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

Btissam, Fatima, and Mohamed: In vitro Study of Antibacterial Activity of Hydro-Alcohol Morrocan Plants Extracts

Authors

INTRODUCTION

In Morocco, as for developing countries, most people, especially in rural areas, use medicinal plants to treat infectious diseases.1 These infectious diseases are a major cause of morbidity and mortality worldwide, but especially in developing countries.2 This situation is aggravated by the high cost of available medicines and the growing number of resistant pathogenic micro-organisms. Few new classes of anti-bacterials are released when older classes lose their effectiveness; as a result, antibiotic resistance has become a growing public health problem. Therefore, the discovery and development of new antimicrobial agents is of crucial priority.3 Thus, our goal is to study the antibacterial properties of crude ethanolic extracts of some Morrocan plant species belonging to different families.

MATERIALS AND METHODS

Chemicals

All reagents (PCA, MH, Agarose, Resazurin, Ethanol, Folin–Ciocalteau reagent, Folin–Denis reagentsodium carbonate, Gallic acid, potassium acetate, Aluminum trichloride, Quercetin), unless otherwise stated, were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

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). Hydro-alcohol 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.4

R=1+PxPY×100
Table 1

Plant species description.

Botanical nameVernacular nameFamilyOriginHarvest periodPart usedWild / cultivated
Geranium roseumLaatarchaGeraniaceaeKhmiss anjraFebruaryLeafed stemsWild
Aloysia triphylla CavLouizaVerbenaceaeKhmiss anjraFebruaryLeavesWild
Laurus nobilisAwrak seydna moussaLauraceaeKhmiss anjraMarchLeavesWild
Lepidium sativumHabb rchadBrassicaceaeMarrakechJulySeedsCultivated
Nigella sativa .LChanoujRanunculaceaeAghraisseAugustSeedsCultivated
Olea europaea. LZaytûnOleaceaeAîn BaydaAprilLeavesWild
Rubia tinctorum LFewwaRubiaceaeMeknèsMarchRootsCultivated
Sesamum indicum. LZenjlanePedaliaceaeBeni MellalNovemberSeedsCultivated
Trigonella foenum graecumHalbaFabaceaeGharbJuneSeedsCultivated
Table 2

The bacterial strains used.

Bacterial strainsCharacteristics of bacteria

According to the requirementAccording to GramAccording to the profile of sensitivity towards antibiotics
Staphylococcus aureus (PN15)  Food isolates
Staphylococcus aureus 25923 Positive 
Listeria monocytogenes 4032   
Bacillus cereus ATCC 14579  Reference strains
Escherichia coli ATCC 25929Non-demanding bacteria  
Escherichia coli (TF2)   
Pseudomonas aeruginosa (P116) Negative 
Pseudomonas aeruginosa 195  Food isolates
Salmonella enterica   

ATCC: American type culture collection

Table 3

Détection qualitative des groupes chimiques dans les extraits éthanoliques bruts.

SpeciesAlcaloïdsLeucoanthocyaninsIridoïdsSaponinsAnthraquinonsAnthocyaninsDeoxyosesAldehydsEssential oils
G. roseum-+++-++++++
A. citriodora--------+
L. nobilis+++++++++-++
L. sativum-+---+-+-
N. sativa+--------
O. europaea++++--+-++++
R. tinctorum+---+++++
S. indicum+--+---++-
T. foenum graecum+++++----++
Total (N)6/95/93/94/92/95/92/95/96/9
Total (%)665533442255225566

+++ : Very abundant; ++ : abundant; + Presence of the metabolite; - Absence of the metabolite

R: Extract yield (%), Px: Extract weight (g), Py: 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 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.

Table 4

The antimicrobial activity of Ees.

