Antibacterial effects of Thymus algeriensis extracts on some pathogenic bacteria

. Despite the presence of many antibiotics, bacterial resistance is growing steadily and some of these antibiotics have become ineffective, which poses a major challenge to the health sector. In this context, this work has demonstrated, in vitro , the inhibitory action of the bacterial growth resulting from methanolic and ethanolic extracts of Thymus algeriensis Boiss. & Reut. , a medicinal plant species harvested from the Algerian South-west area, as well as the determination of the phenolic content of those crude extracts. The methanolic extract of Thymus algeriensis showed a significant antibacterial effect with 16.5 and 19 mm against Pseudomonas aeruginosa and Staphylococcus aureus, respectively. Klebsiella pneumoniae was not inhibited by both tested extracts. Besides, ethanol extract has not prevented the growth of the Enterobacter cloacae. This biological activity can be explained by the appreciable rates noted for both of the plant extracts in terms of total phenolic levels, which ranged between 79.45 and 67.13 mg GAE g -1 dry weight.


Introduction
All over the world and for many centuries, the herbaceous plants are an integral component of everyday life and culture.These plants were used in pharmaceuticals, cosmetics and in food technology as antioxidants.Medicinal plants and herbal medicines form an important part of the treatment in the indigenous medicine systems.A wide variety of modern drugs have been used for treating illnesses including constipation and cancer, which were derived from the plant kingdom.This is why many countries are actively engaged in bio-mining Medicinal plants for therapeutically precious and biologically active phytochemicals (Kumar & Jnanesha, 2016).
The Thymus sp.(Thyme) are small permanent therapeutic botanical herbs native of the Mediterranean basin, south of Italy, and Asia.They belong to the Lamiaceae family, which is one of the largest families among flowering plants, practically, with almost a range of 220 genera and 4000 species in the world (Ameen, 2013, Colpaert 2006, Javed, Erum, Tabassum, & Nikolić et al., 2014).
The thyme has also become one of the most important medicinal plants used for food purposes as a spice for its taste qualities.Thymus algeriensis Boiss.& Reut.(Synonym Thymus hirtus Willd.subsp.algeriensis) is an aromatic plant under the common name of "Mazoukcha".This species is the most widespread North African species.It is characterized by curved stems and white or pink flowers (see Figure 1), essentially used in Algeria both as a popular herb and as a spicy herb.(Hazzit & Baaliouamer, 2007) (Jayari et al., 2018).
Acta Scientiarum.Biological Sciences, v. 41, e48548, 2019 In order to detect new sources of antibacterial agents, we report, in this study, the results of antibacterial effects using the crude extracts obtained.Non volatile hydrophilic fractions were extracted by maceration of Thymus algeriensis aerial parts with two common solvents, ethanol, and methanol.
Therefore, the objectives of this study were (a) to determine the effect of different solvents (methanol and ethanol) on extraction by means of measuring the total phenolic content (TPC), the total flavonoid content (TFC) and the total anthocyanin content (TAC); and (b) to evaluate the antibacterial properties of T. algeriensis.
Plant species was identified by the Laboratory of Biotoxicology, Pharmacognosy and Biological Valuation of Plants, University Dr. Tahar Moulay Saida, Algeria, as target species of interest.Plant material stripped and air-dried at room temperature (not faced to direct sunlight).
The voucher specimen has been deposited at the Herbarium of the University Dr. Tahar Moulay; Saida, Algeria.(Code.T.A.B.R-2016).
Two solvents were used to extract polar fractions of the Thymus algeriensis aerial parts by maceration method.50 g of sample (powdered) soaked under frequent agitation, in either 500 mL of 100% methanol or 500 mL of 100% ethanol.After they allowed standing at room temperature for a period of 3 days, extracts were filtered, concentrated and stored at 4°C until later analysis.(Azwanida, 2015)

