Comparison of The Antimicrobial Activity of Some Scutellaria orientalis L. Taxa Growing in Turkey

: Scutellaria species are commonly used in the treatment of various diseases in traditional medicine. One of the members of Lamiaceae, the genus Scutellaria L. has approximately 471 species on earth. The genus is represented by 39 taxa in Turkey which 17 of them are endemics. In this study, plant samples of 15 S. orientalis subspecies from different regions of our country were collected and methanol extracts were prepared from aerial parts. In vitro antimicrobial activity of these extracts against three gram positive and three gram negative bacteria and against a yeast using broth microdilution method. Methanol extracts of S. orientalis taxa were found to have moderate to low antimicrobial activity compared to the literature.


INTRODUCTION
Due to increasing resistance to antimicrobials and slowdown in the exploration of new ones, combating with infectious diseases is getting harder nowadays. Studies on extracts or compounds from plants remain important to discover new sources as antimicrobial agents. The genus Scutellaria L. (Lamiaceae) contains 471 species throughout the world [1]. Scutellaria species are generally subcosmopolitan plants and are distributed especially in the central Iran-Turanian region of Asia [2]. In Turkey, the genus includes about 39 taxa and 17 of these are endemics (43.6%) [3][4][5][6][7][8][9].
There are many studies on the phytochemical properties of Scutellaria species. Phenolic compounds, diterpenoids were isolated by Ersöz et al. and Rodríguez et al. from S. pontica K.Koch [16,19]. Essential oil compositions of S. albida L., S. diffusa Benth., S. heterophylla Montbret & Aucher ex Benth, S. salviifolia Benth., S. brevibracteata Stapf, S. galericulata L. and S. tortumensis (Kit Tan & Sorger) A.P.Khokhr. were also investigated [20][21][22][23]. Cytotoxic activity of phenylethanoid glycosides isolated from S. salviifolia was determined [24]. The acetylcholinesterase, butyrylcholinesterase and tyrosinase inhibitory activities of methanol extracts and the antioxidant activity of methanol and ethyl acetate extracts were investigated by DPPH and FRAP experiments in a study by Şenol et al. [25]. In recent years, İçen et al.  [29]. In this study, we planned to investigate the antibacterial and antifungal activity of methanol extracts of 15 subspecies of S. orientalis using broth microdilution method.

Plant Material
Aerial Edm.) were collected from common territories of diverse localities in Turkey. All taxa were identified according to "Flora of Turkey and the East Aegean Islands" [3] by Mehmet Çiçek. The voucher specimen are kept in the Herbarium of Ankara University Faculty of Pharmacy, Ankara, Turkey (AEF). The species names, collection localities, dates and herbarium numbers of 15 taxa are given in Table  1.

Preparation of Extracts
Aerial parts of S. orientalis taxa were dried and then powdered. About 5 g powdered samples were extracted with methanol (2x200 ml) in a rotary shaker for 24 h. Extracts were filtered and further concentrated to dryness under reduced pressure at 37ºC using a rotary evaporator (Büchi, Switzerland). Methanol extracts were obtained and kept in the freezer +4°C until the experimental practices.

Preparation of Bacterial and Fungal Suspensions
Microorganisms used in the experiment were gram negative bacteria (Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922; gram positive bacteria Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and yeast Candida albicans ATCC 10231). Microorganisms were obtained from the culture collection of the Ankara University, Faculty of Pharmacy, Pharmaceutical Microbiology Department.
Antibacterial and antifungal activity tests proceeded similarly with CLSI recommendations by broth microdilution method with some modifications [30,31]. Glycerol stocks kept at -80C were inoculated to Sabouraud Dextrose Agar (SDA, Oxoid) medium for yeast and Mueller-Hinton Agar (MHA, Merck) medium for bacteria, incubated for 20 h at 35±2C. Isolated colonies from the second passage of overnight cultures transferred into 0.85% NaCl to obtain turbidity of 0.5 McFarland standards. Sabouraud Dextrose Broth (SDB) medium for yeast and Mueller-Hinton Broth (MHB, Merck) medium for bacteria were used to dilute these suspensions to give a final concentration 2.510 3 CFU/ml for yeast and 510 5 CFU/ml for bacteria.

Broth Microdilution Method
Firstly, two-fold 8 serial dilutions of S. orientalis methanol extracts (1000 to 7.81 µg/ml) were prepared in 96-well microplates using SDB media for yeast and MHB media for bacteria in 100 µl volume. After serial dilution, 100 μl concentration-adjusted microorganism suspensions were transferred to each well and microplates were left for incubation (24 h for yeast and 20 h for bacteria) at 35±2C. Sterility and growth control wells were included. Ciprofloxacin and miconazole were used as positive controls. The well of lowest concentration with no growth was recorded as MIC (µg/ml) value, at the end of incubation time. All experiments conducted in two parallels.

