COMPARATIVE STUDY OF IN VITRO ANTIMICROBIAL ACTIVITIES OF FOENICULUM VULGARE MILL. (UMBELLIFERAE) EXTRACT

The importance to push scientifically the investiga tions on the organic extracts of the plants aromati c s potential source of new antimicrobial compounds com es from the traditional use of the plants. However, the consumption of these natural products requires à th orough research in this field. The antimicrobial ef fect of organic and aqueous leaves extracts of F eniculum vulgare Mill., However, which makes difficult this antimicrobial activity, is the insolubility of orga nic extracts in water. The standard M27-T technique is basically used to cure this problem. The microorgan isms under examination were Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus hirea, Escherichia coli and Candida albicans. The M27-T technique allowed us to determine the Minimum Inhib itory Concentrations (MICs) of different extracts. Therefore, the test’s results showed that the all s amples were clearly different in terms of antimicro bial activities. All extracts of Foeniculum vulgare showed the most activity on all the microorganisms tested. The most significant and active extract under study were methanol and ethyl acetate on all the bacteri a tested in comparaison to the hexane and aqueous ext racts. On the other hand, the results of antimicrob al activity of aqueous extract were more compelling th an the hexane and dichloromethane extracts when use d on Candida albicans (ATCC and CBS) (MIC = 0,78 mg mL ). It then appear that C. albicans ATCC is the least susceptible microorganisms to the ethyl aceta te extract. The chloramphenicol, amoxicillin and amphotericin B were used as standard antibiotics to arry this study.


INTRODUCTION
Foeniculum vulgare Mill., which is commonly known as fennel, belongs to the Apiaceae (Umbelliferae) family. It is an annual, biennial or perennial herbs and typical aromatic plant that grows in several regions all over the world. It is growing to a height ranging from 70 to 200 cm. it grows wild in most regions, especially the west and south regions of turkey.
Fennel, which is largely planted in temperate and tropical regions of the world, is extensively used for medicine purposes and as a culinary spice (Tanira et al., 1996;Beaux et al., 1997;Patra et al., 2002;Barros et al., 2010).
The current study was meant to assess the antimicrobial properties of the extracts of Foeniculum by using the standard M27-T broth microdilution technique of the National Committee for Clinical Laboratory Standards (NCCLS) (Barchiesi et al., 1994;Espinel-Ingroff et al., 1995;Bouamama et al., 2006).
We are more interested here on the results of antimicrobial testing of Foeniculum vulgare extracts against Escherichia coli, Enterococcus hirea, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans.

Plant Material
Leaves of Foeniculum vulgare Mill. were collected in march 2009 from Ouaouizerth, the area of Azilal (Morocco). Specimens were botanically identified at the laboratory of vegetable ecology in the department of Biology, University Cadi Ayyad, Faculty of Sciences Semlalia, of Marrakesh, Morocco.

Extracts Preparations
The extract preparation involves taking dried and finely powdered leaves (100 g) of the fennel and then extracting it with methanol, using Soxhlet apparatus for 48h. This methanol extract is filtered and then evaporated. The process in question is carried out through an rotary evaporator adjusted under a temperature of 45°C. Yielding 24 g (24%). Both extracts were redissolved in distilled water and successively extracted with hexane, dichloromethane and ethyl acetate. Each organic extract was then evaporated to dryness and labeled as indicated in Table 1.
Afterwards, we prepare approximately 100 mg mL −1 of each extract solution in distilled water. The pH was adjusted between 5 and 7. Extracts were sterilized over a membrane filter unit of 0.2 µm of pore size and preserved at +4°C until used.

Antifungal Activity
The same M27-T method, which was described by Bouamama et al. (2006), was used again with some modification in terms of culture medium and the concentrations of the extract and of the Amphotericin B. used the same method M27-T described by Bouamama with some modification on the culture medium and the concentrations of the extract and of the Amphotericin B. The culture medium is the Sabouraud dextrose broth (BIOKAR) buffered to pH 7. Amphotericin B was used as standard antibiotic with concentrations ranging from 25 to 0,024 µg mL −1 . The antifungal concentrations ranged from 100 to 0.09 mg mL −1 . After inoculation, the plates were incubated for 24, 48 and 72 h. at 35 °C.

Antibacterial Activity
We used the same method M27-T for the antibacterial test. The culture medium was the Mueller-Hinton broth. Amoxicillin and chloramphenicol (Sigma) were used as standards. Concentrations ranged from 320 to 0.31 µg mL −1 for amoxicillin and for chloramphenicol. After inoculation, the plates were incubated at 37°C for 24 and 48 h.

