**Wound Healing and Antibacterial Properties of Leaf Essential Oil of** *Vitex simplicifolia* **Oliv. from Burkina Faso**

Magid Abdel Ouoba1, Jean Koudou2\* , Noya Some3, Sylvin Ouedraogo2,3 and Innocent Pierre Guissou1,2 *1Unit of Teaching and Research (UFR) of Health Sciences, University of Ouagadougou, 2Laboratory of Chemistry of Natural Products, Faculty of Sciences, University of Bangui, Bangui, 3Institute of Research in Health Sciences, CNRST, Ouagadougou, 1,3Burkina Faso 2Central African Republic* 

#### **1. Introduction**

108 Chromatography and Its Applications

Zhao, J.L.; Liu, L.; Zhang, H.B.; Wu, Y.C.; Wang, D. & Chen, Y. J. (2006). Three-Component

*Pharmazie,* 337, pp. 127-132*.*

Friedel-Crafts Reaction of Indoles, Glyoxylate, and Amine under Solvent-Free and Catalyst-Free Conditions - Synthesis of (3-Indolyl)glycine Derivatives. *Archiv* 

> *Vitex simplicifolia* Oliv. (Verbenaceae) is a perennial shrub or small tree which grows to a height of aproximatively 8 m and is widely distributed from Egypt to Guinea. In Burkina Faso, the plant is used for internal or external use to treat various diseases like skin diseases, dermatitis, bilharzia, migraines, fever, aches, amoebiasis, sore teeth, colic, infant tetanus(Nacoulma,1996). Our ethnobotanical investigations have revealed that this plant is also used in the treatment of skin infections and wounds healing. In Burkina Faso, infectious diseases are the leading cause of infant mortality (2.37%) and maternal (14.6%), therefore they constitute public health problems. The treatment of skin diseases dates back to ancient times, and many treatments were using medicinal plants. About 30% of traditional remedies are used to treat wounds and skin lesions, compared to only 1-3% of modern drugs (Mantle et al., 2001). The healing process is an immune response that begins after injury and takes place in three stages: vascular and inflammatory stage, phase of tissue repair and phase of maturation. A drug having simultaneously the potential antioxidant and antimicrobial activities may be a good therapeutic agent to accelerate cicatrization and wound healing [Houghton et al., 2005; Phillips et al., 1991; Heike et al., 1999]. Aromatherapy is now considered to be another alternative way in healing people, and therapeutic values of aromatic plants lie in their volatile constituents such as monoterpenoids, sesquiterpenoids and phenolic compounds that produce a definite physiological action on the human body [Bruneton, 1993].To the best of our knowledge, there is no report on pharmacological studies of this plant. The present work reported results of a detailed investigation of

<sup>\*</sup> Corresponding Author

Wound Healing and Antibacterial Properties

**Determination of the strain sensitivity** 

**2.5 Antibacterial activity** 

after treatment. All of the tests were made in duplicate.

coquette, France). All of the tests were made in triplicate.

**Determination of MIC and MBC values** 

on Plate Count Agar (Merck, Germany).

1986]. All tests were performed in triplicate.

**Statistical analysis** 

of Leaf Essential Oil of *Vitex simplicifolia* Oliv. from Burkina Faso 111

Observation of the evolution of wound healing versus time was carried out at 48h and 96h

The test was performed using Müller-Hinton medium for bacteria using disk diffusion method following the National Committee for Clinical Laboratory Standards methods [Kiehlbauch et al., 2000]. Overnight broth cultures of each strain were prepared in nutriment Broth (Diagnostic Pasteur, France). The final concentration of each inoculums was got making dilution of each strain in 9 % NaCl solution. The turbidity of each inoculum was compared with McFarland 0.5 solution. The final concentration of each inoculum (approximatively 5.105 CFU / ml) was confirmed by viable count on Plate Count Agar

Positive and negative growth controls were performed for every test. The plates were incubated aerobically at 30°C or 37 °C for 24 hours. The bacterial sensitivity to the essential oil was assessed by measuring the diameter of inhibition zone and recorded if the zone of inhibition is greater than 9 mm. The inhibition zones were compared with that of ampicilline (BIO-RAD Marne- la- coquette, France) and tetracycline (BIO-RAD Marne- la-

A broth microdilution method was used to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) [Bassolé et al., 2003] All tests were performed in Mueller-Hinton Broth (Becton Dickinson, USA). A serial of double of each essential oil was prepared in 96 well-plates over the range 0.03-8% (v/v). The broth was supplemented with tween 80 at a concentration of 0.1% in order to enhance essential oil solubility. The tween 80 was at the final concentration of 0.001% (v/v). Overnight broth cultures of each strain were prepared in Nutrient Broth (Diagnostic Pasteur, France) and the nal concentration in each well was adjusted to 5 x 105 CFU/ml following inoculation. The concentration of each inoculum was conrmed by viable count

