**3. Results**

### **3.1 Analyses**

GC and GC/MS analyses of the essential oil composition of *Vitex simplicifolia* were as previously described [Ouoba et al., 2009] The oil contained monoterpenoids as predominant (71.02%). Among monoterpene hydrocarbon, myrcene (53.50%) had been found as the major component and four components were detected as predominant: α-pinene (5.13%), β-pinene (2.48%) and β-phellandrene (1.38%). In the oxygenated fraction, 10 monoterpenes (6.32%) and 12 sesquiterpenes (5.58%) were present with linalool (4.70%) and humulen-1,2- epoxyde (1.15%) as the major constituents. Among mono and sesquiterpenes three ketones are detected as minor compounds piperitone (0.05%) cis-jasmone (0.11%) and salvia-4(14)en-1 one (0.07%). No phenolic compound has been detected in the oil.

## **3.2 Wound healing activity**

Experiments on rabbits showed that the oil has a healing effect. As shown in Table1, the different stages of evolution of healing capacity of essential oil in comparison with those of cicatryl and natural immunity of rabbit. The different stages of evolution of healing were characterized by changes in the color of the wound over time, the closure of lacerations and the absence of erythema and edema of the wounds.


Table 1. Different stages of evolution of wounds healing.

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

The results showed that almost of the bacterial strains were sensitive to *Vitex simplicifolia* essential oil (Table2). *Staphylococcus aureus* ATCC9144 BHI (zone of inhibition 34.5mm) was the most sensitive bacteria tested. Only *Bacillus cereus* LMG 13569 BHI was not sensible to *Vitex simplicifolia* (zone of inhibition 8.5mm) (Fig1).

GC and GC/MS analyses of the essential oil composition of *Vitex simplicifolia* were as previously described [Ouoba et al., 2009] The oil contained monoterpenoids as predominant (71.02%). Among monoterpene hydrocarbon, myrcene (53.50%) had been found as the major component and four components were detected as predominant: α-pinene (5.13%), β-pinene (2.48%) and β-phellandrene (1.38%). In the oxygenated fraction, 10 monoterpenes (6.32%) and 12 sesquiterpenes (5.58%) were present with linalool (4.70%) and humulen-1,2- epoxyde (1.15%) as the major constituents. Among mono and sesquiterpenes three ketones are detected as minor compounds piperitone (0.05%) cis-jasmone (0.11%) and salvia-4(14)en-1-

Experiments on rabbits showed that the oil has a healing effect. As shown in Table1, the different stages of evolution of healing capacity of essential oil in comparison with those of cicatryl and natural immunity of rabbit. The different stages of evolution of healing were characterized by changes in the color of the wound over time, the closure of lacerations and

rabbits treated wounds at 48h wounds at 96h wounds from 6 to 10

maturation stage (end of cicatrization) **complete healing** 

tissue repair stage (end) maturation stage (start)

tissue repair stage (end) maturation stage

(start)

The results showed that almost of the bacterial strains were sensitive to *Vitex simplicifolia* essential oil (Table2). *Staphylococcus aureus* ATCC9144 BHI (zone of inhibition 34.5mm) was the most sensitive bacteria tested. Only *Bacillus cereus* LMG 13569 BHI was not sensible to

days

maturation stage (end of cicatrization) **complete healing** 

maturation stage (end of cicatrization) **complete healing** 

one (0.07%). No phenolic compound has been detected in the oil.

the absence of erythema and edema of the wounds.

tissue répair stage maturation stage (start)

tissue répair stage maturation stage (start)

tissue répair stage maturation stage (start)

*Vitex simplicifolia* (zone of inhibition 8.5mm) (Fig1).

**3.3 Determination of the strain sensitivity** 

Table 1. Different stages of evolution of wounds healing.

stage

stage

stage

vascular and inflammatory

vascular and inflammatory

vascular and inflammatory

**3. Results 3.1 Analyses** 

**3.2 Wound healing activity** 

Essential oil

Cicatryl

Rabbits untreated

(1a) *Salmonella enterica*

(1b) *Shigella dysenteria*

(1c) *Staphylococcus aureus*

Fig. 1. Inhibition zones for some bacterial strains.

