Phytochemical Investigation and Evaluation of Anti-Microbial Activities of Some Indigenous Artemisia Spp. of Ethiopia: Rapid Method of Isolation Artemisinin From Artemisia Annua


 Artemisia spp are one of the most important traditional medicinal plants of Ethiopia which are used for the treatment of infection and non-infection health problems. The genus Artemisia (Astraceae) consists of about 500 species worldwide. Previous reports indicated that the different species of Artemisia have a wide array of biological activities including antimalarial, cytotoxic, antihepatotoxic, antibacterial, antifungal and antioxidant activity. In this study, the main aim was to investigate chemical components of Artemisia spp. (A. abyssinica, A. absinthium and A. annua) and evaluate their antimicrobial activities against bacterial strains. The results indicated that the crude extract of these plants were effective against some selected strains of bacterial strains. Here we isolated the well-known antimalarial drug artemisinin (7 mg, 0.004%) from Artemisia annua leaves using a rapid n-hexane fractionation method. The n-hexane extract of A. abyssinica, ethyl acetate extract of A. absinthium and n-hexane of A. annua showed varying degrees of inhibiting effect against bacterial strains such as Staphylococcus aureus ATCC 25923T, Salmonella enteritidis ATCC13076T, Klebsiella pneumoniae ATCC1053T, boydii ATCC1233T, Escherichia coli ATCC 25922T, hospital acquired Acinetobacter baumannii. The ethyl acetate extract of Artemisia absinthium (A.abe) showed the maximum inhibiting effect (35 mm) against A. baumannii. The minimum zone of inhibition (< 3 mm) was recorded for test extract of A.ap against Klebsiella pneumoniae ATCC1053T. Ethyl acetate extract of Artemisia absinthium (A.abe) was more effective against these selected bacterial strains and the zone of inhibition ranged from 5-35 mm. The minimum inhibition zone (8 mm) was detected against S. typhimurium ATCC 13311T for both A.ac and n-hexane- EtOAc fraction (8:2) of Artemisia abyssinica. The maximum zone of inhibition (25 mm) for fraction (A.ach F4) of Artemisia abyssinica obtained by column chromatography was recorded against S. pyogen ATCC 19615. However, there was no zone of inhibition detected for boydii ATCC1233T due to these test extracts. Significant variations (P = 0.887) were observed between all test extracts of these medicinal plants at 95% of confidence intervals. There is no zone of inhibition or growth for negative control. But, clear zones of inhibition were detected for positive control due to some standard impregnated disks. Based on our results we recommend that various species of Artemisia seem to have great potential for in-depth investigation for various antimicrobial activities that assists the effort in searching for antimicrobial lead compounds.