Positive and negative growth controls were included in every test. The tray was incubated aerobically at 30 °C (Reference Gram-negative strain) or 37 °C (Reference and isolated Gram-positive) and MICs were determined. The MIC defined as the lowest concentration of the essential oil at which the microorganism tested does not demonstrate visible growth. To determine MBCs, 10µl suspension were taken from each well and inoculated in Mueller– Hinton Agar (Becton Dickinson, USA) for 24 h at 30 or 37 °C. The MBC is defined as the lowest concentration of the essential oil killing 99.9% of bacteria inocula [Michel Briand,

Data were expressed as mean±SEM. A one way variance was use to analyse data. p<0.01

represented significant difference between means (Duncans multiple range test).

(Merck, Germany). 3µl of essential oil was put on every disk (8 mm diameter).

cicatrization and antibacterial activities of the leaf essential oil with the aim to contributing to the search for beneficial uses of this plant.

## **2. Materials and methods**

#### **2.1 Animals**

The experiments were conducted using six male and female rabbits aged 7 months and weighing between 1±0.05 and 2±0.06 kg, housed alone under 12h light-dark cycle, controlled humidity (75%), and temperature (20-25°C) conditions with free access to food and water [Draize et al., 1944]. In addition, three other rabbits were taken as controls. The experiments were carried out according to the method as described previously [Draize et al., 1944] in accordance with the guidelines for the care of laboratory animals and ethical guidelines for the investigation of experimental pain in conscious animals and revised by Official Journal of France (1971/04/21).

#### **2.2 Plant material and extraction**

The leaves of *V. simplicifolia* Oliv were collected in January 2007 from the Kadiogo region, village of Balgui, 10 km near Ouagadougou, Burkina Faso. A voucher specimen was identified by Pr. Jeanne Millogo, botanist (University of Ouagadougou) and deposited at the herbarium of IRSS of Ouagadougou.

Dried and powdered leaves (500g) were subjected to hydrodistillation for 4h with a clavengertype apparatus. The essential oil was collected and dried, after decantation, over anhydrous sodium sulfate and stored in refrigerator at 4°C for further use [Ouoba et al., 2009].

#### **2.3 Reference bacteria strains**

Microorganisms used in this study were:

*Bacillus cereus* LMG13569BHI, *Listeria innocua* LMG13568BHI, *Staphylococcus aureus* ATCC 25293BHI, *Staphylococcus camorum* LMG 13567BHI, *Staphylococcus aureus* ATCC9144BHI, *Enterococcus faecalis* CIP103907BHI, *Proteus mirabilis* CIP104588, *Shigella dysenteria* CIP5451, *Salmonella enterica* CIP105150, *Escherichia coli* CIP105182. These strains were identified by the conventional methods and tested. Bacteria were obtained from stock cultures of the laboratory of pharmacology and clinic biochemistry of CRSBAN, University of Ouagadougou. The bacteria stock cultures were maintained on Müller-Hinton agar and which were stored at 4°C.

#### **2.4 Wound healing activity**

Assessment of the healing power of the oil was performed using the method [Draize et al., 1944], on 6 male and female rabbits housed in individual cages. Both flanks of each rabbit were shaved, deeply incised prior to application of the essential oil. Rabbits were fixed horizontally from their ears and legs. One flank was covered with a compress soaked with 0.44 mg (0.50 ml) of the pure oil and held by a sticking-plaster, the other untreated flank serving as control. The same operation was repeated with Cicatryl as a reference standard, with a dose of 1 g per flank. The rabbits were returned to their cages after treatment. Observation of the evolution of wound healing versus time was carried out at 48h and 96h after treatment. All of the tests were made in duplicate.

#### **2.5 Antibacterial activity**

110 Chromatography and Its Applications

cicatrization and antibacterial activities of the leaf essential oil with the aim to contributing

The experiments were conducted using six male and female rabbits aged 7 months and weighing between 1±0.05 and 2±0.06 kg, housed alone under 12h light-dark cycle, controlled humidity (75%), and temperature (20-25°C) conditions with free access to food and water [Draize et al., 1944]. In addition, three other rabbits were taken as controls. The experiments were carried out according to the method as described previously [Draize et al., 1944] in accordance with the guidelines for the care of laboratory animals and ethical guidelines for the investigation of experimental pain in conscious animals and revised by Official Journal

The leaves of *V. simplicifolia* Oliv were collected in January 2007 from the Kadiogo region, village of Balgui, 10 km near Ouagadougou, Burkina Faso. A voucher specimen was identified by Pr. Jeanne Millogo, botanist (University of Ouagadougou) and deposited at the

