Jorjani Biomedicine Journal

Introduction
In the new century, the tendency of consumers to use fewer chemicals and preservatives has led to extensive research on the benefits and harms of using natural compounds with antimicrobial properties. Further, the resistance of microorganisms to antibiotics poses problems that have become one of the major concerns of human society and the health care system (1). With the advent of antibiotic-resistant bacteria, the effectiveness of existing drugs has diminished, increasing the failure of antimicrobial treatments. In addition, the widespread use of industrial drugs, as well as the misuse of these drugs, can lead to many side effects (2,3). As a result, one way to prevent the development of pathogenic bacteria resistant to antibiotics is to use new compounds that are fundamentally different from the existing industrial source in terms of basic structure with antimicrobial drugs (4). Plants produce secondary metabolites including alkaloids, flavonoids, tannins, glycosides, terpenes, and phenolic compounds. Some of these compounds have complex molecular structures and antimicrobial properties, therefore, in recent years, there has been a strong desire to discover and introduce antimicrobial agents of plant origin (5). Knowledge of chemical compounds in plants is important not only for the discovery of medicinal drugs but also because the use of chemical products of plant origin that have antimicrobial properties help control resistant strains (6). In this regard, the World Health Organization has recommended the use of natural drugs as a reliable method for the treatment of microbial and non-microbial diseases (7). Numerous plants have been identified that have antimicrobial properties, but most of them have not yet been adequately studied (8). Plants as a source of new drugs remain largely unknown, and screening studies to discover the effects of their biologically active compounds and molecules are necessary. The first step in achieving this goal is to investigate their antimicrobial activity in vitro (9).
Artemisia is one of the medicinal plants that is used in traditional medicine to treat some diseases and so far many studies have been performed on it to detect different degrees of inhibitory and antimicrobial effects. The plant is widely grown in a variety of climates, especially in arid regions where plant growth is somehow difficult due to climatic conditions; one of the reasons for this expansion could be the high ability of this plant to adapt to different climatic conditions. Chemically, Artemisia extracts often contain polypranoids and aromatic compounds that can have antimicrobial effects on a range of bacteria due to their phenolic groups in their structure (10).
Although the determination of the active ingredients of the plants is pharmacologically important, it is necessary to first determine the effectiveness of the extracts obtained from them (11). In this regard, it has been noted that in the early stages of the discovery and production of new drugs, the selection of raw plant extracts may be more appropriate than the screening of compounds for this reason (12). In the present study, we conducted a preliminary screening to determine the antimicrobial effect of aqueous, ethanolic, methanolic, acetone/ethanolic and hydroethanolic extracts of Artemisia aqueous on standard P. aeruginosa, S. pyogenes, and oral bacteria sample from rotten teeth, in order to investigate the antibacterial effect of Artemisia for using as a natural mouth wash or antibacterial agent as preservative material in food, cosmetic, and the health industries.
Materials and Methods
Preparing the plant
Artemisia specimens were collected from the Isfahan Medicinal plants market. After collection, the sample was rinsed several times with water, then dried in the shade, and the sample was powdered with a grinder for easy extraction.
Extraction
The extract of the plant prepared in the separate stages with solvents including water, ethanol, hydroethanol, acetone/ethanol, and methanol. Extraction was done by the maceration method for all solvents, but hydroethanolic extract was performed using the Soxhlet method to determine whether the temperature in the extraction process affects the antibacterial properties of the extracts. To prepare the water extract, 24 grams of plant powder was added into an Erlenmeyer flask and increased the volume with distilled water to 100 ml. It was put in a shaker for 3 days, then the extract was first filtered with a sterile lace cloth and then with filter paper. After smoothing, the solution was poured into a petri dish and placed in an incubator at 37 C to evaporate water and dry completely (13). After drying, the extract was kept away from light. The above method was also used to prepare ethanolic, methanolic, and acetone/ethanolic extracts. For preparing hydroethanolic extracts, 24 g of dried Artemisia powder with 200 ml hydroethanolic solvent was placed in the Soxhlet extractor for 7 hours. The solvent was evaporated slowly and was concentrated at 37 C. Then, 5% of Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, United States) was utilized to prepare the extract. Finally, two types of solvents including water and DMSO were used to dissolve the various extracts. The aqueous extract of Artemisia in water and other extracts in DMSO were completely dissolved and finally filtered with 0.22 μm MS ® MCE Syringe Filter from Membrane Solutions Company (14,15).
