Introduction
Staphylococcus aureus bacteria is responsible for a large number of hospital infections [1]. It has even been proposed as a secondary bacterial infectious agent in the COVID-19 and a cause of death [3]. Pseudomonas aeruginosa bacteria exist in groundwater and swimming pools [5, 6]. it enters plants and foods through the rhizosphere [7]. By contaminating water, it is transferred to cows and their milk and remains even after pasteurization [8]. Proteus vulgaris bacteria is a human pathogen and is isolated from patients with coronary artery disease [9]. Antibiotic resistance of these three bacteria investigated in this research.
Ailanthus altissima is a fast-growing deciduous tree (tree of heaven) from the Simaroubaceae family and grows in almost all regions of Iran [15]. The antibacterial role of Ailanthus altissima leaf extract with the synthesis of zinc oxide nanoparticles on Escherichia coli has been reported [16]. Saponin has antioxidant and antimicrobial properties, and its presence in some medicinal plants has caused it to be used in the manufacture of medicines. This study aims to investigate antiseptic properties of Ailanthus altissima and Salvia nemorosa.
Methods
In this study, two field and laboratory methods were used. In the field method, the plant growth area was visited, samples were collected and their initial preparation was done. In the laboratory method, extracts were obtained from two plants with three different solvents; bacteria were cultivated in a special culture medium; bacteria were treated with the well diffusion method by the extracts, and saponin was extracted from the plants.
The flowering branches of Salvia nemorosa and the leaves of Ailanthus altissima were collected from an area in the south of Karaj and northeast of Mohammadshahr in Karaj, Iran in 2017 and dried in the shade. Bacterial strains were obtained from Iranian Research Organization for Science and Technology.
The extraction was done by Maceration method in the forms of aqueous, methanolic, and hydroalcoholic extract according to the modified method of Azwanida (2015). In Maceration extraction method, 10 grams of the plant was powdered, water was added by 1.5 times and mixed on a shaker. It was passed through Whatman grade 2 filter paper and placed in a rotary machine for 3 hours. In Methanolic method, 10 grams of the plant was powdered. Methanol was added at a ratio of 1:10 and placed on a shaker for 72 hours. Methanol was then added again at a ratio of 1:10 and placed in the rotary machine for 30 minutes. In hydroalcoholic method, 10 grams of the plant was powdered. Methanol and water were then added at a ratio of 80:20 and placed on a shaker for 72 hours. In order to concentrate, it was then placed in the rotary machine for 1 hour.
Nutrient Broth culture medium was used as the main stock for maintaining bacteria. To prepare nutrient agar culture medium, 10 grams of culture medium was dissolved in 500 ml of distilled water. It was cultivated in 4 stages and densely. After 24-48 hours of cultivation [23], a suspension was prepared from the colonies. The density of the suspension was compared with 0.5 McFarland Standard. The 0.5 McFarland suspension had an optical absorbance of 0.08-0.1 at the wavelength of 625 nm. Cultivation was done in Mueller-Hinton agar medium according to the modified method of Golus et al. (2016). Diluted extracts with different concentrations (20-30-40 and 50 mg/ml) were prepared in dug wells. Then, 30 mL were poured into the wells and incubated, and their growth or non-growth halos were examined [25].
Saponin was extracted according to the modified method of Zeb et al. (2014). In this method, 20 grams of the plant was powdered and extracted with 20% ethanol in two steps. After concentration, it was mixed with 20 ml of diethyl ether in a separatory funnel. The resulting solution was shaken with 60 ml of normal butane. The extract was treated with 10 ml of 5% sodium chloride and the resulting solution was concentrated and dried. 
The tests were repeated three times and the results were analyzed using GraphPad Prism v.7 software.
Results
The increase in concentration of extracts for both plants had a significant relationship with the increase in the diameter of the growth halo (p<0.0001). The methanolic extract of Ailanthus altissima with a concentration of 50 mg/ml had the most effect on Staphylococcus aureus bacteria (mean non-growth halo diameter= 16.5±0.5 mm). The effect of maceration extract of Ailanthus altissima leaf on Staphylococcus aureus at concentrations of 50, 40, and 30 mg/ml was higher than that of Salvia nemorosa (Figure 1 and 2).