Plant speciesBacterial strainsØ of the inhibition zone for 50 mg/mlof extract (mm)Qualitative decisionMIC (mg/ml)MBC (mg/ml)Report MBC/MICDecision according to Oursou et al., (2008)
Laurus nobilis (Awrak seydna moussa)E. coli6No inhibitory25502Bactericidal
TF 26No inhibitory501002Bactericidal
S. aureus9No inhibitory1,563,122Bactericidal
PN 156No inhibitory3,126,252Bactericidal
L. monocytogenes11±0.22*Slight inhibitory6,2512,52Bactericidal
P 1165No inhibitory50>100>2Bacteriostatic
P. aeruginosa5No inhibitory100>100>1Bacteriostatic
B. cereus15±0.13*Slight inhibitory0.50.51Bactericidal
S. enterica6No inhibitory6.256.251Bactericidal
Lepidium sativum (Habb Rchad)E. coli6No inhibitory25>100>4Bacteriostatic
TF 26No inhibitory50>100>2Bacteriostatic
S. aureus6No inhibitory251004Bacteriostatic
PN 156No inhibitory50>100>2Bacteriostatic
L. monocytogenes6No inhibitory50>100>2Bacteriostatic
P 1166No inhibitory50>100>2Bacteriostatic
P. aeruginosa6No inhibitory50>100>2Bacteriostatic
B. cereus6No inhibitory251004Bacteriostatic
S. enterica6No inhibitory100>100>1Bacteriostatic
Nigella sativa. L (Chanouj)E. coli6No inhibitory25>100>4Bacteriostatic
TF 26No inhibitory50>100>2Bacteriostatic
S. aureus9No inhibitory12,512,51Bactericidal
PN 1512±0.15*Slight inhibitory25501Bactericidal
L. monocytogenes6No inhibitory251004Bacteriostatic
P 1166No inhibitory100>100>1Bacteriostatic
P. aeruginosa6No inhibitory>100>100>1Bacteriostatic
B. cereus17±0.23**Moderate inhibitory6,2512.52Bactericidal
S. enterica6No inhibitory501002Bactericidal
Olea. europaea.L (Zaytûn)E. coli6No inhibitory251004Bacteriostatic
TF 26No inhibitory251004Bacteriostatic
S. aureus6No inhibitory100>100>1Bacteriostatic
PN 156No inhibitory>100>100>2Bacteriostatic
L. monocytogenes6No inhibitory251004Bacteriostatic
P 1166No inhibitory251004Bacteriostatic
P. aeruginosa6No inhibitory25>100>4Bacteriostatic
B. cereus8±0.2No inhibitory100>1001Bacteriostatic
S. enterica6No inhibitory100>100>1Bacteriostatic
Rubia tinctorum.L (Fewwa)E. coli6No inhibitory251004Bacteriostatic
TF 26No inhibitory50>100>4Bacteriostatic
S. aureus6No inhibitory6,2512,52Bactericidal
PN 156No inhibitory12,51004Bactericidal
L. monocytogenes6No inhibitory6,2512,58Bacteriostatic
P 1166No inhibitory100>100>1Bacteriostatic
P. aeruginosa6No inhibitory>100>100>1Bacteriostatic
B. cereus9No inhibitory25251Bactericidal
S. enterica6No inhibitory12,5502Bactericidal
Sesamum indicum.L (Zenjlane)E. coli6No inhibitory50>100>2Bacteriostatic
TF 26No inhibitory100>100>1Bacteriostatic
S. aureus6No inhibitory50>100>2Bacteriostatic
PN 156No inhibitory50>100>2Bacteriostatic
L. monocytogenes7No inhibitory251004Bacteriostatic
P 1166No inhibitory50>100>2Bacteriostatic
P. aeruginosa6No inhibitory100>100>1Bacteriostatic
B. cereus6No inhibitory50>100>2Bacteriostatic
S. enterica6No inhibitory100>100>1Bacteriostatic
Trigonella. foenum grecum (Halba)E. coli6No inhibitory50>100>2Bacteriostatic
TF 26No inhibitory100>100>1Bacteriostatic
S. aureus6No inhibitory50>100>2Bacteriostatic
PN 156No inhibitory100>100>1Bacteriostatic
L. monocytogenes6No inhibitory251004Bacteriostatic
P 1166No inhibitory100>100>1Bacteriostatic
P. aeruginosa6No inhibitory>100>100>1Bacteriostatic
B. cereus6No inhibitory251004Bacteriostatic
S. enterica6No inhibitory50>100>1Bacteriostatic
Géranium roseum (Laatarcha)E. coli6No inhibitory25502Bactericidal
TF 26No inhibitory501002Bactericidal
S. aureus6No inhibitory25502Bactericidal
PN 156No inhibitory25502Bactericidal
L. monocytogenes10±0.31*No inhibitory12,5252Bactericidal
P 11614±0.21**Slight inhibitory12,51008Bacteriostatic
P. aeruginosa8±0.11*No inhibitory251004Bacteriostatic
B. cereus7±0.5No inhibitory25502Bactericidal
S. enterica6No inhibitory25502Bactericidal
Aloysia triphylla Cav (Louiza)E. coli6No inhibitory100>100>1Bacteriostatic
TF 26No inhibitory>100>100>1Bacteriostatic
S. aureus6No inhibitory25>100>4Bacteriostatic
PN 156No inhibitory50>100>2Bacteriostatic
L. monocytogenes6No inhibitory25>100>4Bacteriostatic
P 1167±0.12No inhibitory25>100>4Bacteriostatic
P. aeruginosa7±0.09No inhibitory50>100>2Bacteriostatic
B. cereus8±0.15*No inhibitory50>100>2Bacteriostatic
S. enterica6No inhibitory50>100>2Bacteriostatic

