In vitro Study of Antibacterial Activity of Hydro-Alcohol Morrocan Plants Extracts

Context: Several aromatic and medicinal Morrocan plants have been used traditionally in pharmaceutical products and traditional medicine for the treatment of several pathologies. Objective: Evaluation of the protective power of nine Moroccan plants ethanol extracts against some strains of bacteria. Method: The antibacterial activities of ethanolic extracts (EE) were evaluated using agar-well diffusion method, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and IC50 against nine foodborne bacteria [Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus (PN15 and 25923), Escherichia coli (TF2 and ATCC 25929), Pseudomonas aeruginosa (P116 and 195) and Salmonella enterica]. Screening of chemical constituents was carried out as well. Results: We noted the presence of leuco-anthocyanins, anthocyanins, essential oils, alkaloids, and aldehydes in the extracts. The strains of S. enterica followed by S. aureus and P. aeruginosa were the most resistants to the extracts effect. The principal component analysis (APC) demonstrated that the highest antibacterial activity was that of L. nobilis and O. europaea ethanol extract (EE), which was directly bactericidal on all the strains tested with the exception of P. aeruginosa. While, R. tinctorum, S. indicum and L. sativum were characterized by the lowest activity. Statistical analysis: Analysis of variance was performed by uni-varied ANOVA in the software SPSS 22 Fr. Conclusion: The active compounds were soluble in ethanol. The antimicrobial activities of L. nobilis and O. europaea may contribute to understand their involvement in pharmaceutical products and traditional medicine against many microbial infections.


Plant collection and extract preparation
Plants were collected in March, May and Jun 2015 from different region of Morocco (Table 1).The selected parts were dried at 40°C for 15 h.All samples were then ground into a fine powder, which was passed through an 80-mesh sieve.Aqueous extracts were obtained by extraction of samples (30 g) with distilled water (300 ml), for 60 min at 80°C (HAE) or 24 h min at 25°C (CAE).Hydroalcohol extracts were obtained by extraction of samples (20 g) with 200 ml of ethanol solution (70%) for 24 h.The extractions were performed three times.After evaporation, the extracts obtained were autoclaved at 121°C for 15 min and stored at 4°C away from light until use.The extracts yield was determined by the following formula.R: Extract yield (%), P x : Extract weight (g), P y : Plant weight (g).

Qualitative analysis of phytochemicals
Different groups of secondary metabolites such as aldehydes, terpenoids, polyphenols including flavonoids and tannins, alkaloids, saponins and quinone substances were investigated as used by. 5

Evaluation of antibacterial activity
Antibacterial activity was evaluated at Laboratory of Microbiology of hygiene and food safety department of the Institute Pasteur Tangier -Morocco.

Microbial Strains and Growth Conditions
Six different reference strains and food-borne isolates were used for assessing the plant antimicrobial properties; including Gram-positive and Gram-negative bacteria (Table 2).Fresh cultures were prepared by

According to Gram
According to the profile of sensitivity towards antibiotics

Pseudomonas aeruginosa 195
Salmonella enterica ATCC: American type culture collection transferring a loop of cells from the agar slant to a test tube containing 5 ml of brain heart infusion (BHI) (BioRad) and then incubated overnight at 37°C.

Disk Diffusion Assay
Disc-diffusion assay was used to determine growth inhibition caused by plant extracts. 14For each strain, inoculums (10 6 -10 8 CFU per milliliter), was spread on Mueller-Hinton Agar (MHA) (BioRad).Enumeration of bacteria was performed by measuring turbidity at 550 nm (VARIAN Cary 50 UV-Vis).Sterile Whitman's filter discs (N°40; Ø =6 mm), impregnated with 10 µ l of different extracts dilutions from the initial concentration of 50 mg/ml, were deposited on the surface of each petri dish.In parallel, an empty disc and an antibiotic disc were used as a negative and positive control respectively.The petri dishes were kept at 4°C for 15 to 20 min to allow the diffusion of the extract, then incubated at 37°C for 18 to 24 h, under normal atmosphere, after which, inhibition zones around each disc (> 6 mm) were measured (disc diameter included).
Each test was performed in triplicate.

Determination of the minimum inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC) of ethanol extracts was determined by the method of Mann and Markham, (1998), 6 using resazurin as viability indicator.Different dilutions of the extracts (50; 25; 12.5; 6.25; 3.12; 1.56; 0.8; 0.4; 0.2 and 0.1 mg/ml) were prepared from a stock solution (100 mg/ml).To each well containing 50 µ l of the mixture, was added 50 µ l of the bacterial suspension (10 6 to 10 8 CFU/ml) prepared in Mueller-Hinton Broth medium (MHB).Plate was then incubated at 37°C for 18 to 20 h.After the first incubation step, 5 µ l of resazurin (1 mg/ml) was added to each well.Reading results was carried out after further incubation for 2 h at 37°C.The MIC corresponds to the lowest extract concentration, which does not produce change of resazurin staining.Then, the optical density at 550 nm was measured (Epoch BioTek UV-Vis) for IC 50 determination.The following formula was used to calculate the survival germs percentage. 6

S d d
S: survival percentage of germs, di: densimat value of experimental tube before incubation, Di: densimat value control tube before incubation, Df: densimat value after incubation control tube, df: densimat value of experimental tube after incubation.