Inoculum Preparation
Yeast colonies, which obtained from 24h cultures, were taken to another environment called Sabouraud dextrose broth at 35°C under agitation. The first suspensions were used for the inocula preparation in the same culture medium. Thomas hematimeter was utilised to determine the concentrations of yeast cells. The concentrations of yeast cells were determined using a Thomas's hematimeter. Budding organisms were counted as two. The original concentrations were adjusted to a concentration of 10 4 -10 5 colony forming units (CFU)/ml with broth used in the susceptibility test.
Stock bacterial inocula suspensions were obtained from 18 h culture on Mueller-Hinton broth at 37°C. The suspensions finally yielded serve as the inocula preparation. The cell density of each suspension was determined using a Thomas hematimeter and then adjusted to a concentration of 10 5 -10 6 CFU mL −1 by dilution with Mueller-Hinton broth.

Readings and Control
The experiments were repeated three times and the results were determined as an average value. The result readings were made visually. The MIC was considered as the lowest drug concentration of antifungal or antibacterial agent inhibiting the total growth of microorganisms. MIC was detected by lack of visual turbidity (matching the negative growth control). Subcultures were made from the clear wells which did not show any growth after incubation during the MIC assays on nutritive agar with 2% for bacteria and Potato Dextrose Agar (PDA) (BIOKAR) for fungi.

RESULTS
The Table 1 shows that the extracts tests had an antimicrobial activity. This activity depends on the nature of the extract, there concentration and bacterial or fungal strain. These results are indeed in line with the previous analysis of the leaves extracts of Foeniculum vulgare Mill., which possessed antimicrobial properties with MIC values ranging from 0.78 to 6.25 mg mL −1 for bacteria and 0.39 to 25 mg mL −1 for fungi.
Extract Regarding the fungi studied, we showed that all tested extracts are inhibitory effects on Candida with CMI which vary from 0.39 to 6,25 mg mL −1 . F. vulgare extracts have the most compelling inhibition activities against Candida. The species Candida CBS is strongly inhibited by P4 and P5 (MIC = 0,78 mg mL −1 ) and P1 (MIC = 0, 39 mg mL −1 ). C. albicans CBS and ATCC are the least sensitive strain to the F. vulgare extracts P2 and P3 (MIC = 6, 25 mg mL −1 ).

DISCUSSION
For many age, medicinal plants have been used to cure diseases. Herbal medicines have increasingly been used to treat effectively infections that are difficult to manage. We are fully aware that plants perfectly produce certain natural chemicals that are toxic to bacteria. A large body of literature has validated the antimicrobial activity of plant extracts (Basile et al., 1999;Pesewu et al., 2008;Babri et al., 2012).

Antibacterial Activity
The results indicated that all extracts have antibacterial activity against examined Gram negative and Gram positive bacteria. Compared with other studies, our data show better antimicrobial activities for Foeniculum species. While observing the therapeutic activity against most of the pathogenic bacteria, gram-positive bacteria were more sensitive to extract than gram-negative bacteria. Gnan and Demello (1999) reported an antimicrobial activity of Goiaba leaves extract at a concentration of 6, 5 mg mL −1 against Staphylococcus aureus. Chandrasekaran and Venkatesalu (2004) reported that MIC values of Syzygium jambolanum seed extracts ranged between 0,0031 and 0,5 mg m mL −1 against E. coli, S.aureus, P. aeruginosa and C. albicans. Tanis et al. (2009) reported that chloroform and methanol extract of N. arvensis and N. unguicularis were the least effective against the microorganisms. These data coincide with those of Okoli and Iroegbu (2004), who reported that water and methanol extracts of some plants displayed a significant antimicrobial activities. Similarly, Basri and Fan (2005) had reported that the aqueous and acetone extracts of galls of Quercus infectoria (Oak) displayed similarities in antimicrobial activity on the bacterial species and as such, it is the potentially source of antimicrobials.

Antifungal Activity
Compared to Amphotericin B, organic extracts of fennel leaves (as shown on the table above) have an effective antifungal activity. these results are perfectly in accordance with those of Zahid et al. (2012) who had reported that the aqueous extract of fennel (Foeniculum vulgare Mill.) had potential antifungal activity against three soil borne: fungi namely: Macrophomina phaseoli, Rhizocotina Solani and Fusarium moniliforme.

CONCLUSION
To sum up, we basically use the standard M27-T technique to survey the organic and aqueous leaves extracts of Foeniculum vulgare Mill. (Apiaceae) used in traditional medicine, for their antimicrobial properties. Our study has emphasized the fact that the antimicrobial activities are much more significant at Foeniculum species. The leaves extracts of F. vulgare species displayed the inhibitory activities against some of the microorganisms involved in many infections and skin diseases.
Although fungi have been the main source of antibiotics up to now, the discovery of new plant products with potential antimicrobial application is of considerable interest in view of the increasing antibiotic resistance to many microorganisms.
This study enables us for further attention of a phytochimical analysis of different extracted and research to identify the active compounds responsible for the biological activity of this plant. More studies t conducted to explicate the exact mechanism of action by which extracts exert their antimicrobial effect.