Disk Diffusion Assay

Disc-diffusion assay was used to determine growth inhibition caused by plant extracts.14 For each strain, inoculums (106108 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 (106 to 108 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 IC50 determination. The following formula was used to calculate the survival germs percentage.6

S=dfd1DfD1×100

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.

Table 5

Study of the antibacterial effect by comparison of IC50 of EEs, between the nine plant species.

 Mean Ps (S) ± Er.StdMean Ps (R) ± Er.StdMean EC (S) ± Er.StdMean EC (R) ± Er.StdMean SA (S) ± Er.StdMean SA (R) ± Er.StdMean Sal (S) ± Er.StdMean Bac (S) ± Er.StdMean Lis (S) ± Er.Std
G. roseum19,47±0,28 (c)68,58±0,21 (d)27,61±0,27 (f)33,98±0,45 (e)29,71±0,33 (b)29,55±0,44 (a)10,28±0,17 (a)5,86±0,24 (a) (b)14,90±0,19 (e)
A. citrodora15,24±0,23 (b)23,50±0,34 (b)22,15±0,29 (e)24,54±0,21 (d)58,39±0,22 (e)131,45±0,29 (g)121,41±0,34 (f)17,56±0,31 (d)13,26±0,25 (d)
L. nobilis16,20±0,18 (b)42,74±0,24 (c)7,44±0,13 (b)19,23±0,29 (b)39,12±0,33 (c)42,44±0,13 (b)26,62±0,29 (b)7,3±0,18 (b)23,29±0,22 (f)
L. sativum102,42±0,36 (e)246,86±0,78 (g)11,31±0,30 (c)21,79±0,35 (c)686,78±0,31 (i)1536,27±0,79 (i)1334,008±0,96 (h)66,97±0,64 (g)126,64±0,31 (i)
N. sativa115,42±0,68 (f)435,13±0,71 (h)20,62±0,39 (d)34,49±0,43 (e)91,76±0,58 (g)116,20±0,82 (f)28,34±0,55 (b)24,27±0,25 (f)29,84±0,34 (g)
O. europaea5,51±0,15 (a)4,72±0,27 (a)3,38±0,22 (a)10,75±0,19 (a)23,17±0,25 (a)61,52±0,27 (d)38,92±0,30 (c)5,37±0,19 (a)1,47±0,16 (a)
R. tinctorum103,36±0,42 (e)122,66±0,24 (f)117,47±0,47 (h)131,99±0,56 (h)41,68±0,25 (d)54,05±0,46 (c)48,25±0,29 (d)20,49±0,28 (e)11,54±0,28 (c)
S. indicum214,25±0,17 (g)857,21±0,30 (i)27,57±0,30 (f)63,45±0,19 (f)463,91±0,33 (h)1213,65±0,55 (h)435,94±0,45 (g)12,03±0,43 (c)8,97±0,18 (b)
T. foenum graecum78,34±0,25 (d)82,46±0,41 (e)65,29±0,32 (g)82,11±0,34 (g)81,69±0,34 (f)104,06±0,24 (e)63,50±0,20 (e)65,54±0,28 (g)48,54±0,20 (h)
TOTAL74,47±6,84202,04±27,1433,65±3,6246,92±3,91168,47±23,82365,46±57,86234,16±34,2825,04±2,4230,94±3,84
FISHER39228,86 (p<0,000) **399567,45 (p<0,000) **13166,2 (p<0,000) **11791,48 (p<0,000) **471130,42 (p<0,000) **1325772,26 (p<0,000) **904313,22 (p<0,000) **5000,34 (p<0,000) **24019,31 (p<0,000) **

