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 field shifted methyne carbon signal at 105.4 ppm and a quaternary carbon signal at 93.7 ppm indicated the compound is highly oxygenated. The 13C-, DEPT, 1H -NMR spectral data of the compound was identified by comparison of its 1H, 13C NMR spectral data with those reported for artemisinin (Cafferata et al. 2009; Margueritte et al. 2018). Its melting point was also comparable with those previously reported for the compound by 153-154°C (Klayman et al. 1984), 150- 152°C (Acton et al. 1993) and 148-151°C (Dahnum et al. 2012). There were different methods of extraction and purification for artemisinin. Dahnum et al. (2012) isolated artemisinin using extraction of A. annua with methanol while stirring, portion of the methanol extract with n-hexane followed by column chromatography. It has been confirmed that artemisinin can be purified from A. annua L. using (Appalasamy et al. 2014) Pressurized Hot Water, Soxhlet extraction and maceration with method at 60°C followed by HPLC (Hao et al. 2002; Sixt and Strube 2017). Tzeng et al. (2007) obtained pure artemisinin by normal column chromatography of ethanol modified SC-CO2 extractions of whole parts of A. annua. The percentage yield of artemisinin (0.004%) we obtained in this study is lower than those reported 0.016% by Dahnum et al. (2012), 0.12% by (ElSohly et al. 2004; ElSohly et al. 2004; ElSohly et al. 1990). Our methods of purification are fastest with less solvent and purification processes and thus economical for production of the antimalarial drug from A. annua leaves when compared with previously reported methods. In the current study, the artemisinin compound was obtained by using Separatory funnel. However, Hao with his co-authors (Hao et al. 2002) obtained artemisinin compound from extract of A. annua L by using Microwave-assisted extraction and Soxhlet method. The same authors employed chloroform, cyclohexane, ethanol, n-hexane petroleum ether, petroleum ether and trichloromethane for artemisinin extraction.
The 1H –NMR spectral data of the compound also indicated the presence of protons on olefinic proton at 5.65 ppm, methyl protons at 0.92 ppm (d, J = 6.6 Hz), 1.15 ppm (d, J = 7.1 Hz), and 1.75 ppm (s) appeared as a. Based on the above spectral data the structure of the AAN-7 was identified as a sesquiterpene lactone dibydro-epideoxyartannrrin B. The compound was previously reported from the stem and leaves Artemisia annua (Brown 2004). The 1H and 13C NMR data of AAN-7 was in a good agreement with those reported by Brown (1992). It has been reported that (Foglio et al. 2002) dibydro-epideoxyartannrrin B when administered orally (100 mg/kg) on the indomethacin ulcer model inhibited the ulcerative lesion index with ED50 values of 55.6 mg/kg indicating the compound have a relationship with an increase prostaglandin synthesis.
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, flavonols, 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 flavonoids (O- and C-glycosylated) and hydroxycinnamic acids derivatives were detected for Artemisia argentea. These phenolic compounds are extracted by using methanol and measured by the Folin-Ciocalteu method (Gouveia-Figueira and Castilho 2011). These authors added that these phenolic compounds have antioxidant capacity. Similar results were observed with other species of Artemisia. Carvalho et al. (2011) isolated both phenolic content and flavonoid compounds from Artemisia species. These compounds are determined by using Folin-Ciocalteau’s reagent and methanolic AlCl3·6H2O, respectively (Carvalho et al. 2011). Similarly, study has shown that the phenolic compounds have been separated by using HPLC method when the Artemisia argentea extracts had been performed by using alcoholic methods of extraction (Gouveia-Figueira and Castilho 2011). For instance, the alcoholic extract of Artemisia argentea was shown to contain a 152.8 mg 100g DW-1 total phenolic content and a 109.20 mg 100g DW-1 flavonoid content i for a given plant material (Carvalho et al. 2011). The phenolic compounds are the dominant antioxidants that show scavenging efficiency 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 identified 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 specifically Artemisia annua. The presence of natural antioxidants such as alkaloid, flavonoids, 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 finding to our suggestion. Other compound with tR=5.0 min had been identified 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 identified as borneol (7.5%), camphor (22.8–42.6%), β-caryophyllene (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 1-epi-cubenol (0.7–5.2%). linalool (0.1–11.9%), β-pinene (6.5%), sabinene (8.2%), and β-thujone (9.8%) were identified from an extract of A. annua. These chemical compositions were characterized by using two-dimensional GC time-of-flight mass spectrometry (MS) (Abad et al. 2012; Ma et al. 2007; Padalia et al. 2011). After GC-MS and GC analysis had been performed, major components of essential oils such as myrcene, trans-thujone and trans-sabinyl acetate with 10.8, 10.1 and 26.4%. percentage yield were obtained from A absinthium, respectively (Lopes-Lutz et al. 2008). These essential oils showed moderate inhibitory effects against certain microbial cells such as Candida albicans and Staphylococcus aureus. However, the same essential oils were shown weak activities against Escherichia coli, Proteus vulgaris, and Salmonella typhimurium (Abad et al. 2012; Ma et al. 2007; Padalia et al. 2011). These discrepancies in terms of degree of inhibition effect could be due to variation of chemical composition found in these essential oils.