Dried and powdered leaves (500g) were subjected to hydrodistillation for 4h with a clavengertype apparatus. The essential oil was collected and dried, after decantation, over anhydrous

*Bacillus cereus* LMG13569BHI, *Listeria innocua* LMG13568BHI, *Staphylococcus aureus* ATCC 25293BHI, *Staphylococcus camorum* LMG 13567BHI, *Staphylococcus aureus* ATCC9144BHI, *Enterococcus faecalis* CIP103907BHI, *Proteus mirabilis* CIP104588, *Shigella dysenteria* CIP5451, *Salmonella enterica* CIP105150, *Escherichia coli* CIP105182. These strains were identified by the conventional methods and tested. Bacteria were obtained from stock cultures of the laboratory of pharmacology and clinic biochemistry of CRSBAN, University of Ouagadougou. The bacteria stock cultures were maintained on Müller-Hinton agar and

Assessment of the healing power of the oil was performed using the method [Draize et al., 1944], on 6 male and female rabbits housed in individual cages. Both flanks of each rabbit were shaved, deeply incised prior to application of the essential oil. Rabbits were fixed horizontally from their ears and legs. One flank was covered with a compress soaked with 0.44 mg (0.50 ml) of the pure oil and held by a sticking-plaster, the other untreated flank serving as control. The same operation was repeated with Cicatryl as a reference standard, with a dose of 1 g per flank. The rabbits were returned to their cages after treatment.

sodium sulfate and stored in refrigerator at 4°C for further use [Ouoba et al., 2009].

to the search for beneficial uses of this plant.

**2. Materials and methods** 

of France (1971/04/21).

**2.2 Plant material and extraction** 

herbarium of IRSS of Ouagadougou.

**2.3 Reference bacteria strains** 

which were stored at 4°C.

**2.4 Wound healing activity** 

Microorganisms used in this study were:

**2.1 Animals** 

#### **Determination of the strain sensitivity**

The test was performed using Müller-Hinton medium for bacteria using disk diffusion method following the National Committee for Clinical Laboratory Standards methods [Kiehlbauch et al., 2000]. Overnight broth cultures of each strain were prepared in nutriment Broth (Diagnostic Pasteur, France). The final concentration of each inoculums was got making dilution of each strain in 9 % NaCl solution. The turbidity of each inoculum was compared with McFarland 0.5 solution. The final concentration of each inoculum (approximatively 5.105 CFU / ml) was confirmed by viable count on Plate Count Agar (Merck, Germany). 3µl of essential oil was put on every disk (8 mm diameter).

Positive and negative growth controls were performed for every test. The plates were incubated aerobically at 30°C or 37 °C for 24 hours. The bacterial sensitivity to the essential oil was assessed by measuring the diameter of inhibition zone and recorded if the zone of inhibition is greater than 9 mm. The inhibition zones were compared with that of ampicilline (BIO-RAD Marne- la- coquette, France) and tetracycline (BIO-RAD Marne- lacoquette, France). All of the tests were made in triplicate.

#### **Determination of MIC and MBC values**

A broth microdilution method was used to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) [Bassolé et al., 2003] All tests were performed in Mueller-Hinton Broth (Becton Dickinson, USA). A serial of double of each essential oil was prepared in 96 well-plates over the range 0.03-8% (v/v). The broth was supplemented with tween 80 at a concentration of 0.1% in order to enhance essential oil solubility. The tween 80 was at the final concentration of 0.001% (v/v). Overnight broth cultures of each strain were prepared in Nutrient Broth (Diagnostic Pasteur, France) and the nal concentration in each well was adjusted to 5 x 105 CFU/ml following inoculation. The concentration of each inoculum was conrmed by viable count on Plate Count Agar (Merck, Germany).

Positive and negative growth controls were included in every test. The tray was incubated aerobically at 30 °C (Reference Gram-negative strain) or 37 °C (Reference and isolated Gram-positive) and MICs were determined. The MIC defined as the lowest concentration of the essential oil at which the microorganism tested does not demonstrate visible growth. To determine MBCs, 10µl suspension were taken from each well and inoculated in Mueller– Hinton Agar (Becton Dickinson, USA) for 24 h at 30 or 37 °C. The MBC is defined as the lowest concentration of the essential oil killing 99.9% of bacteria inocula [Michel Briand, 1986]. All tests were performed in triplicate.

#### **Statistical analysis**

Data were expressed as mean±SEM. A one way variance was use to analyse data. p<0.01 represented significant difference between means (Duncans multiple range test).

Wound Healing and Antibacterial Properties

of Leaf Essential Oil of *Vitex simplicifolia* Oliv. from Burkina Faso 113

(1a) *Salmonella enterica*

(1b) *Shigella dysenteria*

(1c) *Staphylococcus aureus*

Fig. 1. Inhibition zones for some bacterial strains.