Introduction
The practice of exploiting natural products, the ora and fauna, to alleviate pain and to cure diseases of humans as well as domestic animals is as old as the It has been recognized that certain traditional medicinal plants have been employed and act as a rst line difference for microbial pathogens. These medicinal plants have been commonly used for treatment of high fever that occurred due to malaria in countries such as Ghana, Mali, Nigeria and Zambia (World Health Organization. Programme on Traditional Medicine 2002). It has been con rmed that the Artimisia spp have contained essential oil that able to inhibit certain pathogenic Bacteria cells such as Staphylococcus aureus, and Staphylococcus epidermidis). Certain fungi species such as Aspergillus niger, Candida albicans, Cryptococcus neoformans, Microsporum canis, and Microsporum gypseum, Trichophyton rubrum, have also been found to be inhibited by essential oil obtained from Artimisia spp in Canada (Lopes-Lutz et al. 2008a) Artemisia abyssinica (known as 'chikugn' in Ethiopia) is an erect, annual or short-lived perennial herb, 30-60cm high. It is quite commonly used in traditional medicine and in rituals. It has been reported for treatment of disease such as rabies, tonsillitis, gonorrhea, cough, syphilis and leprosy. The fresh roots of the same herbs are used to treat epilepsy in domestic animals (Geyid et al. 2005;Yineger et al. 2007b).
Artemisia annua is an erect aromatic annual herb of up to 2 m in height. It is a common weed over large parts of Eastern Europe and Asia, and has become naturalized in North America. It is cultivated on commercial scale in eastern China, in the Balkans and more recently in India and Africa (Van Wyk 2011). It is an exotic species introduced ten years ago from abroad and currently widely cultivated in southern Ethiopia mainly for its traditional anti-malarial "herbal tea" consumption and as remedy for Hemorrhoid, Asthma and Common cold. It is a source for the production of artemisinin, a sesquiterpene lactone with antimalarial effects against susceptible and multi drug resistant Plasmodium species (Hailu et al. 2013;Nibret and Wink 2010).
The volatile components of Artemisia absinthium, A. abyssinica, A. afra, and A. annua have been characterized using GLC/MS (Nibret and Wink 2010) from extracts of their leaves and aerial parts. Study conducted by Lopes-Lutz et al. (2008) have shown that the extracts of essential oils for these medicinal plants of Artemisia species have been characterized by using GC-MS and GC. It was found that dichloromethane extract of A. absinthium contain camphor (38.73%) major as a major components of compound (Nibret and Wink 2010). Some antioxidant such as beta-carotene or linoleate and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical have also been reported by using GC-MS and GC for extracts of Artemisia absinthium (Lopes-Lutz et al. 2008). Certain natural products or volatile component such as Octa-3,5-diene-2,7-dione,4,5-dihydroxy (A. abyssinica, 54.95%), Epoxylinalool (A. afra, 29.10%) and Deoxyqinghaosu (A. annua, 20.44%) have been obtained from dichloromethane extract for Artemisia species (Nibret and Wink 2010).
A large number of medicinal plant consents such as essential oils, terpenes, sesquiterpenes and alkaloids have been shown to be found in Artemisia species associated with anti-protozoal, antimicrobial and antifungal activities (Bakhiet and Adam 1995). Nevertheless, a considerable amount of research is still needed to explain the curative effect associated with traditional herbal remedies, to identify simple technology that could produce therapeutic agents at low cost for the alleviation of suffering and infectious disease widespread in the world. The wide utilization of the above three Artemisia species for various diseases of infectious and non-infectious origins triggered us to investigate their chemical compositions and evaluate their in vitro effects against some group of infectious bacteria.

Sample collection
The samples of Artemisia absinthium which locally known as "Arity" were collected from around Bale Robe, Indato Gasera (Fig. S1a) and A. annua were obtained from Wondogenet (Fig. S1c) research center, respectively. The people living around Robe Bale commonly use Artemisia absinthium (Fig. S1b) against different ailments. Artemisia absinthium were highly available and purchased from different markets in Ethiopia.

Extraction and fractionation of Artimisia abyssinica
The leaves of Artemisia abyssinica (Local name: Ajoo in Afan oromo) (5 g ) were air dried and extracted with n-hexane/Ethyl acetate (8:2) for one day at room temperature which were then ltered and concentrated using rotary evaporator to yield 2 g blackish oil crude extract. The crude extract (1 g) was then subjected to column chromatography using increased polarities of the n-hexane/ethyl acetate solvent system and yielded fractions 1-6 ( Table 1).

Steam distillation of leaves of Artimisia abyssinica
A 100 g of leaves of the plant was ground and steam distilled for 3 hrs and the distillate was then poured into a Separatory funnel with 100 ml of chloroform.
The organic phase was separated from the aqueous phase by using the Separatory funnel. The organic layer was dried using anhydrous Na 2 SO 4 and ltered.
The oil extracted had been concentrated using Rotary Evaporator and yielded yellowish oil (0.02 g).

Extraction of leaves of and isolation of compounds from Artimisia annua
Isolation of artemisinin from A. annua leaves Ground leaves of A. annua (200 g) were extracted with distilled water (150 ml), while shaking for 8 hrs and ltered using Whatman No. 1 lter paper. The ltrate was then added into a Separatory funnel and fractionated with 150 ml of n-hexane three times. The n-hexane was then concentrated using a rotary evaporator and yielded a white crystal coded as AANH-1 (7 mg, 0.004%).