Wound Healing and Antibacterial Properties

[Mertz et al., 1993].

of Leaf Essential Oil of *Vitex simplicifolia* Oliv. from Burkina Faso 115

and reconstruction of anatomical and functional disturbances of the skin[Chattopadhyay et al., 2002]. The repair of damaged tissues occurs as a sequence of events that included inflammation, proliferation and migration of different cell types [Sidhu et al., 1999]. At the dose of 0.44 mg used only once the essential oil healed wounds for 96h. Essential oil of *Vitex simplicifolia* accelerated the three stages of cicatrization process: vascular and inflammatory, tissue repair and maturation. While at the dose of 1000 mg used only once, cicatryl exhibited a complete healing with for 7 days against 10 days for the effect of natural immunity of rabbit. In the phase of maturation a renewal of the skin was seen, the old skin started to fall and made way for the new. The essential oil of *Vitex simplicifolia* exhibited a stronger healing effect than cicatryl and natural immunity. This effect could be due to the presence of ketones in the oil that activated the healing process with stimulating of new cell growth, reducing old scare tissue in wound and were highly immunostimulatory [Willem, 2004]. The presence of minor compounds as aldehydes and sesquiterpenes activated anti inflammatory, calming and sedative effects. Thus, their low proportion allowed to consider possible synergistic effects of these compounds in the oil. The significant presence of monoterpenoids in the oil might cause analgesic, antioxidative, antiseptic effects and stimulating the immune system

In other hand, the skin infections are in most cases due to staphylococci with the pathogenic species is Staphylococcus aureus. It is responsible for suppurative infections, widespread and food poisoning. Thus, wound infections are most common in developing countries because of poor sanitation. *Staphylococcus aureus, Shigella dysenteria*, *Salmonella enterica*, *Escherichia coli* are important microorganisms causing an infection of the wound [Mansouri et al., 2011]. The best sensitivity to *Vitex simplicifolia* essential oil was, respectively, obtained on *Staphylococcus aureus* ATCC 9144 BHI (34.5mm), *Salmonella enterica* CIP 105150 (28mm), *Shigella dysenteria* CIP 5451(24.5mm), *Staphylococcus aureus*  ATCC 25293 BHI (24.5mm), *Enterococcus faecalis* CIP 103907 BHI(21mm). Following the results in Table 2 the different strains were less sensitive to V.s than ampicilline, while *shigella dysenteria* CIP 5451 was sensitive to V.s. The most important information was that essential oil exhibited more activity on *Staphylococcus aureus* ATCC 9144 BHI (34.5mm), *Salmonella enterica* CIP 105150 (28mm) *Shigella dysenteria* CIP5451(24.5mm) than tetracycline and ampicilline (*Shigella dysenteria* CIP 5451, 11mm). The essential oil failed to inhibit *Staphylococcus aureus* ATCC 9144 BHI at the lowest MIC 0.25%. The essential oil was bacteriostatic for *Staphylococcus aureus* ATCC 9144 BHI, *Escherichia coli* CIP 105182 and *Listeria innocua* LMG 135668. The most resistant strains with highest MBC (8%) were *Enterococcus faecalis* CIP 103907, *Staphylococcus aureus* ATCC 25293 and *Staphylococcus camorum* LMG 13567. Considering MICs and MBCs no significant difference could be seen between Gram-positive and Gram-negative bacteria. The chemical composition of the oil consisted of various constituents. Therefore, the determination of the component responsible for activity was very difficult. Furthermore the essential oil consists of complex mixture of numerous constituents. Major or minor compounds might cause the bacteriostatic and cicatrization activities exhibited, terpinene-4-ol and other monoterpenes in essential oil may act as antiseptic, anti-inflammatory and antimicrobial: myrcene, sabinene, terpinene, cadinene and limonene [Sinan Dayisoylu et al. 2009]. In addition, the presence of α-pinene, β-pinene [Houghton Peter, 2004] terpinen-4-ol [Lee et al., 2001] and


V.s.: *Vitex simplicifolia* Amp: ampicilline Te: tetracycline

Table 2. Diameter of inhibition zone (mm) of bacteria growth.