Antimicrobial Activity Results
In this study, in vitro antibacterial and anti yeast activity of methanol extracts of fifteen S. orientalis taxa was evaluated using broth microdilution assay against the above mentioned panel of human pathogenic strains of three gram positive bacteria, three gram negative bacteria and a yeast. The results are given in Table 2 as Minimum Inhibitory Concentrations (MIC).
To the best of our knowledge and according to the literature survey, there is no report on comparative antimicrobial activity of S. orientalis subspecies growing naturally in Turkey. This study is the first to demonstrate that 15 subspecies of S. orientalis possessed in vitro antibacterial activity.
According to the results of our study, antibacterial activity values of all methanol extract of S. orientalis taxa were found to be between 250-62.5 μg/ml. Antimicrobial activity scale of MIC values 500 to 100 μg/ml were evaluated as moderate, and MIC values less than 100 μg/ml were considered to be good according to the concentration ranges stated by Morales et al. (2008) [32]. As seen in Table 2, it was found that methanolic extracts obtained from the aerial parts of S. orientalis taxa have moderate to good antimicrobial activity. All of the species (except S. orientalis subsp. virens (125 μg/ml MIC value) showed stronger activity (62.5 μg/ml MIC value) against Gram negative P. aeruginosa ATCC 27853. Other gram negative bacteria, K. pneumoniae ATCC 13883 and E. coli ATCC 25922 were affected more than gram positive bacteria. Least affected bacteria were S. aureus ATCC 29213 and E. faecalis ATCC 29212.
Among the extracts, S. orientalis subsp. orientalis, S. orientalis subsp. santolinoides and S. orientalis subsp. haussknechtii were the most effective ones. Also, all of the extracts showed good activity with a value of 62.5 μg/ml against C. albicans ATCC 10231, except for S. orientalis subsp. virens, S. orientalis subsp. santolinoides and S. orientalis subsp. bornmuelleri since their activities were moderate with a value of 125 μg/ml. According to these results, all methanol extracts of S. orientalis taxa showed higher antimicrobial activity against yeasts than bacteria. Dereboylu et al. (2012) investigated antimicrobial activity of Scutellaria cypria var. cypria, S. cypria var. elatior and S. sibthorpii essential oils against B. subtilis ATCC 6633, S. aureus ATCC6538-P, E. faecalis ATCC 29212, Salmonella typhimurium CCM 5445, K. pneumoniae CCM 2318, E. coli ATCC 12228, P. aeruginosa ATCC 27853 and C. albicans ATCC 10239. They reported the antibacterial activity in the range of 20-10 mg/ml and antifungal activity 20 mg/ml. [33].
In a study conducted in 2017, antimicrobial activity of S. salviifolia methanol extract was tested with the disc diffusion and microdilution methods. MIC results of methanol extract varied between 12,5-25 mg/ml against E. coli, K. pneumoniae, S. enteritidis P. aeruginosa, S. aureus [27].  2019) found that aqueous, methanol and n-butanol extracts from roots and methanol and n-butanol extracts from aerial parts of S. salviifolia, S. diffusa and S. pontica and n-hexane extracts from root parts of S. diffusa showed low or no antibacterial activity (1024 μg/ml). Aqueous extracts from the aerial parts showed moderate activity with the values of 1024-512 μg/ml; chloroform, ethylacetate and n-hexane extracts of roots and aerial parts showed low to moderate or no activity with the values of 1024-256 μg/ml, 1024-256 μg/ml, 1024-256 μg/ml, respectively. Their anti yeast fungal activity results ranged between 1024-32 μg/ml. Chloroform extracts from roots and n-hexane extracts from aerial parts of S. salviifolia and chloroform and aqueous extracts from the aerial parts of S. pontica had good antifungal activity against some Candida spp. with values between 64-32 μg/ml. And totally they found higher antifungal activity than antibacterial, in accordance with our results [11].

CONCLUSION
This study is the first report on comparative antimicrobial activity of S. orientalis taxa from Turkey with methanol extracts by microdilution method to the best of our knowledge. It was determined that the methanol extracts of these 15 taxa had similar levels of activity with slightly different values, which ranged between 250-62.5 μg/ml against all microorganisms. In the future, we plan to investigate antibacterial and anti yeast tests of different Scutellaria species and compare their results.