Extraction of A. abyssinica and A. absinthium leaves
Leaves of A. abyssinica (50 g) and A. absinthium (50 g) were extracted with distilled water (50 ml) by shaking for 8 hrs. The extracts were then ltered using lter paper. The ltrate was then added into a Separatory funnel containing 50 ml of n-hexane each. The organic (n-hexane) layer was then fractionated from the aqueous layer three times. The n-hexane was then concentrated using a rotary evaporator and yielded white solid (30 mg, 0.06%) and (50 mg, 0.1%), respectively. These extracts were then compared with that of n-hexane of A. annua (Fig. S1d) using TLC plate, solvent system n-hexane/EtOAc (7:3).
Methanol extraction and isolation of compounds from leaves of A. annua Methanol extraction of leaves of A. annua Fresh leaves of A. annua were dried, powdered (Fig. S1d) air dried for a week. A 100 grams of powdered leaves of Artemisia annua macerated using methanol three times and ltered. The extract was then evaporated using a rotary evaporator vacuum at a temperature of 40°C. Distilled water (50 ml) was added to the methanol crude extract and partitioned using 50 ml n-hexane three times using a Separatory funnel. The aqueous layer was then further fractionated using 50 ml of ethyl acetate three times. Each extract was concentrated using a rotary evaporator at a temp of 40 o C and yielded n-hexane fraction (21 g, 21 %) and ethyl acetate fractions (9 g, 9%).
Puri cation of n-hexane fraction of leaves of A. annua.
The most viscous n-hexane extract of the plant was fractionated by column chromatography using silica gel 60 as the stationary phase and a mixture of ethyl acetate-hexane with increasing polarity (gradient elution) as a mobile phase. Fractions 1-3 were obtained using n-hexane as an eluent and fraction 4-7 were collected using n-hexane-EtOAc (97:3) and fraction 8-10 were collected using n-hexane/EtOAc (9:1). Fractions 6 and 7 were found as amorphous compounds after concentrated with a rotary evaporator and showed single spots with the same Rf values with n-hexane-EtOAc (97:3). Then fraction 7 was submitted for NMR analysis. Fraction 6 was ( Fig. 4c) stored at 4°C and used for antimicrobial activity (antibacterial and fungal) and antimalarial study.
Characterization of isolated compounds from A. annua The 1 H and 13

Antibacterial activities test
Disk diffusion method and agar well diffusion were used to detect antimicrobial activities of crude extracts, fractions and essential oils leaves of the plants using a Muller Hinton agar media (Merck, Germany) (Beef extract, 2 g; Acid Hydrolysate of Casein, 17.5 g; starch, 1.5 g; Agar, 17 g).

Disk diffusion method
Disk diffusion method of antibacterial activities was performed (Bauer et al. 1966). Brie y, Muller Hinton agar media was used as a culture medium.
Concentrations of 1-2x10 8 CFU/ml of bacterial inoculate were used. McFarland 0.5 was used as standard controls for bacterial inoculum. The media were poured onto 90 mm diameter petri plates until the thickness of the agar was 4 mm so that possible problems of diffusion of the tested products could be prevented. A 0.1 ml of each bacterial solution was inoculated and uniformly distributed onto the plates by means of sterile swabs. Plates were allowed to stand for 15 min. At the same time, 6 mm diameter disks were soaked/ impregnated with the crude extracts, fraction and essential oils of Artemisia spp using different concentrations. The impregnated disks were symmetrically placed onto the medium by using sterile tweezers. One of the disks was soaked with sterile distilled water and used as a negative control. The plates were incubated for 24±2h at 37ºC under anaerobic conditions. The results were evaluated by measuring the areas with no bacterial growth. These experiments were carried out in triplicates and control cultures were prepared for all the strains.

