Nowadays new bacterial resistance to commonly used chemical drugs has turned into a widespread phenomenon. Enterococcus faecalis is one of these bacteria. E. faecalis can be a cause of bacteremia, meningitis, septicemia, endocarditis, wound infection, infant infections and urinary tract infections Kafil and Asgharzadeh, 2014. In addition to vancomycin-resistant enterococci (VRE), this bacterium has been reported to acquire resistance to daptomycin, which has aroused concerns Werth et al., 2014.

With increasing drug resistance among bacteria, efforts are being made to seek out new therapies. Phytotherapy is one of the most promising therapies for many diseases Asadi-Samani et al., 2017Bahmani et al., 2016Kooti et al., 2016Moradi et al., 2017Rahimi-Madiseh et al., 2017Rahimifard et al., 2017Rouhi-Boroujeni et al., 2017Sarrafchi et al., 2016. Indeed, the collection and screening of medicinal plants can be helpful in areas with high potential for growth of medicinal plants Bahmani et al., 2014. Therefore, the present study was conducted to investigate the antibacterial effects of Medicago sativa, Onosma sericeum, Parietaria judaica L., Phlomis persica and Echinophora platyloba DC, and to determine their phenolic and flavonoid contents. These plants are grown in the Chaharmahal and Bakhtiari provinces of Iran, which are areas with high potential for the growth of medicinal plants. Understanding their antibacterial effects would address the suitable candidates for phytotherapy and the feasibility for production of new antibiotics.

M. sativa, also known as alfalfa and lucerne, comes from the Fabaceae family Sadowska et al., 2014. M. sativa is used as a food additive in the United States, Russia, North Africa and China because of their high vitamin content Shi et al., 2014. It produces secondary metabolites, such as coumarins, isoflavones, naphthoquinones, alkaloids and saponins, that have nematocidal, cytotoxic and antimicrobial effects Sadowska et al., 2014.

O. sericeum is a perennial plant that grows naturally in Iran and is a member of the Boraginaceae family Gharehmatrossian et al., 2016Naz et al., 2006. In addition to the roots of this herb (which is a dye and used in cosmetics), other properties have been reported for this plant, including antitumor, anti-inflammatory, antipregnancy, antimicrobial, cardiotonic and antiviral effects Gharehmatrossian et al., 2016.

P. judaica (Urticaceae) grows abundantly in urban areas of the Mediterranean region. P. judaica is a perennial herb, with individual plants consisting of many shoots emerging from a common rootstock Fotiou et al., 2011Mozaffarian, 2015. P. persica is from the family Lamiaceae. The genus Phlomis consists of 100 species worldwide, with 17 species in Iran. This herb in phytotherapy is used to treat topical wounds and respiratory diseases. Some other properties including analgesic, anti-diarrheatic, hemorrhagic ulcer-treating, antimalarial, anti-inflammatory, antimicrobial and immunosuppressive effects have also been reported for this plant Sarkhail et al., 2006. Moreover, some sources have reported tonic, diarrhea and free radical-inhibitory effects Hussain et al., 2010.

The Echinophora genus consists of 10 species of which Echinophora orientalis, Echinophora sibthorpiana, Echinophora cinerea and E. platyloba are present in Iran Shahneh et al., 2013. E. platyloba is used more often as a food additive in Iran Avijgan et al., 2010. This plant has been reported to exhibit antifungal, antioxidant and antibacterial properties Sharafati-chaleshtori et al., 2012.

M. sativa extract could inhibit E. faecalis at 512 μg/ml. At 1024 μg/ml, M. sativa extract could destroy the bacteria. O. sericeum displayed more potent bacteriostatic activity against E. faecalis than M. sativa; the bactericidal activity of O. sericeum was the same as M. sativa. P. judaica showed MIC and MBC of 128 and 512 μg/ml, respectively. Moreover, 32 μg/ml of P. persica and E. platyloba inhibited E. faecalis, while 128 μg/ml of P. persica and E. platyloba eliminated E. faecalis.

The least potent bactericidal effects among the studied plants was seen by M. sativa and O. sericeum. Lastly, P. persica and E. platyloba showed the most potent antibacterial effects on E. faecalis ( Table 1 and Table 2 ).

According to Table 3 , all plants contained phenols and flavonoids. P. judaica had the highest total phenolic and flavonoid content. The plant with the least flavonoid content was E. platyloba ( Table 3 ).

This present study was conducted to investigate the antibacterial effects as well as the total phenolic and flavonoid contents of a number of medicinal plants collected from the Chaharmahal and Bakhtiari provinces of Iran. The aim was to evaluate which plants could be considered as suitable alternatives for development of antibiotics to fight treatment-resistant bacteria.

