Antibacterial Activity of Lyophilized Aqueous Extract of Coriaria Myrtifolia from Northern Morocco

280 | P a g e J U L Y 1 8 , 2 0 1 4 Antibacterial Activity of Lyophilized Aqueous Extract of Coriaria Myrtifolia from Northern Morocco Karima Hezbri, Maha Hafse, Abdellah Farah, Imed Sbissi, Maher Gtari and Kawtar Fikri Benbrahim 1. Laboratory of Microorganisms and Active Biomolecules. Sciences Faculty of Tunis. TUNISIA. 2. Laboratory of Microbial Biotechnology. Faculty of Science and Technology Saiss. Sidi Mohamed Ben Abdellah University. Fez. MOROCCO. 3. Laboratory of Aromatic Plants, Medicinal and Natural Products, National Institute of Medicinal and Aromatic Plants. Sidi Mohamed Ben Abdellah University, Fez. MOROCCO. *Corresponding author:


INTRODUCTION
The infectious diseases induce more than 17 million deaths every year throughout the world from which more than half are registered in Africa [1]. They constitute an important concern of the public health because of their frequency and gravity [2]. During the research for alternative and effective medicine against these pathologies, scientists showed an interest more and more growing for medicinal plants [3,4].
Throughout the world and especially in Morocco, medicinal plants play an important role in therapy, in both urban and rural areas and are an inexhaustible source of new antibacterial agents. Medicinal plants could provide the treatment or prevention of chronic and / or serious infectious diseases and solve the problem of bacterial resistance for current antibacterial agents. To check better the bacterial infections, it is thus urgent to highlight new effective antibacterial agents and it's in this context that this work is considered.
Coriaria myrtifolia is an evergreen shrub, exceptionally in winter, up to 2-3 meters, very common in the Mediterranean region. This plant can be easily adapted to different soil types and climates [9] and can colonize even nitrogen-poor soils because its roots can form nitrogen-fixing nodules [4].
In this study, we evaluated the antibacterial activity of lyophilized aqueous extract of Coriaria myrtifolia against twenty three Gram positive and Gram negative bacterial strains involved in several pathologies and / or implicated in the process of food spoilage.

Preparation of extract
100g of leaves powder was mixed with 2 liters of distilled water and left for 48 hours to prepare maceration. The obtained solution was carefully filtered several times on hydrophilic cotton then on filter paper Whatman n°1. The filtrate was frozen at -30 ° C and lyophilized at -45 ° C with a Telstar lyophilisator type [7]. 400 mg of freeze-dried aqueous extract was dissolved in 1 ml of sterile distilled water and served as a stock solution. A serial dilution ranging from 400 to 25 mg/ml was then realized.

Evaluation of the antibacterial activity of the extract
The screening of antibacterial activity was realized by the disk diffusion method. Whatman paper disks of 6 mm diameter were sterilized and placed on Petri dishes previously inoculated with 100μl of the bacterial strains (10 8 UFC/ml) investigated.
The discs were then impregnated with 10 µl of the lyophilized aqueous extract at different concentrations. A paper disk impregnated with distilled water served as control [8].
All inoculated plates were incubated at 37°C for 24 h, then inhibition zone diameters observed and measured.

Results and Discussion
The study of the antibacterial activity was realized by the disc diffusion technique. Results are expressed by the appearance of inhibition zones around the disks on solid medium (LB). The inhibition zones diameters are presented in Table 2.
The results indicate that the aqueous extract has a dose-dependent antibacterial activity, because the inhibition is proportional to the extract's concentration and that the bacterial sensibility varies from a tested strain to another one.
The extract is active against three species of Bacillus studied: Bacillus cereus B. circulans and B. thuringiensis.
B. circulans with inhibition zones diameters from 20 mm (400mg/ml) to 12.3 mm (25mg/ml) is the most sensitive Bacillus tested strain. While the fourth Bacillus species, B. coagulans was resistant to the same extract.
Salmonella typhimurium (ATCC 14028) and Salmonella Braenderup (H9812) from the same origin, show a different behavior against the extract, the first one is resistant while the second presents an important sensibility. J U L Y 1 8 , 2 0 1 4

Bacterial strains
Several Gram-positive and Gram-negative bacteria from different origins were used (Table 1)  Three strains of E. coli from different origins were used. The extract has an important antibacterial activity against E. coli (DH5α) while it has no effect against E. coli (ACC25922) and E. coli (I2CF1). This suggests that the antibacterial activity of the aqueous extract of C. myrtifolia varies according to the tested strain and that mutation can lead to loose of a strain resistance.
These last observations can let us suggest the existence of bacterial intraspecific variability for the extract's inhibition power. J U L Y 1 8 , 2 0 1 4 The aqueous extract presents no effect against Enterococcus strain tested, which is consistent with a previous study that showed resistance of Enterococcus feacalis against methanol extract of Padina pavonica [10]. However, ethanolic and aqueous extracts of Acacia aroma [11] and methanolic extract of Rhus corriaria are active against this strain [12].
The C. myrtifolia aqueous extract is active against Pseudomonas aeruginosa, Enterobacter hafniya, Listeria monocytogenes, Shigella sp. and Serratia sp. Previous studies showed that these strains were also sensitive towards other plant's extracts [10,13].  Hyper sensibility noticed for Gram + strains can be explained by the presence of a single peptidoglycan layer in their cell wall and the absence of outer double membrane [15]. Moreover, they are easily influenced by the external environmental changes such as temperature and pH [16]. Furthermore, the Gram-negative strains are more resistant due to the nature of their outer membrane, containing lipopolysaccharids and proteins, that is impermeable to most of the biocides [17].

CONCLUSION
Medicinal plants could provide prevention or alternative treatment for chronic and / or severe infectious diseases, and solve the problem of bacterial resistance against existing antibacterial agents.
This work is a contribution to the study of antibacterial activity of lyophilized aqueous extract of Coriaria myrtifolia from the northern Morocco. The study revealed that the extract is active against most tested strains.
The results of this work show that Coriaria myrtifolia can be exploited as an inexhaustible source of new natural antibacterial agents.
Moreover, they open large perspectives to study other Coriaria myrtifolia extracts inhibitory effect and to try to determine the most active bio molecule implied in this antibacterial activity.

AUTHOR'S CONTRIBUTIONS
KH executed the laboratory work in Tunisia. MH executed the laboratory work in Morocco, contributed to plant material collection and drafted the manuscript. AF revised the manuscript. IS contributed to plant material collection and to extract preparation. MG contributed to choose the plant material and provided a part of required chemicals and laboratory consumables. KFB collected the plant, designed the protocol, provided the other part of required chemicals and laboratory consumables, read and substantially revised the manuscript. All authors read and approved the manuscript.