Agar well diffusion
Agar well diffusion was also conducted following the methods of (Sz et al. 2016) with some modi cation. Brie y, about 15-20ml of Mueller Hinton agar was poured on to glass plates of the same size and allowed to solidify. Agar surface of each plate was streaked by a sterile cotton swab with the reference bacterial strain. Agar plates were punched with a sterile cork borer of 6 mm diameter size using cork borer and al diameter of three agar wells were formed. A 100 µl of each sample of diluted crude extracts, fraction and essential oils of the plants were added into respective agar wells. The plates were allowed to standby for 30 min. Different Impregnated antibiotics disks or standard antibiotics were also used as positive control ( Table 4). Three of the disks were soaked with sterile distilled water as a negative control. The plates were incubated at 37°C for 48 h. The results were recorded by measuring the areas with no bacterial growth. These results were obtained after using the following formula: Inhibition value = Inhibition diameter in mm -Disk diameter (6 mm)/2. These experiments were carried out in triplicates and control cultures were prepared for these bacterial strains.

Data analysis
One way ANOVA was used to analyses zone of inhibition due to some test extract obtained from Artemisia spp. Some data was also computed into table and Excel. Post Hoc test was used to determine whether chemosuppression induced by each of the plant extracts was signi cantly different from the chemosuppression in the positive and negative control group.

Result
Thin Layer Chromatography Analysis of Artemisia species leaves extracts About 10-20µL sample of test extract compound was spotted on to TLC plate prepared using silica gel and allowed to run in the solvent system consisting of ethyl acetate and n-hexane (3:7) for 40 min at room temperature. TLC pro le and their Rf values for A. abyssinica, A. absinthium that may contain artemisinin or other sesquiterpene lactones closer in structures with artemisinin were evaluated and required for further analysis.

