minimum inhibitory concentration of antibiotic modulation in combination with essential oil of*  Mesosphaerum suaveolens*. PA 24,* Pseudomonas aeruginosa *24; EC 06,* Escherichia coli *06; SA 10,*  Staphylococcus aureus *10; EOMS, essential oil of* Mesosphaerum suaveolens*.*

**Figure 4.** *Map of the collection of the species* Mesosphaerum suaveolens *in the municipality of Quixelô—CE, Brazil.*

#### **4.3 Phytochemical analysis of essential oil by gas chromatography (GC-FID)**

For gas chromatography (GC), the Agilent Technologies 6890 N GC-FID system, equipped with DB-5 capillary column with the following specifications: 30 m of length, 0.32 mm and 0.50 μm of film thickness was used, which was connected to an FID detector. The temperature ramp consisted of: Initial temperature of 60°C for 1 min and was raised to 3° C/min until reaching 180°C [8].

#### **4.4 Identification of the components**

As for the identification, the terpenes were identified as to the of retention index (RI). In addition, they were compared with two spectral libraries, Nist and Wiley, and data in the literature [23].

**147**

*Chemical Composition and Antibacterial Activity of the Essential Oil of* Mesosphaerum…

*Escherichia coli* 06 Urine culture Cephalothin, cephalexin, cefadroxil, ceftriaxone, cefepime, ampicillin-sulbactam

*Source: Laboratory of Microbiology and Molecular Biology—LMBM—regional University of Cariri—URCA.*

*Isolated clinical bacterial strains used for MIC and modulation tests with their antibiotic resistance and origin* 

Uroculture Amikacin, imipenem, ciprofloxacin, levofloxacin, piperacillintazobactam, ceftazidime, meropenem, cefepime

> Cefadroxil, cephalexin, cephalothin, oxacillin, penicillin, ampicillin, amoxicillin, moxifloxacin, ciprofloxacin, levofloxacin, ampicillin-sulbactam, amoxilin/ac. Clavulanic, erythromycin, clarithromycin, azithromycin, clindamycin

For the antibacterial tests, standard strains were used to determine minimum inhibitory concentration (MIC), being *Escherichia coli* ATCC 25922, *Pseudomonas aeruginosa* ATCC 25853 and *Staphylococcus aureus* ATCC 25923. While for the modulation and also MIC tests, strains resistant cells (**Table 3**), being *Escherichia coli* 06,

As for the culture medium for the antibacterial assays, Brain Heart Infusion (BHI) was prepared according to the measures recommended by the manufacturer. While for *in vitro* modulation assays, the drugs used were Gentamicin from class aminoglycoside, Norfloxacin, belonging to the classes of fluoroquinolones and

It was followed the methodology employed in the work Bezerra et al. [3] for the determination of the Minimum Inhibitory Concentration (MIC). In this study, concentrations ranging from 1 to 1024 μg/mL of the essential oil of *M. suaveolens* against pathogenic bacteria were evaluated. For that, the inoculants of the strains were mixed with BHI (10%), being distributed in microdilution plates with the natural product. After 24 hours of microbial growth at a temperature of 37°C, the

To assess the modulating effect of essential oil, sub-inhibitory concentrations (MIC/8) of the product against multidrug-resistant bacteria were used. For that, concentrations of standard antibiotics (1–1024 μg/mL) were added to microdilution plates containing BHI (10%) and bacteria inoculum, as well as volatile *M. suaveolens* terpenes in sub-inhibitory concentrations. After 24 hours in a bacteriological oven

The results were analyzed in the GraphPad Prism program, version 6, in which the data were analyzed by Anova One-way and followed by post hoc Tukey test and

*DOI: http://dx.doi.org/10.5772/intechopen.92704*

**Bacteria Origin Resistance profile**

Rectal swab culture

**4.5 Antibacterial activity**

*Pseudomonas aeruginosa* 03

*Staphylococcus aureus* 10

**Table 3.**

*profile.*

*4.5.1 Bacterial strains, culture media and drugs*

Imipenem of the carbapenem class.

*4.5.3 Effect modulator of antibiotics*

were considered significant when *p* < 0.05.

**4.6 Statistical analysis**

*4.5.2 Minimal inhibitory concentration (MIC)*

MIC was evaluated with the addition of resazurin.

(37°C), a resazurin solution was added to determine the MIC [7].

*Pseudomonas aeruginosa* 03 and *Staphylococcus aureus* 10.

*Chemical Composition and Antibacterial Activity of the Essential Oil of* Mesosphaerum… *DOI: http://dx.doi.org/10.5772/intechopen.92704*


#### **Table 3.**

*Essential Oils - Bioactive Compounds, New Perspectives and Applications*

*Minimum inhibitory concentration of antibiotic modulation in combination with essential oil of*  Mesosphaerum suaveolens*. PA 24,* Pseudomonas aeruginosa *24; EC 06,* Escherichia coli *06; SA 10,* 

Staphylococcus aureus *10; EOMS, essential oil of* Mesosphaerum suaveolens*.*

**4.3 Phytochemical analysis of essential oil by gas chromatography (GC-FID)**

*Map of the collection of the species* Mesosphaerum suaveolens *in the municipality of Quixelô—CE, Brazil.*

1 min and was raised to 3° C/min until reaching 180°C [8].

**4.4 Identification of the components**

and data in the literature [23].

For gas chromatography (GC), the Agilent Technologies 6890 N GC-FID system, equipped with DB-5 capillary column with the following specifications: 30 m of length, 0.32 mm and 0.50 μm of film thickness was used, which was connected to an FID detector. The temperature ramp consisted of: Initial temperature of 60°C for

As for the identification, the terpenes were identified as to the of retention index (RI). In addition, they were compared with two spectral libraries, Nist and Wiley,

**146**

**Figure 4.**

**Figure 3.**

*Isolated clinical bacterial strains used for MIC and modulation tests with their antibiotic resistance and origin profile.*