Only the hydroalcoholic extract of Ailanthus altissima had significant effect on Proteus vulgaris (mean non-growth halo diameter= 6.5 mm), but the Salvia nemorosa extracts had no significant effect (Figure 3). The amount of saponin was high in two plants; it was higher in Salvia nemorosa (28.20±0.15%) than in Ailanthus altissima (24.01±0.10%).




Discussion
Even though the methanolic extract of both Ailanthus altissima and Salvia nemorosa had strong effect on Staphylococcus aureus, but the highest effect and the largest diameter of non-growth halo was related to Ailanthus altissima. Staphylococcus aureus is an opportunistic pathogen with extensive activity in most places; Ailanthus altissima can be used as a good disinfectant to remove this omnipresent bacterium in public places with minimal side effects. But, chlorination is currently used for disinfection of swimming pools, and ultraviolet rays are used in hospitals for disinfection of beds, walls, and floors.
Comparison of the effects of all concentrations of aqueous, alcoholic and hydroalcoholic extracts of Ailanthus altissima on Staphylococcus aureus showed that the largest diameter of non-growth halo was observed after treatment with methanolic extract (16.5±0.5). Poljuhaa et al. also reported the methanolic extract of Ailanthus altissima as a valuable antibacterial source (Escherichia coli) and suggested the phenolic and flavonoid compounds such as glycosides and quercetin-3-o-glucoside to be the factors of this characteristic [28].
The assessment of the effect of aqueous extract of Ailanthus altissima and Salvia nemorosa on Pseudomonas aeruginosa also showed an interesting result: only Salvia nemorosa had a significant effect in all tests (with a non-growth halo diameter of 26±1 mm). Therefore, regardless of the type of extract (aqueous, alcoholic, hydroalcoholic), the type of plant, in addition to the type of bacteria, plays a role in influencing and inhibiting the growth of bacteria. Ivanov et al. investigated the effect of different solvents for extracting Salvia nemorosa and introduced it as a natural bioactive therapeutic agent with antioxidant, antidiabetic and antimicrobial properties [29].
Methanolic and hydroalcoholic extracts of Ailanthus altissima were more effective on Pseudomonas aeruginosa and Staphylococcus aureus, where the methanolic extract (with non-growth halo diameter of 17.5±0.5 mm) was higher effect. Ailanthus altissima hydroalcoholic extract was effective against Proteus vulgaris and had an antimicrobial effect. Al-Snafi also reported that the Ailanthus altissima plant is capable of destroying bacteria that are resistant to several drugs [30] (Figure 4 and 5). 








The saponin amount in Ailanthus altissima plant was 24.01±0.10% while it was 28.20±0.15% in Salvia nemorosa plant. In Chenopodium quinoa, saponin amount is 2.03±0.29% and in Launaea sarmentosa, which is an anti-cancer drug, it is 10.80% [32]. The high amount of saponin in the study plants is undoubtedly one of the most important reasons for their antimicrobial properties. The extracts of two plants can be used to disinfect hospital equipment surfaces, swimming pools, and milking equipment in livestock farms where the three Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus vulgaris bacteria are found.
Ethical Considerations
Compliance with ethical guidelines
 There were no ethical considerations to be considered in this research.
Funding
This study was extracted from the MA. thesis of the first author in Yadegar-e-Imam Khomeini Yadegar emam Branch, Islamic Azad University.
Authors' contributions
All authors equally contributed to preparing this article.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
The authors would like to thank Yadegar-e-Imam Khomeini Shahre Rey Branch, Islamic Azad University for their financial support, and Dr. Falsafi and the staff of the laboratory in Yadegar-e-Imam Khomeini Shahre Rey Branch (Khosravi and Etemadi) for their cooperation.

 

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