#### **3.4 Determination of antibacterial activity**

The MICs and MBCs of *Vitex simplicifolia* essential oil were consigned in Table3. Five bacterial strains were selected and tested because of their highest sensitivity to essential oil. The oil inhibited the growth of these bacteria with MIC of 0.50% except for *Staphylococcus aureus* ATCC 9144BHI that was more sensitive with MIC of 0.25%. The results of MBC demonstrated a bacteriostatic effect.


Table 3. Minimum inhibitory concentration, minimum bactericidal concentration data (%v/v) obtained by microdilution method.

#### **4. Discussion**

Wound healing is very complex, it involves a sequence of multifactorial events including several cellular and biochemical processes. These processes aim to ensure the regeneration

The MICs and MBCs of *Vitex simplicifolia* essential oil were consigned in Table3. Five bacterial strains were selected and tested because of their highest sensitivity to essential oil. The oil inhibited the growth of these bacteria with MIC of 0.50% except for *Staphylococcus aureus* ATCC 9144BHI that was more sensitive with MIC of 0.25%. The results of MBC

Reference strains Origin MIC MBC MBC/MIC

0,5

8

4

4

8

8

1

0,5

0,5

0,5

0,5

0.25

Wound healing is very complex, it involves a sequence of multifactorial events including several cellular and biochemical processes. These processes aim to ensure the regeneration

Table 3. Minimum inhibitory concentration, minimum bactericidal concentration data

*Enterococcus faecalis* CIP 103907 BHI *Bacillus cereus* LMG 13569 BHI *Listeria innocua* LMG 13568 BHI *Staphylococcus aureus* ATCC 25293 BHI *Staphylococcus camorum* LMG 13567 BHI *Staphylococcus aureus* ATCC 9144 BHI

V.s.: *Vitex simplicifolia* Amp: ampicilline Te: tetracycline

**3.4 Determination of antibacterial activity** 

demonstrated a bacteriostatic effect.

*Enterococcus faecalis*

*Escherichia coli* CIP

CIP 103907

105182 *Listeria innocua* LMG 135668 *Staphylococcus aureus* ATCC 25293 *Staphylococcus camorum* LMG

13567

BHI

*Staphylococcus aureus* ATCC 9144

**4. Discussion** 

Table 2. Diameter of inhibition zone (mm) of bacteria growth.

CIP

CIP

LMG

ATCC

LMG

ATCC

(%v/v) obtained by microdilution method.

*Escherichia coli* CIP 105182 *Proteus mirabilis* CIP 104588 *Shigella dysenteria* CIP 5451 Salmonella enterica *CIP 105150*

**Reference strains Origin V. s Amp Te** 

CIP LMG LMG ATCC LMG ATCC CIP CIP CIP CIP

21 8.5 16.5 24.5 19.5 34.5 10.5 11.5 24.5 28

16

8

8

16

16

4

and reconstruction of anatomical and functional disturbances of the skin[Chattopadhyay et al., 2002]. The repair of damaged tissues occurs as a sequence of events that included inflammation, proliferation and migration of different cell types [Sidhu et al., 1999]. At the dose of 0.44 mg used only once the essential oil healed wounds for 96h. Essential oil of *Vitex simplicifolia* accelerated the three stages of cicatrization process: vascular and inflammatory, tissue repair and maturation. While at the dose of 1000 mg used only once, cicatryl exhibited a complete healing with for 7 days against 10 days for the effect of natural immunity of rabbit. In the phase of maturation a renewal of the skin was seen, the old skin started to fall and made way for the new. The essential oil of *Vitex simplicifolia* exhibited a stronger healing effect than cicatryl and natural immunity. This effect could be due to the presence of ketones in the oil that activated the healing process with stimulating of new cell growth, reducing old scare tissue in wound and were highly immunostimulatory [Willem, 2004]. The presence of minor compounds as aldehydes and sesquiterpenes activated anti inflammatory, calming and sedative effects. Thus, their low proportion allowed to consider possible synergistic effects of these compounds in the oil. The significant presence of monoterpenoids in the oil might cause analgesic, antioxidative, antiseptic effects and stimulating the immune system [Mertz et al., 1993].

