The Effect of Some Verbascum Plant Extracts on Cytoplasmic Membrane of Multidrug Resistant Bacteria by Flow Cytometry

— Verbascum species have been the focus of antioxidant and antimicrobial studies thanks to their secondary metabolites, especially saponins. While disk diffusion and dilution methods are generally used within the scope of antimicrobial studies, the Flow cytometry method is not well known. The ability of six Verbascum species to increase the permeability of multidrug resistant bacterial cells was conducted by flow cytometric assay on Listeria innocua and Escherichia coli. Fluorescence based flow cytometry is a technique for measuring characteristics of cells or biological particles and using flourescent dye as fluorogenic substrate provides. Propidium iodide (PI) is a fluorescent molecule and it can be used to stain cells. The SYTO dyes can be used to stain nucleic acids in both live and dead cells, as well as in Gram-positive and Gram-negative bacteria.


I. INTRODUCTION
The natural compounds also known as secondary found in medicinal plants belong to various chemical structures and some of these compounds have anticancer, antioxidant, and antimicrobial activity. However, little is known about the antibacterial drug resistance mechanisms of these compounds.. The Verbascum genus is known as "Mullein" has more than 2500 species in the world. This plant species in Turkey has about 233 units and 196 of these types indicate an endemic feature. Verbascum is a plant species used for a variety of medical studies in Turkey. Leaves and flowers of Verbascum species are used as an expectorant and diuretic as sweaty [1]. The antibacterial antioxidant, anticancer, antimalarial and cytotoxic activities of Verbascum species have been the subject of many studies. [1][2][3][4][5]. Mirjalili et al reported the antibacterial and antioxidant properties of Verbascum species belonging to various regions of Iran [4]. Soltaninejad and Akhgar have obtained a report on these components by isolating some secondary metabolites from a Verbascum genus. Biologically active components of plants of the Verbascum genus are saponins, iridoid, and phenylethanoid glycosides, neolignan and monoterpene glucosides, phenolic and fatty acids, spermine alkaloids and steroids, and flavonoids [6][7][8][9][10]. Since verbascum seeds contain saponin, they show a poisonous effect. For this reason, it is used especially in fishing on the Black Sea coast. In the same study, antimicrobial properties of three Verbascum species (Verbascum Olympicum Boiss., Verbascum Prussianum Boiss. And Verbascum Bombyciferum Bois) were reported. [11]. The antioxidant and phenolic structure of the extract of Verbascum Glomeratum Linneus species  6 36 was observed by HPLC [12]. Anticarcinogenic and antioxidant properties of Verbascum thapsus L. species have been reported in the literatüre. Verbascum thapsus L. leaf was extracted with different solvents, and its antibacterial activity was investigated. In addition, in these studies, it was stated that microbial biofilms are also very important in ensuring that microorganisms live in a complex structure. [13][14][15][16]. It has been reported that the leaves of Verbascum Thapsus have antibacterial properties against Escherichia Coli, Yersinia Pestis, Bacillus Cereus, Pseudomonas Aeruginosa, Listeria Monocytogenes and Staphylococcus Aureus bacteria.
Similarly, it has been reported in the literature that the roots of Verbascum Undulatum also exhibit antibacterial properties. [17][18]. CuO nanoparticles synthesized from Verbascum thapsus leaves showed both photocatalytic and antibacterial activity against Gram-positive Staphylococcus aureus and drug-resistant Gram-negative Escherichia coli bacteria. Such approaches also help to develop new biofilm strategies from plants. [19][20]. The pharmacological properties of Verbascum species have been revealed in many studies such as those described above and took their place in the literature. In conclusion, Verbascum species have an important place in the treatment of diseases due to their antioxidant and antibacterial properties [16,[21][22][23]. The pharmacological properties of Verbascum species have been revealed in many studies such as those described above and took their place in the literature. In conclusion, Verbascum species have an important place in the treatment of diseases due to their antioxidant, antibacterial and biofilm activity properties [3,16,[21][22][23][24][25][26][33][34][35][36][37][38][39][40]. In this study, the antibacterial effects of six Verbascum species (Verbascum tripolitanum, Verbascum sinuatum, Verbascum caesareum, Verbascum gaillardotti, Verbascum pinetorum and Verbascum antiochium) against Listeria innocua and Escherichia coli bacteria were investigated by Flow cytometry.

Plant Material
Verbascum

Preparation of the methanol extracts
The plant sample, weighing about 100 g was extracted with methanol at 40-45ºC for 2 hours (3 times). The filtrates were combined and concentrated in vacuo at 45ºC. Finally, the extracts were then lyophilized and kept in the dark at 4ºC until tested.