Evaluation of the antibacterial activity of the extract
In the present study, P. aeruginosa and S. pyogenes, and the oral bacteria sample were used to evaluate the antibacterial activity of Artemisia extract. The oral bacterial sample was randomly chosen by a general sampling of a decayed tooth and no specific strains identification was done. P. aeruginosa and S. pyogenes were clinical strains and were provided from the Al-Zahra University Hospital of Isfahan, Iran. First, equal volumes of each bacterial suspension were prepared according to standard 0.5 McFarland (16) vy7`. The bacteria were first cultured on Mueller Hinton Agar medium (QUELAB's, Canada). To investigate the antibacterial activity of each extract on studied bacteria, extracts were used at a concentration of 50 mg/ml. Dilution antimicrobial experiments were performed twice and in accordance with the recommendations of the Standards Committee of Clinical Laboratories (CLSI) (17). For this purpose, 15 ml of sterile culture medium with an appropriate temperature was transferred into 8 mm Petri dishes. The surface of the culture medium was covered with bacterial suspension, then 5 well with a diameter of 6 mm were made in each plate using a sterile Pasteur pipette. To assure the accuracy of the test and comparison conditions, description gentamicin and nalidixic acid antibiotic discs (Padtan Teb Company, Iran) were settled in two wells (positive control). DMSO was also utilized as a negative control sample in one of the wells. The plant extract suspension was loaded into two wells. Each extract was analyzed on P. aeruginosa, S. pyogenes, and the oral bacteria sample. Finally, all the plates were incubated at 37 ˚C for 24 hours.
After finding the Artemisia extracts that had antibacterial effects, the different concentrations of those extracts were prepared and poured in the prepared wells on Mueller Hinton Agar medium. Seven vials were prepared. 900 microliters of distilled water was poured into each vial. About 100 µl was obtained from the main vial with a concentration of 50 mg/ml and added to 900 µl of distilled water in the first vial and well mixed. Next, 100 µl were removed from the first vial and added to the second vial, and this process continued until the seventh vial. Finally, 100 µl was removed from the last vial and poured out. In this way, a series of dilutions from the 50 mg/ml of extracts were prepared. An equal amount of the extract in each vial was transferred to wells in Mueller Hinton Agar medium containing the cultured bacteria to determine the antibacterial properties of each concentration. All plates incubated at 37 ° C for 24 h. Then the lowest concentration that caused the non-growth zone was considered as the minimum inhibitory dosage. Units from the non-growth area of each dilution were collected and to ACC at the position 1003. In fact, nucleotide transitional mutation of G to A has caused alanine 335 amino acid to be replaced by threonine.
Result
The gained results have revealed that aqueous extract of Artemisia had no effects on P. aeruginosa and S. pyogenes but it has an inhibiting effect on oral bacteria sample. The ethanolic and hydroethanolic extract of Artemisia did not indicate any anti-bacterial effects on all three mentioned bacteria strains. The result of the methanolic extract has shown more impact on different bacteria.
As demonstrated in Table 1, the methanolic extract had a significant antibacterial effect on S. pyogenes and the oral bacteria sample. Also, the acetone/ ethanolic extract had the most inhibiting effect on S. pyogenes.
50 mg/ml of Artemisia extracts was the minimum concentration, which had inhibitory and bactericidal effects. None of the fewer concentrations had an antibacterial effect. (Table 2)

Gentamicin	Nalidixic acid	Hydro ethanolic extract (Soxhlet) 50 mg/ml	Acetone/ ethanolic extract (Maceration) 50 mg/ml	Methanolic extract (Maceration) 50 mg/ml	Ethanolic extract (Maceration) 50 mg/ml	Aqueous extract (Maceration) 50 mg/ml	Concentration                 (mg/ml(       Bacteria Strain
25	0	0	0	0	0	0	P. aeruginosa
30 ± 0.14	15 ± 0.15	0	16 ± 0.09	15 ± 0.21	0	0	S. pyogenes
N/D	N/D	0	0	15 ± 0.37	0	8 ± 0.1	oral bacteria sample

Hydroethanolic extract	Acetone/ethanolic extract	Methanolic extract	Ethanolic extract	Aqueous extract	Substance    Bacteria Strain
-	-	-	-	-	P. aeruginosa
-	50	50	-	-	S. pyogenes
-	-	50	-	50	oral bacteria sample

Discussion
Artemisia extract has been reported to contain many antimicrobial compounds such as phenols, cineol, ketones, camphor, and thymol, all of which interfere with the action of membrane enzymes by destroying cell walls and bacterial proteins; in fact, the presence of antibacterial activity of this plant can be attributed to the presence of these compounds (10,19,20). In Artemis species, there are high concentrations of beneficial substances along with antioxidants that are effective in maintaining the natural flora of the body. These antimicrobial effects are likely to be mediated by a variety of mechanisms, including disruption of the mitochondrial-related proton chain, disruption of membrane proteins and lipids, and inhibition of the sarcoplasmic reticulum calcium pump (21).