Isolation of artemisinin from A. annua leaves
In the current study, Artemisinin compound ( Fig. 1a) was extracted from the leave part of A. annua using ethyl acetate extract, n-hexane, and petroleum ether. While extraction, a white crystal (mp 153-154ºC) designated as AANH-1 was isolated from n-hexane extract of the plant and analyzed by TLC and showed a single spot. The 1 H-NMR of the compound showed highly down eld shifted signal 5.88 ppm which appeared as a singlet is due to a presence of proton attached to a carbon containing two oxygen ( Table 2, Fig. 2a).
Additionally 1 H NMR spectrum indicated the presence of three methyl protons two appeared as doublet and one signal singlet in the compound. The compound has 15 carbon signals (3 methyl signals, 4 methylene, 5 methine and 3 quaternary carbons) which indicated the compound is a sesquiterpene ( Table 2, Fig. 2b).
Characterization of AAN-7A Fraction 7 obtained from CC of n-hexane extract of A. annua as colorless oil. It's TLC showed a single spot indicating the compound is pure. It was then analysed by 1 H, 13 C-NMR and DEPT which showed the compound has 15 carbons out of which 3 methyl carbons, 4 methylene carbons, 5 methine carbons and 3 quaternary carbons. 13 C and DEPT spectra of the compound showed it has ester carbonyl carbon at 179.5 ppm and ole nic carbon at 142.3 and 121.8 ppm. The compound has also oxygenated quaternary carbon at 83.2 ppm.
In this Study, sesquiterpene lactone dibydro-epideoxyartannrrin B. was identi ed and designed as AAN-7 (Fig. 1b) (Table 4). During our study, the survey showed that the rural community used to treat different infection using these traditional medicinal plants. These infections may be due to bacteria or fungi. Specially, Artemisia abyssinica which is a native medicinal plant to Ethiopia in the area is a well-known traditional medicine for healing of some infections.
Our study showed that test extract of Artemisia annua and Artemisia absinthium have oil. It was proved that these plants are used to produce a considerable amount of essential oil which might be used to inhibit growth of some hospital acquired bacterial pathogens such as hospital acquired A. baumannii (Fig. 3a). In this study, the crude extract of Artemisia absinthium ethyl acetate extract (A.ab e ) showed the maximum inhibiting effect (35mm) ( Table-4) and followed by n-hexane extract of Artemisia annua (A.ah) against Hospital acquired A. baumannii in which zone of inhibition was 34 mm (Fig. 3a). The minimum zone of inhibition was recorded for ( Table 4) test extract of A.ap.
The minimum inhibition zone (8mm) was detected against S. typhimurium ATCC 13311 T for both A.ac and EtOAc oils (8:2) of fractionated test extract. The maximum zone of inhibition (25mm) for fractionated test extract of Artemisia abyssinica (A.ach F4) was recorded against S. pyogen ATCC 19615 (Fig. 3b). Signi cant variations (P=0.887) were observed between all test extracts of these medicinal plants at 95% of con dence intervals. During this study, the minimum zones of inhibition were detected for the bacterial strains that were sensitive to the test extract of Artemisia spp in the disk diffusion assay. However, the maximum zone of inhibition was detected for the same medicinal plants in the agar well diffusion assay.
In the present study, our nding showed that Hospital acquired A. baumannii is highly sensitive for test extract of A.ah and A.abe with a range of zone of inhibition (15-35mm) in diameter (Fig. 3c). The same bacterial species is highly sensitive for some standard impregnated commercial disks (Fig. 3d). In the current study, A.abe is especially more potent against the staphylococcus aureus (Fig. 3e&f) In this study, the maximum zone of inhibition was 19.67 mm and the minimum inhibition zone was 12 mm against Escherichia coli ATCC 25922 due to A.ah (Fig. 4a). For A.ach oil, the maximum zone of inhibition was recorded for Escherichia coli ATCC 25922 and followed by A.achF4 which is 15 mm in diameter (Fig. 4b).
The test extract of A.ah, A.ap and A.abe were also used to inhibit the growth of Klebsiella pneumoniae ATCC1053 T which is a food borne bacterial pathogen.
In the present study, the minimum zone of inhibition (<3mm or range between <3mm -5mm) due these test extract is recorded for Klebsiella pneumoniae ATCC1053 T . This bacterial spp may develop resistance against the test extract of A.ah A.ap and A.abe. In this study, both disk and agar well diffusion was conducted for Shigella boydii ATCC1233 T However, there was no zone of inhibition detected for Shigella boydii ATCC 1233 T (Fig. 4a). In this study, the test extract of A.ah and A. abe have synergistic effect on hospital acquired A. baumannii, a hospital acquired pathogenic bacterial spp (Fig. 4b).
In the present study, the distilled water was used as negative control using agar well diffusion and disk diffusion against Salmonella enteritidis ATCC13076 T . Standard impregnated disks were also used as positive control. There is no zone of inhibition or growth for negative control (Fig. S2a). But, a very clear zone of inhibition was detected for positive control due to some standard impregnated disks (Fig. S2b). There was a best synergistic effect for Tetracycline, Cipro oxacin and Chloramphenicol against Salmonella enteritidis ATCC13076 T . At the same time, the test extract of Artemisia species such as A.ah, A.ap, A.ac and A.abe were used against the same bacterial species. A test extract of A.ap has inhibitory effect against Salmonella enteritidis ATCC13076 T with a very clear zone of inhibition (34 mm) (Fig. S2c). For instance, traditionally the community uses A. abysinthium for treatment of gastric pain. As shown in Fig. 4d, no inhibition effect against Shigella boydii by A.ap. extracts.