Phytochemical screening
Extract yield (%) was determined as described in Yihune and Yemata (2019), as follows: Then, to determine the total phenolic content, 200 μL of the sample extract was mixed with 1 mL of Folin-Ciocalteu's reagent.After 4 min., 800 μL of 7.5% Na2CO3 solution was added, and the mixture was incubated for 120 min.in the dark.
The reaction mixture absorbance was measured at 760 nm, and the reaction mixture without the sample was used as a blank.Gallic acid was used as a standard.The TPC was expressed as Gallic acid equivalents for dry powder (mg GAE g -1 ) (Ćujić et al., 2016).
The TFC was determined with aluminum chloride test; 1 mL of diluted plant extract was mixed with 1 mL of 2% AlCl3 methanolic solution.After incubation at room temperature for 10 min., the absorbance of the reaction mixture was measured at 430 nm.Quercetin was used as a standard, and the TFC was expressed as mg quercetin equivalents g -1 for dry weight (mg QE g -1 ) (Ghedadba, Bousselsela, Hambaba, Benbia, & Mouloud, 2014).
The pH differential method used to deduce the TAC, which consists in measuring the absorbance of the mixture at two wavelengths: 510 and 700 nm via two buffer systems at pH 1.0 and at pH 4.5.Cyanidin-3glucoside was chosen as a standard, and the TFC was expressed as mg cyanidin-3-glucoside equivalents g -1 for dry weight (mg C3G g -1 ).

TAC= [(A x MW x DF) / MA] x 100
Where: A: absorbance; MW: molar mass; DF: dilution factor; MA: molar absorption.All previous measurements were carried out in triplicate.

Assay for the antibacterial potential
To evaluate the susceptibility of bacterial strains to plant extracts the disc-diffusion method was used.The bacteria cultures were grown in nutrient broth liquid medium at 37°C.After 24 hours of growth, each bacterium, at a concentration of 10 6 cells mL -1 , was inoculated on the surface of Mueller-Hinton agar plates.Subsequently, filter paper discs (6 mm in diameter) impregnated with extract (50 μL) were placed on the surface of each inoculated plate.
Empty standard antibiotic disks were used as a negative control.The plates were incubated at 37°C for 24 hours.
The antibacterial properties of Thymus algeriensis extracts were compared with those of the following positive controls: Rifampicin (RF); Gentamicin (GN ) and Ampicillin (AP).
The extracts that showed antibacterial effect were tested to determine the Minimal Inhibitory Concentration (MIC) defined as the lowest concentration capable of inhibiting the growth of bacterial strains, where standard bacteria strains were used in the modulation (Santos et al., 2019).
To do this, a dilution method in solid medium was used.It consists of putting a standardized bacterial inoculum in direct contact with a range of plant extracts increasing concentrations.(Burnichon & Texier, 2003).
After 24 hours of incubation at 37°C, the MIC of each sample was determined by the absence of bacterial growth in each plate, comparing the sample readout with the was not inoculated nutrient medium.Analyses were done in triplicate.