Experimental Stage
Initially optimum conditions were determined. Verbascum concentrations were arranged 6 mg/ml-3mg/ml-1,5mg/ml in 10% dimethylsulfoxide. 200 μl of the amount of extract for added in bacterial culture. PI (propidium iodide) or Fluorophores SYTO 9 concentration was applied at 10% in PBS buffer in Listeria Innocua bacteria, while PI was applied as 10% SYTO undiluted in Escherichia coli. In the experiments for Listeria Innocua bacteria, 2.5 μL PI (diluted) and 2.5 μl SYTO (diluted) were applied in a 500 μl bacteria culture. Operations were carried out in a 1-hour time unit at 70° C. The incubation process is for positive control. In the negative control, 200 μl of 10% dimethylsulfoxide was added. This application was applied in a period of 25°C. 2.5 μl PI (diluted) and 2.5 μl SYTO (undiluted) was applied in 500 μL bacteria culture for Escherichia coli. The incubation process was applied in one hour at 70°C. This process is for positive control. In the negative control, 200 μl 10% dimethylsulfoxide was added. The incubation was carried out at 25°C for 1 hour. All these trials were performed by incubating 6 mg/ml-3mg/ml-1,5mg/ml 10% Dimethylsulfoxide at 25°C for 1 hour for Listeria Innocua and Escherichia coli bacteria.  (Figures 2-24, even-numbered figures). When the extract concentration in the bacterial culture reduced, the number of living cells increased.

Lischeria Innocua tests
Verbascum tripolitanum According to Figure 2, when the drug concentration is 6 mg/mL, it is understood that 95.01% of the living Listeria innocua cells die and this ratio decreases as the concentration decreases. This ratio is 78.93% while the concentration is 1,5 mg/mL. According to Figure 4, when the drug concentration is 6 mg/mL, it is understood that 86,13% of the living Listeria innocua cells die and this ratio is 54,02% while the concentration is 1.5 mg/mL. According to Figure 6, when the drug concentration is 6 mg/mL, it is understood that 93,86% of the living Listeria innocua cells die and this ratio is 82,38% while the concentration is 1.5 mg/mL.

Fig.7: Negative (A) and positive (B) control of Listeria innocua
According to Figure 8,  innocua cells die and this ratio is 70,97% while the concentration is 1.5 mg/mL. According to Figure 10, when the drug concentration is 6 mg/mL, it is understood that 94,02% of the living Listeria innocua cells die and this ratio is 75,85% while the concentration is 1.5 mg/mL. According to Figure 12, when the drug concentration is 6 mg/mL, it is understood that 95,22% of the living Listeria innocua cells die and this ratio is 76,96% while the concentration is 1.5 mg/mL.  According to Figure 14, when the drug concentration is 6 mg/mL, it is understood that 83,72% of the living Escherichia coli cells die and this ratio is 46,17% while the concentration is 1.5 mg/mL According to Figure 16, when the drug concentration is 6 mg/mL, it is understood that 54,32% of the living Escherichia coli cells die and this ratio is 18,25% while the concentration is 1.5 mg/mL. According to Figure 14, when the drug concentration is 6 mg/mL, it is understood that 64,14% of the living Escherichia coli cells die and this ratio is 36,75% while the concentration is 1.5 mg/mL. According to Figure 20, when the drug concentration is 6 mg/mL, it is understood that 80,41% of the living Escherichia coli cells die and this ratio is 38,54% while the concentration is 1.5 mg/mL. According to Figure 22, when the drug concentration is 6 mg/mL, it is understood that 75,21% of the living Escherichia coli cells die and this ratio is 40,42% while the concentration is 1.5 mg/mL. According to Figure 24, when the drug concentration is 6 mg/mL, it is understood that 79,94% of the living Escherichia coli cells die and this ratio is 40,74% while the concentration is 1.5 mg/mL. Verbascum species studied in this study showed antibacterial effects for both Listeria innocua and Escherichia coli. However, when looking at the results given in Table 1 and

IV. CONCLUSION
It can be said that the Verbascum species extract may disrupt the membrane barrier and allow exogeneous solutes such as PI and SYTO 13 to permeabilize into the bacterial cells. Secondary metabolites in the Verbascum species may have the ability to increase the permeability of bacterial membranes and allow antibiotics to access the bacterial targets. These results show that the flow cytometry method is more advantageous than traditional methods, such as disk diffusion and mikro dilution, for quickly generating a large amount of data. This technique can serve as a powerful tool in optimally combining different preservative factors in order to design an effective antimicrobial system for selected foods. As a result, This studyshows that methanolic extracts of Verbascum species is a potential source of natural antioxidants and antimicrobial agent and can form the basis for pharmacological studies.

ACKNOWLEDGMENTS
Authors would like to thank Germany Flensburg University of Applied Sciences Biotechnology for their contribution in conducting this study.