According to the findings of a survey, the quality of natural extracts and their antioxidant effects depend not only on storage time, geographical origin, and harvest time but also on the environment and technological factors used for the extraction process. Also, the type of solvent used in extraction is one of the other important factors that are important in causing antioxidant effects (22). The results obtained by Appalasamy et al. Also showed that the extract obtained from Artemisia at a concentration of 6 mg/ml led to inhibitory effects on Escherichia coli and Staphylococcus. This result proves that the active ingredients in the crude extract have synergistic and intensifying effects, thus resulting in a higher overall antimicrobial activity (23). Another study by Jutea et al. revealed that Artemis extract significantly inhibited the growth of Enterococcus hirae (24). Findings of a study also stated that various extracts prepared from Artemis leaves have significant antioxidant properties compared to the standard curve of vitamin C and ferrous sulfate (25).
In a study conducted on Artemisia from Kerman province of Iran, it was found that the essential oil of this plant has antibacterial and fungal effects. Minimum inhibitory concentrations were obtained for Staphylococcus aureus (4 µg/ml), Salmonella typhi (32 µg/ml), E. coli (32 µg/ml), Candida albicans (8 µg/ml), and Aspergillus niger (4 µg/ml) (26). Another study on Artemisia from the city of Babol in Iran also confirmed that all aqueous, ethanolic, and methanolic extracts of Artemisia were able to have significant antibacterial properties in comparison with the blank and antibiotic sample. Aqueous extract with a 30% antibacterial effect of ciprofloxacin on E. coli and methanolic extract with the highest antimicrobial activity with 71% of vancomycin showed an inhibitory effect on the growth of Enterococcus faecalis. Although the aqueous extract had the least inhibitory effect on E. coli and Enterococcus faecalis, the inhibitory effects of this extract were significant compared to the blank sample (27). Based on an in vitro study on aqueous, ethanolic, and methanolic of Artemisia from Markazi province of Iran, it was identified that their MIC were 6250, 3125, and 6250 µg/ml, respectively, so it was concluded that different extracts affect Candida albicans growth and prevent its growth (28). The results of an investigation on Artemisia From the western regions of Isfahan province, Iran, determined that flower essential oil had an inhibitory effect on the growth of pathogenic bacteria, especially gram-positive types (Staphylococcus aureus, Enterococcus faecalis, E. coli) because the diameter of the non-growth zone for was in the range of 8.3-45 mm and the least MIC was 2.5%. However, this essential oil did not affect P. aeruginosa (29).
In the present study, the bacterial effects of different concentrations of aqueous, ethanolic, methanolic, acetone/ethanolic, and hydroethanolic extracts of Artemisia under standard laboratory conditions on P. aeruginosa, S. pyogenes, and oral bacteria sample were investigated.
In most Iranian previous studies, one or two types of solvents have been used for extraction, while in the present study, attempts were made to use different types of Artemisia extracts for investigation. Also, to select the bacterial strain, the species that had not been used in Iranian studies on Artemisia were selected.
Taking into account these two factors (the simultaneous study of several types of extracts and selection of untested strains), it was tried to obtain new and complete information about the antibacterial effect of this plant and to use it for pharmaceutical studies.
The findings showed that aqueous, methanolic, and acetone/ethanolic extracts of Artemisia had significant antibacterial effects on S. pyogenes. All effective extracts at a concentration of 50 mg had antimicrobial effects. Therefore, by using different extraction methods and various solvents, it may be possible to more effectively obtain biomaterials with antimicrobial properties from this plant. Considering that both in this study and in one of the previous studies on the essential oil of the Iranian Artemisia plant, it was found that they have less antimicrobial effects on P. aeruginosa, it seems that in future studies, higher concentrations of these extracts are better to be used for assessment. But overall, it can be concluded with certainty that this plant has a significant effect on gram-positive bacteria and is, therefore, a good choice for use in the food and pharmaceutical industries.
The oral bacterial sample was also randomly selected by a general sampling of a decayed tooth and no specific identification of the strains was performed to evaluate Artemisia antimicrobial overall effect on tooth decaying bacteria. And because of the positive results obtained from inhibitory tests, Artemisia extracts may be used in general for producing all types of mouthwashes and toothpaste. It is a better option compared to the chemical compounds used in these products.
Conclusion
It can be deduced that aqueous, methanolic, and acetone/ethanolic extracts of Artemisia have antibacterial effects on both S. pyogenes bacteria and the oral bacteria sample. This study also revealed that none of the extracts of Artemisia had an antibacterial effect on P. aeruginosa. According to the findings of this study, Artemisia plant extracts can be used in food, cosmetics, and pharmaceutical industries due to its antibacterial properties.