Lopes-Lutz et al. (2008) investigated the chemical composition and antimicrobial activity of essential oil isolated from aerial parts of A. absinthium, A. biennis, A. cana, A. dracunculus, A. frigida, Artemisia longifolia Nutt. and A. ludoviciana using GC/MS. The same authors confirmed that Artemisia oils had inhibitory effects on the growth of some pathogenic bacteria and fungi. These pathogens are Escherichia coli, Aspergillus niger, Candida albicans, Cryptococcus neoformans, Fonsecaea pedrosol. Microsporum canis, Microsporum gypseum, Staphylococcus aureus, Staphylococcus epidermidis, and Trichophyton rubrum (Lopes-Lutz et al. 2008).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 finding 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 inflammatory response.
Some unidentified 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 25923T, Pseudomonas fluorescens, 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 finding, an ethyl acetate oil extract of Artemisia absinthium (A.abe) 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 briefly reported that Artemisia oils able to inhibit growth of pathogenic bacteria such as Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis.
Fungi species such as Candida albicans and Cryptococcus neoformans were similarly inhibited by extract oil of Artemisia). Aspergillus niger, Fonsecaea pedrosol Microsporum canis, Microsporum gypseum, and Trichophyton rubrum are well known dermatophytes that were inhibited by Artemisia oils (Lopes-Lutz et al. 2008). It was found that the A. absinthium essential oil contained component such as β-thujone (10.1%), myrcene (10.8%), and trans-sabinyl acetate (26.4%). A. biennis extract oil contains the acetylenes (Z), (E)-en-yn-dicycloethers (11%), cis-β-ocimene (34.7%), and trans-β-farnesene (40%). A. dracunculus oil contained methyl chavicol which is predominant phenylpropanoids and methyleugeno (16.2%) (Abad et al. 2012b).
It has been reported that water, methanol, ethanol, or acetone extracts of artemisinin which derived from Artemisia annua L. have ability for anti-inflammatory, antioxidant and antimicrobial. The acetone extract is the most candidate of inhibitory effect on lipopolysaccharide-induced nitric oxide (NO), prostaglandin E2 (PGE2), and proinflammatory 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 finding (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 micro-organisms 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 findings 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), Bacillus cereus LMG 13569 (39 mm), Enterococcus faecalis CIP 103907 (39 mm), Shigella dysenteria CIP 5451 (31 mm), Staphylococcus camorum LMG 13567 (30 mm). The other strains had sensitivities between 15-28 mm. The best zone of inhibition of methanol extract of Boswellia papyrifera were obtained for Enterococcus faecalis CIP 103907 (30 mm), Bacillus cereus LMG 13569 (27 mm). The other strains had sensitivities between 6-24 mm (Abdoul-latif et al. 2012).
The bacteria are resistant to test extract of all Artemisia spp. This might be due inappropriate concentration of test extract. Or the test extract might be inefficient to inhibit this bacterial strain. Abdoul-latif et al. (2012) is also stated that Proteus mirabilis CIP 104588 is resistant to the methanol extracts of Boswellia sacra and Boswellia papyrifera while Shigella dysenteria CIP 5451 is resistant to the methanol extract of Boswellia sacra only. Essential oils of Boswellia sacra and Boswellia papyrifera present an antimicrobial activity stronger than the ticarcycline for Enterococcus faecalis CIP 103907T, Escherichia coli CIP 105182T, Shigella dysenteria CIP 5451T, Staphylococcus camorum LMG 13567T, Pseudomonas aeruginosa and Proteus mirabilis CIP 104588T (only for essential oil of Boswellia sacra). The methanol extracts of Boswellia sacra and Boswellia papyrifera have an antimicrobial activity weaker than the ticarcycline except for Escherichia coli CIP 105182 (Abdoul-latif et al. 2012) .
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, flavonoids, 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 specifically shown inhibiting effects against certain bacterial cells (Kalemba et al. 2002). In our study, it was confirmed 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 finding, (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 ATCC13076T and S. aureus ATCC 25923T. 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 ATCC1053T, Salmonella enteritidis ATCC13076T and S. aureus ATCC 25923T. 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 classified 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 (Nibret and Wink 2010b)