Discussion
Our Samples had been characterized by using NMR. The carbonyl signal at 172.1 ppm indicated the compound has an ester group. A highly down eld shifted methyne carbon signal at 105.4 ppm and a quaternary carbon signal at 93.7 ppm indicated the compound is highly oxygenated. The 13 C-, DEPT, 1 H -NMR spectral data of the compound was identi ed by comparison of its 1 H, 13  In the present study, artemisinin was obtained from the test extract of A.ap and A.ah.. All test extracts contain certain essential oil. These test extracts may also contain some other aromatic compounds. These Artimisia species extracts were also shown to inhibition effects. These inhibition effects could be due to the presence of artemisinin, sesquiterpene and other aromatic compounds. In line with this study, it has been reported that (Hanscheid and Hardisty 2018) artemisinin that extracted from A. annua have shown antimalarial therapy, The same authors stated that microbial cells might be developed resistance against artemisinin compounds if it will be added into the list of choice drugs. Similarly in our study, no inhibition was observed for Shigella boydii ATCC1233 T suggesting that this pathogenic bacterial strain may developed resistance toward A. annua extracts that predicted to be among artemisinin compound. It was found that the essential oils derived from A. absinthium were extracted using microwave assisted process, distillation in water and direct steam distillation methods. These extracts of Artimisia species were shown for their relative toxicity against ascaricides and spider mite, Tetranychus urticae (Chiasson et al. 2001). Study were shown that a sesquiterpene (C15H24) compound that were derived from Artemisia absinthium by using present direct steam distillation (DSD) contained essential oil after the Chromatographic analysis had been performed. These oil have shown lethal effect against adult Tetranychus urticae (Chiasson et al. 2001).
Certain total phenolic content have been detected for A. absinthium leaves extracts. This extraction was determined by using the Folin-Ciocalteu (FC) method. These phenolic compounds were included such as benzoic acid, Catechins, avonols, hydroxycinnamic acids, hydroxybenzoic acids, and Gallic acid (Carvalho et al. 2011). The same authors predicted that these phenolic compounds were used as antioxidants. A reversed-phase high-performance liquid chromatography method (RP-HPLC) coupled with diode-array detection (DAD) and electrospray ionization mass spectrometry (ESI/MS) analysis have shown that certain phenolic compound such as avonoids (O-and C-glycosylated) and hydroxycinnamic acids derivatives were detected for Artemisia argentea. . The phenolic compounds are the dominant antioxidants that show scavenging e ciency due to the presence of free radical compounds which is a reactive oxygen species. These free radicals are commonly reported for diversity of plant species (Prior et al. 2000). For instance, (Chialva et al. 1983) studied that about 19 samples of A. absinthium these collected from France, Romania, Siberia and Italy, found to contain free radicals compounds.
Artemisinin has been identi ed as the anti-malarial principle of the plant. The artemisinin derivatives are nowadays established as anti-malarial drugs with activity towards drug-resistant Plasmodium infections (Klayman 1993). Other natural products may be existed within these Artemisia species in addition to artemisinin compound that detected for speci cally Artemisia annua. The presence of natural antioxidants such as alkaloid, avonoids, phenolic compounds, and terpenes in the aerial parts of A. abysssinica party elaborates the observed effects of plant extract (Taramelli et al. 1999) which is a similar nding to our suggestion. Other compound with tR=5.0 min had been identi ed as 5-O-caffeoylquinic acid Artemisia argentea (Gouveia-Figueira and Castilho 2011). The same authors reported catechins, ferulic and caffeic acid from A. argentea and other six related species.
It has been reported that certain chemical components of the essential oil (91-97.1%) were predicted for A. annua. These essential oil components were found to be varying from 0.3-0.7% during the growth period. The major compositions were identi ed as borneol (7.5%), camphor (22.8-42.6%), βcaryophyllene (2-9.2%), 1,8-cineole (3.7-8.4%), (E)-β-farnese (1.3-8.5%), and germacrene D (0.5-7.3%). Meanwhile, other chemical components such as 1epi-cubenol (0.7-5.2%). linalool (0.1-11.9%), β-pinene (6.5%), sabinene (8.2%), and β-thujone (9.8%) were identi ed from an extract of A. annua. These chemical compositions were characterized by using two-dimensional GC time-of-ight mass spectrometry (MS) ( .Certain fungi disease