Groups with the same letters do not differ significantly by tukey test. Er.Std: standard error; R: Resistant; S : Sensitive;

** Very highly significant difference.

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. IC50 value were determined by regression analysis. The values p ≤0.05 were considered significant.

RESULTS AND DISCUSSION

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.7

Alkaloids play an important role in biological structures and well known for their high antibacterial power.8 Antibiotic, antifungal, antiviral activities have been reported about saponins.9,10 While for essential oils, their presence is in general equated with a bacteriostatic effect.11,12

Antibacterial 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 IC50 analysis by the tukey test showed a strong antibacterial effect in O. europaea which, on the other hand, has no inhibitory effect on solid medium. The extract of this specie has a bacteriostatic effect on all strains, except for S. aureus (R) and S. enterica which were more susceptible to G. roseum effect. The highest values were those of S. indicum in the case of P. aeruginosa (S and R), R. tinctorum in the case of E. coli (S and R) and L. sativum in the case of other strains (Table 5).

Table 6

Comparison of two to two means of the EEs extracts on the different bacterial strains.

Paired samplest studentSig.

P. aeruginosa (S)P. aeruginosa (R)6,22p<0,000 **
E. coli (S)E. coli (R)13,66p<0,000 **
S. aureus (S)S. aureus (R)5,74p<0,000 **
P. aeruginosa (S)E. coli (S)5,94p<0,000 **
E. coli (R)4,48p<0,000 **
S. aureus (S)4,62p<0,000 **
S. aureus (R)5,41p<0,000 **
S. enterica (S)3,87p<0,000 **
B. cereus (S)7,39p<0,000 **
L. monocytogenes (S)5,98p<0,000 **
P. aeruginosa (R)E. coli (R)6,2p<0,000 **
S. aureus (S)5,9p<0,000 **
S. aureus (R)1,7p<0,091
S. enterica (S)3,39p<0,0001**
B. cereus (S)0,56p<0,572
L. monocytogenes (S)6,59p<0,000 **
E. coli (S)S. aureus (S)6,36p<0,000 **
S. aureus (R)5,41p<0,000 **
S. enterica (S)5,63p<0,000 **
B. cereus (S)4,52p<0,000 **
L. monocytogenes (S)2,16p<0,032
E. coli (R)S. aureus (S)0,47p<0,633
S. aureus (R)4,95p<0,000 **
S. enterica (S)5,45p<0,000 **
B. cereus (S)4,23p<0,000 **
L. monocytogenes (S)5,19p<0,000 **
S. aureus (S)S. aureus (R)2,7p<0,008 **
S. enterica (S)2,97p<0,003 **
B. cereus (S)6,32p<0,000 **
L. monocytogenes (S)6,49p<0,000 **
S. aureus (R)S. enterica (S)5,24p<0,000 **
B. cereus (S)5,98p<0,000 **
L. monocytogenes (S)6,03p<0,000 **
S. enterica (S)B. cereus (S)4,99p<0,000 **
L. monocytogenes (S)5,07p<0,000 **
B. cereus (S)L. monocytogenes (S)2,62p<0,01 *

In general, the lowest values of antibacterial parameters were obtained with B. cereus, S. aureus followed by L. monocytogenes and were therefore the most sensitive strains. However, strains of P. aeruginosa, E. coli and S. enterica remain the most resistant to the effect of extracts, with high MIC and IC50 values. This corroborates with the results of Sqalli et al., (2008).13

In fact, some studies do not reveal any selective antimicrobial activity against Gram (+) or Gram (-) bacteria.14 On the other hand, other studies have highlighted the high sensitivity of Gram (+) bacteria compared to Gram (-).15,16 This can be attributed to the difference in the outer layers of Gram (-) and Gram (+) bacteria.