Statistical analysis
Data were subjected to statistical analysis using one-way analysis of variance (ANOVA) followed by the least significant difference test of Fisher (LSD) was employed and the differences between individual means and each means used were deemed to be significant at p < 0.05.
Both of the plant extracts had higher total phenolic content, but the T. algeriensis methanol extract had significantly (p < 0.05) higher TPC than its ethanol extract, the TPC was 79.45 and 67.13 mg GAE g -1 dry weight.TFC and TAC recorded for ethanol extract were 25.04 (mg QE g -1 ) 8.14 (mg C3G g -1 ), respectively.Conversely, they were 36.18(mg QE g -1 ) and 6.98 (mg C3G g -1 ) methanol extract.
A large value of Thymus algeriensis TPC was detected in another study in the order of 81.5 mg GAE g -1 (Brahmi et al., 2015).In addition, TPCs estimated for leaves polar fraction (Methanolic water -1 ) of Thymus algeriensis and Thymus capitatus were 248.8 and 240.3 (mg GAE g -1 DW), respectively.
However, TFCs were 15.36 and 14.94 catechin equivalents (mg CE g -1 DW), in the mentioned order.(Megdiche-Ksouri et al., 2015).Moreover, our results are in accordance with the results obtained from other studies and which were conducted on Lamiaceae family (Salvia officinalis L.).
These have revealed that the methanol/water extract also had high TPC and TFC with 323.47 mg g -1 and 218.59 mg g -1 of dried extract (Martins et al., 2015b).
The multiple studies that were conducted have established that the Thymus species are rich and promising sources of phenolics and flavonoids.The phenolic content of plant extract depends on various parameters such as genetic and ecological factors, the part of the plant used, the extraction method employed and even plant age (Amarti et al. 2010;Gharibi, Tabatabaei, & Saeidi, 2015).Msaada et al. (2016), have revealed that both total phenols and flavonoids varied significantly among the region of collection, probably due to the dissimilarities in the soil, climate, solar lighting, humidity, and temperature, which could be affecting samples in each studied region.
TPC were ranged from 8.44 to 18.40 mg GAE g -1 DW and from 26.83 to 63.64mg CE g -1 DW, for TFC.In addition to this, the aerial part extraction of Haloxylon scoparium with different solvents showed the highest yields for water and then for methanol.The high yield of extraction in polar solvents exhibited rich polar constituents of the plant aerial part (Lamchouri et al., 2012).

