Phytochemical evaluation and antibacterial effects of Medicago sativa, Onosma sericeum, Parietaria judaica L., Phlomis persica and Echinophora platyloba DC. on Enterococcus faecalis

Background: Since drug resistance has become one of the predominant problems of health worldwide, it is necessary to use new methods to combat drug-resistant bacteria. In this regard, medicinal plants are considered one of the richest sources to produce antibiotics. The aim of this study was to investigate the antibacterial effects as well as total phenolic and flavonoid contents of a number of medicinal plants collected from the Chaharmahal and Bakhtiari provinces of India, in order to investigate their potential use for the production of new antibiotics. Materials and Methods: In this experimental study, the maceration method was used to prepare hydroalcoholic extract of Medicago sativa, Onosma sericeum, Parietaria judaica L., Phlomis persica and Echinophora platyloba DC. The effect of these plants on Enterococcus faecalis (ATCC 29212) was investigated. To determine the antibacterial effect of the extracts, broth microdilution in sterile 96-well plate was used according to the McFarland standard (105 CFU/ml). The total phenolic content was assayed by using the Folin-Ciocalteu colorimetric method and expressed in terms of gallic acid equivalent. The total flavonoid content was assayed by aluminum chloride colorimetric method and expressed in terms of rutin equivalent. ! Biomed Res Ther 2018, 5(1): 1941-1951 !1941 DOI: 10.15419/bmrat.v5i1.408 ISSN: 2198-4093 www.bmrat.org Results: Based on the results of this study, the 512, 256, 128, 32 and 32 μg/ml doses were determined to be the minimum inhibitory concentrations (MICs), and the 1024, 1024, 512, 128 and 128 μg/ml doses were derived as the minimum bactericidal concentration (MBCs) of M. sativa, O. Sericeum, P. judaica, P. persica and E. platyloba, respectively. E. faecalis and P. judaica contained the highest total phenolic content and flavonoid content, respectively. Conclusion: Given the comparatively higher antibacterial effect of P. persica and E. platyloba, as well as the presence of phenolic and flavonoid compounds in these plants, it is recommended that these plants be further investigated in feasibility studies for the production of new antibiotics.


Introduction
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 Bahmani et al., 2016;Kooti et al., 2016;Moradi et al., 2017;Rahimi-Madiseh et al., 2017;Rahimifard et al., 2017;Rouhi-Boroujeni et al., 2017;Sarrafchi 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., 2016;Naz 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, antiinflammatory, 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., 2011;Mozaffarian, 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).

Extraction
Extraction was conducted by maceration of Medicago sativa, Onosma sericeum, Prietaria judaicaa L., Phlomis persica shoots, and from aerial parts of Echinophora platyloba D.C; these were done in triplicates (for 72h each time). In this method, water and butyric acid-free bitter ethanol at 30/70 ratio were used. The resulting extract was filtered using filter paper and evaporated under nextto-vacuum pressure and 40°C by a rotary evaporator to concentrate. The resulting solution was stored at -20°C until later use.

Preparing different dilutions of extract
The extracts were prepared using dimethyl sulfoxide (DMSO) and distilled water. Different dilutions (4,8,16,32,64,128,256,512 and 1024 μg/ml) of the extracts were prepared using Mueller-Hinton broth agar. The maximum concentration of DMSO was 0.2% in the final concentration.

Preparing standard bacterial strains
Enterococcus faecalis (ATCC 29212) was purchased as lyophilized from Iranian Research Organization for Science and Technology.

Preparing microbial suspension
To prepare a microbial suspension equivalent to 0.5 McFarland standard (10 5 CFU/ml), a 24-hour culture of the bacteria was performed on blood agar, and then a suspension with 0.5 McFarland turbidity was prepared in normal saline.

Determining minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs)
The antibacterial effects of the extracts were determined by broth microdilution in a sterile 96-well plate with reference to 0.5 McFarland standard (10 5 CFU/ml). In this method, the first well was considered "culture medium + extract" (negative control), and the second well was considered "culture medium + bacterium" (positive control). After adding the culture medium at 95µl and the extracts at 100µl to microplate wells and diluting them, we incubated the samples at 37°C for 24h. The concentration of the last (most diluted) well without turbidity was considered MIC (Andrews, 2001). To determine MBC, we subsequently performed a culture of the samples of each tube at 10µl on Mueller-Hinton agar and left them at 37°C to incubate for 24h. The lowest concentrations of the extract in which the bacteria could not grow were considered MBCs. The tests were performed to determine the MICs and MBCs, and were conducted in triplicates (National Committee for Clinical Laboratory Standards, 2001).

Determining total phenolic and flavonoid content
Total phenolic content was measured by Folin-Ciocalteu colorimetric assay and expressed in terms of gallic acid equivalent. Total flavonoid content was measured by aluminium chloride colorimetric method and expressed in terms of rutin equivalent (Dulf et al., 2016;Karimi and Moradi, 2015;Singleton et al., 1999).  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 (Tables 1 and 2). 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). 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 (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)  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.

Conclusion
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.