In other hand, the skin infections are in most cases due to staphylococci with the pathogenic species is Staphylococcus aureus. It is responsible for suppurative infections, widespread and food poisoning. Thus, wound infections are most common in developing countries because of poor sanitation. *Staphylococcus aureus, Shigella dysenteria*, *Salmonella enterica*, *Escherichia coli* are important microorganisms causing an infection of the wound [Mansouri et al., 2011]. The best sensitivity to *Vitex simplicifolia* essential oil was, respectively, obtained on *Staphylococcus aureus* ATCC 9144 BHI (34.5mm), *Salmonella enterica* CIP 105150 (28mm), *Shigella dysenteria* CIP 5451(24.5mm), *Staphylococcus aureus*  ATCC 25293 BHI (24.5mm), *Enterococcus faecalis* CIP 103907 BHI(21mm). Following the results in Table 2 the different strains were less sensitive to V.s than ampicilline, while *shigella dysenteria* CIP 5451 was sensitive to V.s. The most important information was that essential oil exhibited more activity on *Staphylococcus aureus* ATCC 9144 BHI (34.5mm), *Salmonella enterica* CIP 105150 (28mm) *Shigella dysenteria* CIP5451(24.5mm) than tetracycline and ampicilline (*Shigella dysenteria* CIP 5451, 11mm). The essential oil failed to inhibit *Staphylococcus aureus* ATCC 9144 BHI at the lowest MIC 0.25%. The essential oil was bacteriostatic for *Staphylococcus aureus* ATCC 9144 BHI, *Escherichia coli* CIP 105182 and *Listeria innocua* LMG 135668. The most resistant strains with highest MBC (8%) were *Enterococcus faecalis* CIP 103907, *Staphylococcus aureus* ATCC 25293 and *Staphylococcus camorum* LMG 13567. Considering MICs and MBCs no significant difference could be seen between Gram-positive and Gram-negative bacteria. The chemical composition of the oil consisted of various constituents. Therefore, the determination of the component responsible for activity was very difficult. Furthermore the essential oil consists of complex mixture of numerous constituents. Major or minor compounds might cause the bacteriostatic and cicatrization activities exhibited, terpinene-4-ol and other monoterpenes in essential oil may act as antiseptic, anti-inflammatory and antimicrobial: myrcene, sabinene, terpinene, cadinene and limonene [Sinan Dayisoylu et al. 2009]. In addition, the presence of α-pinene, β-pinene [Houghton Peter, 2004] terpinen-4-ol [Lee et al., 2001] and

Wound Healing and Antibacterial Properties

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γ-terpinene[Sonboli et al., 2005] were responsible of antioxidative and antiseptic activities of essential oils studied. However, caryophyllene oxide, E-nerolidol humulene epoxide-1,2 possessed antiinflammatory activity [Chavan et al., 2010; Yu-Tang et al., 2008; Wanjohi Mwangi et al., 2009] Possible synergistic and antagonistic effects compounds in *V. simplicifolia* essential oil should also be taken into consideration. These reports are compatible with our results in the present study.

## **5. Conclusion**

This study shows in *vivo* wound healing activity and in *vitro* bacteriostatic effect of *Vitex simplicifolia* essential oil. The oil demonstrates the strongest wound cicatrization activity than cicatryl and natural immunity. In addition the oil may help to prevent wound infections and others such diarrhoea, dysentery and skin diseases. These results indicate that the plant could be use as a natural potential remedy for healing wounds and antiseptic agent. Further investigations will be performed by determination of analgesic, antioxidant and anti inflammatory activities of the essential oil and to expand to other *Vitex* species.