Assay for the antibacterial potential
The disc-diffusion method is the most common technique used to test the antibiotic properties of crude extracts.In this case, plant extracts were tested against bacterial strains.The data pertaining to the antibacterial potential of the plant extracts are presented in Tables 1 and 2.  As can be noted from Table 1, assayed extracts showed antibacterial potential, especially against the highly pathogenic germs P. aeruginosa, S. typhimurium, and E. coli.Those estimates are promising since Gram-negative bacteria are generally more resistant than Gram positive ones (Siri et al., 2004).
Ethanolic extract has no effect on the growth of E. cloacae while it inhibited widely the growth of both the bacterial strains tested E. faecalis and S. aureus inside zones with diameters 17 mm, 15.5 mm, respectively (p<0.05).
This observation confirmed the evidence presented in a previous study that reported that plants synthesize an array of molecules with very diverse structures such as polyphenols, flavonoids, and terpenoids with low antibiotic activity compared to those produced by microorganisms (Sarker, 2005).
On the other hand, the absence of activity against Gram-negative bacteria could be related to the outer membrane and its permeability properties that perform the crucial role of providing an extra layer of protection against potentially harmful compounds.(Delcour, 2009), (Zgurskaya, López, & Gnanakaran, 2015), (Wiener & Horanyi, 2011).
The results are in agreement with those of Martins et al. (2015a) using methanol water -1 extract of thyme that related higher antibacterial capacity with higher contents in phenolic.
Ethanolic extract (1:1) of Thymus vulgaris presented an important antibacterial activity, It was able to inhibit (50 %) types of susceptible microorganisms tested, among them: P. aeruginosa; and Proteus spp.
According to Al-Bayati (2008), Thymus vulgaris essential oil was found to be active against all the pathogenic bacteria tested except P. aeruginosa.The strongest antibacterial activity was seen against S. aureus with a MIC value of 31.2 µg mL -1 followed by E. coli (62.5 µg mL -1 ), S. typhimurium (125.0 µg mL -1 ) and K. pneumoniae (500.0 µg mL -1 ).
Except P. aeruginosa at 10 mm, methanolic extract of thyme had no inhibitory action against the microorganisms tested: K. pneumoniae, S. aureus, and E. coli.(Albayrak et al., 2013).
The inhibitory effects of Thymus pubescens methanolic extract indicated significant bacterial growth inhibition zone diameters ranging from 8 to 16 mm against Gram-positive bacteria including S. aureus, methicillin-resistant S. aureus and E. faecalis.However, it showed no activity against Gram-negative bacteria (Mehrgan, Mojab, Pakdaman, & Poursaeed, 2008).
Further results recorded for Thymus daenensis methanol extract by Mojab, Poursaeed, Mehrgan, and Pakdaman (2008), indicated a significant antibacterial activity against Gram-positive bacteria including S. aureus, MRSA and E. faecalis but it has shown no activity against Gram-negative bacteria.The produced zone of inhibition ranged from 8 to 29 mm.
Some researchers noticed that the major activity of polar extracts is mainly due to their richness with active compounds, essentially terpenes and phenols (Bekhechi, Atik-Bekkara, & Abdelouahid, 2008).However, the others have explained the absence of antibacterial activity in polar extracts by the fact that the compounds constituting the apolar fractions are at the origin of the antibacterial action.
Another study concluded that medicinal plants essential oil contained more antimicrobial compounds than other types of plant extracts like methanol, ethanol, water, and hexane (Şahin et al., 2004).
In the study done on the antibacterial effects of T. algeriensis essential oil, Bacillus subtilis was more resistant with a concentration of inhibition of 1/250 v v -1 , while other bacteria were inhibited from 1/500 v v -1 the case of Escherichia coli, Micrococcus luteus and Staphylococcus aureus.
The inhibition zones observed in another study of antibacterial activity of the T. algeriensis essential oil were 13 mm against P. aeruginosa, 12 mm against S. aureus, 28 mm against E. coli and 20 mm against S. typhimurium.(Jayari et al., 2018).
However, Thymus ciliatus oil exerted a strong antibacterial activity where the concentration of 1/2000 v v -1 was sufficient to inhibit the growth of Escherichia coli, while S. aureus was more sensitive with an inhibition concentration of 1/3000 v v -1 (Amarti et al., 2010).
For many research groups, the inhibitory effect of T. algeriensis essential oil could be resulted from its composition of linalool and camphor, known to have excellent antibacterial properties or could be related to monoterpene hydrocarbons and oxygenated monoterpenes which are able to affect cell integrity, conducting to both the inhibition of the respiration and an alteration of the permeability.(Jayari et al., 2018).Kabouche et al. (2005), reported that very high antibacterial potential (inhibition zones) of the essential oils of Thymus numidicus from 34 to 66 mm and 26-54 mm with Thymus fontanesii, antagonistic toward E. aerogenes, E. coli, K. pneumoniae, P. aeruginosa, S. aureus, but S. typhimurium was not affected.
Plant extracts have high potential as antibacterial agents, thus, they can be used in the treatment of infectious diseases.T. algeriensis methanolic and ethanolic extracts showed an exceptional richness in phenolic compounds, approved by the appreciable contents measured by the various tests but also by the well-known positive impact of such molecules on human health.
The results, as well as literature data, revealed the great potential of medicinal plants for therapeutic purposes, although they have not been completely investigated.So, more studies need to be conducted to search for new active compounds.

Conclusion
The results indicated that both methanolic and ethanolic extracts of T. algeriensis displayed strong antibacterial ability against the most pathogenic strains tested.It can, therefore, be inferred that those extracts could be useful as a natural antibacterial agents.
Additionally, the findings of this study could be important for further studies to identify, purify and elucidate the exact role of bioactive molecules responsible for such activity and determine possible applications for both food preservation and pharmaceutic purposes.
This study showed that the Algerian flora can constitute an important reserve of interesting plant species, including active compounds that can be used in several fields such as pharmaceutical industries.
More studies about the use of plants for therapeutic issues should be emphasized, especially those relevant to prevent the proliferation of antibiotic-resistant microorganisms.
3 and 70.4 mg GAE g -1 DW of TPC for Lamiaceae family plants methanol extracts.

Table 1 .
Antibacterial activity caused by Thymus algeriensis extracts through Agar Diffusion Method.
Note: MIC -Minimal Inhibitory Concentration; (-): Absence of activity; Negative controls did not show any activity.Capital letters (A-B) and lowercase letters (a-b) indicate significant differences at p < 0.05.