Our findings showed the antibacterial effects of M. sativa on E. faecalis, with MIC of approximately 512 μg/ml and MBC of 1024 μg/ ml for this plant. Chavan et al. conducted a study to investigate the antibacterial properties of M. sativa. The methanol extract of this plant created inhibition zones of 23, 22 and 23 mm in diameter for Escherichia coli, Pseudomonas aeruginosa and Streptococcus aureus, respectively. The authors also reported an MIC of 37 μg/ml for E. coli, 12.03 mg/ml for P. aeruginosa and 111 μg/ml for S. aureus Chavan et al., 2015. The antibacterial effect of this plant could be due to their phenolic compounds. Notably, our study and the study of Chavan et al. demonstrate the optimal antibacterial effects of M. sativa.

Another plant investigated in our study was O. sericeum; its antimicrobial effect on E. faecalis was demonstrated- with MIC and MBC of 256 μg/ml and 1024 μg/ml, respectively. A study conducted by Naz et al., to examine the antimicrobial effects of another species from the Onosma genus (Onosma hispidum), showed that this plant could inhibit the growth of various bacteria and could have an antibacterial effect on E. faecalis Naz et al., 2006. The study by Vukic et al. on Onosma visianii showed that main components of this plant extract were 5,8-O-dimethyl isobutyrylshikonin, acetylshikonin, α-methylbutyrylshikonin, β-hydroxyisovalerylshikonin, isobutyrylshikonin, deoxyshikonin, and 5,8-O-dimethyl deoxyshikonin. Indeed, O. sericeum plant had a potent antibacterial effect not only on Enterococcus faecalis but also Bacillus megaterium, Micrococcus luteus, Staphylococcus epidermidis and Stenotrophomonas maltophilia Vukic et al., 2017. The results of the current study and other studies, thus, show that the species of the Onosma genus can have antibacterial effects on E. faecalis, which can be due to the presence of phenolic compounds in their aerial parts.

P. judaica, which was observed to have the highest phenolic and flavonoid content among the studied plants, also exhibited robust antibacterial effects on E. faecalis. Fares et al. who investigated the antibacterial effects of extracts of wild plants in Palestine showed that P. judaica had antibacterial effect on Streptococcus pneumoniae; the aqueous and ethanol extracts of this plant had an MIC of 3.125 and 100 mg/ml, respectively Fares et al., 2013. Our present study and other studies have demonstrated the antibacterial effects of this plant. The antibacterial effects of this species, therefore, is not an unexpected observation due to the presence of phenolic and flavonoid compounds. Moreover, it is not a surprising observation due to the fact that extracts from this plant can be isolated for its antibacterial compounds.

P. persica and E. platyloba in our study were found to exert the most potent antibacterial effect on E. faecalis when compared to the other studied plants. A phytochemical study showed the presence of relatively equal amounts of phenolic content in both plants. Amor et al. studied the antibacterial effects of Phlomis crinite. They reported that the essential oil of its flower had an inhibitory effect on S. aureus, E. faecalis and Salmonella typhimurium- with the MIC ranging between 39 μg/ml and 625 μg/ml Amor et al., 2008. Sarkhail et al. (2006) showed that P. persica shoot extract had chrysoeryol-7-O-β-D-glucoside, verbascoside and two other glycosidic flavonoids to which the antibacterial effects of this plant can be attributed Sarkhail et al., 2006. Besides that, Ranjbar and Babaei (2016) studied the antimicrobial properties of E. platyloba on different Salmonella species and concluded that this plant, at 150 mg/ml, exerted antifungal effect on Salmonella enteritidis and Salmonella typhi and, at 250 mg/ml, on Salmonella choleraesuis Ranjbar and Babaie, 2016. Entezari et al. (2009) investigated the antibacterial effects of E. platyloba, and reported that the methanol extract of this plant had an inhibitory effect on S. aureus and P. aeruginosa. Thus, at high doses, the E. platyloba plant could completely inhibit the growth of these bacteria. An investigation of the chemical compounds of the essential oil of E. platyloba also showed that monoterpenes and sesquiterpenes are the major compounds of this plant Rahimi-Nasrabadi et al., 2010. As it has been clearly shown in this study and other studies, E. platyloba is a plant that can be an appropriate choice to be further studied for feasibility as an antibacterial agent; it has potent antibacterial effects on different bacterial species as well as phenolic and flavonoid compounds.

All five plants were collected from the Chaharmahal and Bakhtiari provinces, had relatively equal amounts of phenolic compounds, and showed antibacterial effects on E. faecalis. It is recommended that these plants be further investigated in feasibility studies for the production of new antibiotics.

DMSO: Dimethyl sulfoxide

DW: Dry weight

GAE: Gallic acid equivalent

MIC: Minimum inhibitory concentration

MBC: Minimum bactericidal concentration

RU: Rutin

All authors equally contributed on all experiments; designed the study; wrote the manuscript; approved the manuscript for publication.