By the tukey test, it appeared that the difference was highly significant in most cases. The graphical IC50 means representation of these nine species EEs showed a remarkable susceptibility of B. cereus, followed by L. monocytogenes, which showed a non-significant difference to E. coli (S) (Table 5). Comparing IC50 of the other bacterial strains, paired two by two, showed a highly significant difference (p <0.000). Also, for a threshold α = 5%, the Fisher Table provided large critical values, which means that the significant difference observed between IC50 means depended on the species used.

According to the graph of Figure 1, S. aureus (R) followed by S. enterica and P. aeruginosa (p> 0.05), showed a marked resistance to the effect of EEs used. Also, resistant strains had significantly higher IC50 than sensitive ones. However, there were high standard errors in the case of S. aureus, P. aeruginosa (R) and S. enterica, which means that there was a significant difference in the survival of these strains from one specie to the other. This can be explained by the difference in the phytochemical composition of each specie and the concentration in these compounds, since they belong to different plant families.

Analysis of the total variance showed that the percentage of inertia around axis 2 was 77.09% (Table 7). Projection of variables and active species of the PCA on the factorial graph showed the strains distribution into three groups. Groups 1 and 2 were on the positive side and strongly characterized G. roseum, N. sativa and A. citrodora. The species O. europaea and L. nobilis acted on strains of group 1, 2 and 3 at the same time.

The species S. indicum, L. sativum, R. tinctorum followed by T. foenum graecum remained the plants with the lowest growth inhibitory activity of all the bacteria tested (Figure 2).

Comparison of the numerical values of our study with other publications is often qualitative, since the authors express their results with different units making the quantitative comparison difficult.17 Qualitatively, our results were correlated with those of literature.

Kroum., (2009).18 study showed that methanolic and aqueous extracts of T. foenum graecum seeds were not good antibacterial agents. According 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.

Table 7

Principal component analysis of the total variance explained.

ComponentInitial valuesSum of factors squares selected for rotation

Total% of variance% cumulatedTotal% of variance% cumulated
14,5750,8250,824,5750,7750,77
22,3626,2677,092,3626,3177,09

Extraction method: Principal component analysis.

Figure 1

The IC50 means representation of EEs of the nine plant species for the nine strains studied. S : Sensitive ; R : Resistant.

PharmacognJ-10-519-526_g001.jpg
Figure 2

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

PharmacognJ-10-519-526_g002.jpg

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.22 While, the study.23 showed 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.24

In 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.25 The 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.26,27

Several 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.29 These 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.

GRAPHICAL ABSTRACT

PharmacognJ-10-519-526_g003.jpg

SUMMARY

  • Ethanol extracts of the nine plant species were rich of leuco-anthocyanins, anthocyanins, essential oils, alkaloids, and aldehydes

  • The ethanol extract of L. nobilis and O. europaea was directly bactericidal on all the strains tested with the exception of P. aeruginosa.

  • The principal component analysis demonstrated that L. nobilis and O. europaea had the highest antibacterial activity. While, R. tinctorum, S. indicum and L. sativum were characterized by the lowest activity.

ACKNOWLEDGEMENT

We thank Dr. Bendahou Abdelrazak (Department of hygiene and food safety, Pasteur Institute of Morocco) for providing Bacillus and Salmonella strains.

Notes

[6] Conflicts of interest CONFLICT OF INTEREST The authors declare no conflict of interest.

ABBREVIATIONS USED

EE

Ethanolic Extract

PCA

Principal component analysis

MIC

minimum inhibitory concentration

MBC

minimum bactericidal concentration

BHI

brain heart infusion

MH

Mueller-Hinton

PCA

Plate Count Agar

MAPs

medicinal and aromatic plants

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