may be targeted due to some natural products these available within part of Artimisia species. In agreement with this prediction, the dried leaves of Artemisia annua (Jiao et al. 2018) have been shown to be effective against avian coccidiosis which is a fungi disease. These natural products may be existed within leave or root parts of Artimisia species. For instance, in the current study the maximum inhibition zone were detected for ethyl acetate extract using dried and powdered A. annua leave part, a similar nding to (Jiao et al. 2018). The same authors stated that Artemisinin and Artemisia annua leaves alleviate Eimeria tenella infection by facilitating apoptosis of host cells and suppressing in ammatory response.
Some unidenti ed bioactive compound may be used to damage certain structure of bacterial cells. Test extract of Artemisia absinthium ethyl acetate extract (A.abe) was more effective against these selected bacterial strains and their zone of inhibition was ranged from 5-35mm. It was found that the whole part of Artemisia absinthium ethylacetate and chloroform extracts used to inhibit test microorganisms such as Staphylococcus aureus ATCC 25923 T , Pseudomonas uorescens, Bacillus brevis FMC and Bacillus megaterium DSM with 8-16 mm/20ml inhibition zone (Erdogrul 2002) which is strongly in agreement with our current results.
The essential oil of one of A. absinthium, also showed antibacterial activity against commonly known pathogens like Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus (Blagojević et al. 2006). In agreement with this nding, an ethyl acetate oil extract of Artemisia absinthium (A.ab e ) tends to show the maximum inhibiting effect (35 mm) against Hospital acquired A. baumannii. This extract might have contained inhibiting bioactive compound that able to target this pathogenic strain. In line with this study, it was stated that the GC/MS used to show the chemical composition and its antimicrobial activity of essential oil extracted from aerial parts of A. absinthium, A. cana, A. biennis, A. dracunculus, A. frigida, Artemisia longifolia Nutt, and A. ludoviciana of wild sages from western Canada (Lopes-Lutz et al. 2008). The same authors brie y reported that Artemisia oils able to inhibit growth of pathogenic bacteria such as Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis.
It has been reported that water, methanol, ethanol, or acetone extracts of artemisinin which derived from Artemisia annua L. have ability for anti-in ammatory, antioxidant and antimicrobial. The acetone extract is the most candidate of inhibitory effect on lipopolysaccharide-induced nitric oxide (NO), prostaglandin E2 (PGE2), and proin ammatory cytokine (IL-1β, IL-6, and IL-10) production. However, the ethanol extract have the best antioxidant activity due to its highest free radical scavenging activity (91.0±3.2%), similar to α -tocopherol (99.9%) (Kim et al. 2015).
The Methanol extract of the Artemisia vulgaris showed the highest antioxidant and antibacterial properties when compared to the essential oil of the same plants. It has also been stated that the artemisinin compound the ability of inhibitory effect against actinomycete mcomitans, Aggregatibacter,, Fusobacterium nucleatum subsp. animalis, Fusobacterium nucleatum subsp. polymorphum, periodontopathic and Prevotella intermedia microorganisms. For instance, methanol extract used to inhibit F. nucleatum subsp. Polymorphum and Prevotella intermedia which is similar with the current nding (Kim et al. 2015). Johnson et al. (2013) further stated that the methanol solutions of the extracts were found to have a broad spectrum activity against all the microorganisms tested. In the present study, the maximum zone of inhibition for Staphylococcus aureus ATCC 25923 (Fig. 3) was 20.33 mm and 20 mm in diameter (Table 4&5) due to A.abe and A.ach F4, respectively which is more potent than the ndings of Johnson et al. (2013).
The interaction in the oil constituents was resulted in synergy effect on microbial spp. except for Salmonella typhi and Escherichia coli ATCC 25922 with zone diameter of 6 mm each (Johnson et al. 2013). The same author further stated that a minimum zone diameter (6mm) observed for Salmonella typhi and Escherichia coli strains. However, the maximum zone of inhibition were recorded for Candida albicans and Candida albicans ATCC 90028 (30 mm) strains when Tangerine oil extract had been used (Johnson et al. 2013).