Determination of the minimal bactericidal concentration (MBC)
Plate counting agar (PCA) (BioRad) was seeded with 10 µ l of samples from plate wells where there was no resazurin color change.Dishes were then incubated for 18 to 20 h at 37°C.The MBC corresponds to the lowest extract concentration that gives no growth.Moreover, the ratio MBC/CMI of each sample was calculated to assess the antibacterial power.

Statistical Analysis
All in vitro experiments were conducted in triplicate and results were expressed as mean ± SD. Analysis of variance was performed by uni-varied ANOVA for determination of phenolic, flavonoid and tannin contents.Statistical analysis of the antibacterial activity was performed by analysis of variance with two factors in the software SPSS 22 Fr.IC 50 value were determined by regression analysis.The values p ≤0.05 were considered significant.

Detection of chemical groups
The chemical groups screening showed the presence of essential oils, saponins, iridoïds, alkaloids, anthocyanins, and aldehydes (Table 3).In general, the distribution of secondary metabolites differs between species.Laurus nobilis and G. roseum have shown the presence of the majority of the screened chemical compounds.The harvest area and other parameters as the pH and its richness in organic matter, influence greatly the production of chemical compounds in the plant. 7lkaloids play an important role in biological structures and well known for their high antibacterial power. 8Antibiotic, antifungal, antiviral activities have been reported about saponins. 9,10While for essential oils, their presence is in general equated with a bacteriostatic effect. 11,12tibacterial activity Among the EEs of the investigated species, only L. nobilis and G. roseum showed a bactericidal effect on all the strains, except those of P. aeruginosa for which the effect was bacteriostatic.In addition, there was a strong significant activity on the solid medium, with a mild to moderate inhibitory effect in the case of B. cereus and L. monocytogenes in L. nobilis; P. aeruginosa (S and R) and L. monocytogenes in G. roseum; B. cereus and S. aureus (R) in N. sativa.The other species showed a bacteriostatic effect with high MIC and MBC values (Table 4).
The IC 50 analysis by the tukey test showed a strong antibacterial effect in O. europaea which, on the other hand, has no inhibitory effect on  to Essawi et srour (2000), 19 the seeds of N. sativa have not demonstrated antibacterial activity.Ogunsola., (2014) 20 tested the antibacterial activity of aqueous and ethanolic extract of S. indicum seeds and as in our case, the aqueous extract was inactive on the bacteria tested ; the antibacterial activity of these species is may be in their aerial parts.According to, 21 the effect of infusion and decoction of L. nobilis on 6 strains was inactive on all strains tested, which is in contrast with our results where L. nobilis EE was active on S. aureus and on B. cereus.In a recent study, L. nobilis EE was highly active against Gram + and Gram-bacteria. 22While, the study. 23showed that EE of the same specie has a very low antibacterial activity against E. coli and P. aeruginosa (MIC = 100 mg/ml) in comparison with previous study.The antibacterial potential of L. nobilis was attributed to its constitutional richness in terpenoids, glycosides, anthocyanins and essential oils. 24n general, results revealed variable responses according to the strains and their resistance, the type of the extract and its concentration, which was in agreement with the results of. 25The difference in the action between these EEs is probably due to the difference in the chemical composition, the nature and composition of the microorganism's membrane and the influence of the reaction medium. 26,27everal classes of polyphenols such as tannins and flavonoids such as epigallocatechin, catechin, myricetin, quercetin, 15 luteolin and flavanones, 28 are very active antibacterial substances.Their absence of an extract could justify its weak activity.
In addition, recent results have shown that saponins are the most remarkable antibacterial compounds compared to polyphenols and flavonoids.Alkaloids, in turn, are recognized for their high antibacterial potency. 29hese alkaloids concentrated in our EEs could be partly responsible for the antibacterial activity obtained.Oxygenated terpenes and especially terpene alcohols are also very active antimicrobial agents. 30

CONCLUSION
The antimicrobial activity evaluation of the hydro-ethanolic extracts of nine plant species showed the presence of a moderate activity in all the investigated species.The best effect was noticed in L. nobilis.The most sensitive strains were S. aureus and B. cereus with a dose-response relationship, while the most resistant were P. aeruginosa and E. coli (R).
Comparison of our results with those of the literature showed that the antibacterial activity of the plant extracts was very variable depending on the phytochemical composition of the plant, the solvents used for the extraction, and the bacteria tested.
The susceptibility of germs to EEs may justify their use in the traditional treatment of some microbial diseases in different regions of Morocco.These plants seem to have a broad spectrum of antibacterial activity.As a result, these extracts would present major targets, safe and effective in antibacterial therapy and for the preservation of food, and can be used in antiseptic and disinfectant formulations, as well as in chemotherapy.

Figure 1 :
Figure 1: The IC 50 means representation of EEs of the nine plant species for the nine strains studied.S : Sensitive ; R : Resistant.

Figure 2 :
Figure 2: Projection of different variables of the Principal Component Analysis (PCA) on the factorial graph.S : Sensitive ; R : Resistant.

Table 5 : Study of the antibacterial effect by comparison of IC 50 of EEs, between the nine plant species.
Groups with the same letters do not differ significantly by tukey test.Er.Std: standard error; R: Resistant ; S : Sensitive ; **: Very highly significant difference.