In line with this study, (Patil et al. 2011) extracted and obtained physiologically active composition in pure or mixture form from some Artemisia sp. such as A. dracunculus, A. herba-alba, A. judaica, A. vulgaris, A. abysinica, A. absynthicum, A. afra, A. cannariensis, A. pallens, A. annua, A. abrotanum, A. ludoviciana, and A. capillaris or A. scoparia (Patil et al. 2011). Moreover, the same author stated these plants used to prevent or treat (pre) diabetes and associated accompanying diseases or secondary diseases. The best zone of inhibition for essential oil of Boswellia papyrifera for bacteria was obtained for Salmonella enterica CIP 105150 (40 mm In Afro-Asian countries, many species of Artemisia such as A. abyssinica are used in folk medicine as anthelmintics, antispasmodics, antirheumatics and antibacterial agents due to the presence of the presence of certain natural products such as alkaloids, anthraquinones, avonoids, sterols, tannins, and volatile oils (Adam et al. 2000). Certain essential oils are also reported from Artemisia asiatica Nakai. These essential oils have shown antibacterial and antifungal effects. They include such as 1,8-cineole, selin-11-en-4alpha-ol and monoterpene alcohols fraction. These essential oils have been found to be effective against Bacillus subtilis, Aspergillus fumigatus, Candida albicans, Escherichia coli, Rhodotorula rubra, Pseudomonas aeruginosa and Staphylococcus aureus. The monoterpene alcohols have speci cally shown inhibiting effects against certain bacterial cells (Kalemba et al. 2002). In our study, it was con rmed that Artemisia annua have shown antimicrobial properties. It could be due to the presence of secondary metabolites, a work similar to Appalasamy et al. (2014). In agreement with our nding, (Appalasamy et al. 2014) extracted the main bioactive compound which is artemisinin from A. annua L. that collected from Malaysia. due to the tropical hot climate, A. annua could not be planted for production of artemisinin, the main bioactive compound. The same authors found out that the A. annua L. leave extract is able to inhibit certain gram-negative and Gram-positive and Gram-negative bacteria. However, the leaf extract of A. annua L is unable to inhibit Candida albicans (Appalasamy et al. 2014). It was found that aerial parts of A. abyssinica and A. herba-alba extracts are also employed and shown in molluscicides effect. It was suggested that the molluscicides are due to the presence of sesquiterpene lactones and terpenoid compounds in these medicinal plants (Segal 1985;Watt and Breyer-Brandwijk 1962).However, in our study, inhibition activities were detected for Artemisia annua petroleum ether extract, Artemisia absinthium ethyl acetate extract and A. abyssinica were detected against certain medically important bacterial pathogens such as E. Coli ATCC 25922 T , Hospital acquired A. baumannii, Salmonella enteritidis ATCC13076 T and S. aureus ATCC 25923 T . This could be due to the presence of Artemisinin and other sesquiterpene compounds. These compounds may target certain bacterial structures such as cell wall, cell membrane, genetic material or ribosome that are used for protein synthesis. It has been observed that the extract compounds obtained from Artemisia species have shown insecticidal and repellent activities (Malik and Mujtaba Naqvi 1984).
In the current study, these Artemisia absinthium, a traditionally known as Ariti have been shown inhibitory effects against certain pathogenic bacterial cells such as E. Coli ATCC 25922 T , Hospital acquired A. baumannii, Klebsiella pneumoniae ATCC1053 T , Salmonella enteritidis ATCC13076 T and S. aureus ATCC 25923 T . Mostly, in Ethiopia these Artemisia species are employed for ritual during solemn ceremonies.
The Artemisia abyssinica have shown inhibitory effects against certain bacterial pathogens. These Artemisia species are traditional referred to as Ajo. It is highly grown some highland environments. These plants are commonly employed for house cleaning rather than as traditional medicinal plants. It has repellent and pungent odors. The same Artemisia species traditionally known as Ather in Saudi Arabia (Adam et al. 2000), where these plants are abundantly grown. The same authors classi ed Artemisia abyssinica under the family of Asteraceae. Furthermore, it was stated that A. abyssinica have shown certain effects on the growth, haematological (treatment of the blood) and organ pathology in rats at a low concentration (Adam et al. 2000). The test extract of Artemisia species such as A.ah, A.ap A.ac and A.abe were used against certain bacterial species. A test extract of A.ap has inhibitory effect against Salmonella enteritidis with a very clear zone of inhibition (34 mm) (Fig. S2c). It was found that leaves and aerial parts of extract for Artemisia species such as Artemisia absinthium, A. abyssinica, A. afra, and A. annua have been found to be effective against Trypanosoma brucei brucei with in Ethiopia (

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.