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. 2024 Jul 10;9(29):31508–31520. doi: 10.1021/acsomega.4c01096

Antibacterial and Cytotoxicity of Extracts and Isolated Compounds from Artemisia abyssinica: A Combined Experimental and Computational Study

Dawit Tesfaye †, Milkyas Endale ‡, Venkatesha Perumal Ramachandran †, Emebet Getaneh §, Guta Amenu §, Leta Guta §, Taye B Demissie ∥, Japheth O Ombito ∥, Rajalakshmanan Eswaramoorthy ⊥, Yadessa Melaku †,*
PMCID: PMC11270564  PMID: 39072116

Abstract

graphic file with name ao4c01096_0004.jpg

Artemisia abyssinica is a widely cultivated hedge plant in Ethiopia. Traditionally, they have been used to treat a variety of health conditions, including intestinal problems, infectious diseases, tonsillitis, and leishmaniasis. Silica gel chromatographic separation of the methanol and ethyl acetate extracts of the leaves, roots, and stem barks of A. abyssinica led to the isolation of 12 compounds, labeled as 1–12. Among these, compounds 1, 3, 4, 5, and 7–11 are reported as new to the genus Artemisia. The extracts and isolated compounds from A. abyssinica were evaluated for their in vitro antibacterial activity against four bacterial strains: Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, using the disc diffusion assay. All of the extracts displayed weak antibacterial activity, with inhibition zone diameters (IZDs) ranging from 6.10 ± 0.3 to 9.30 ± 0.20 mm. The isolated compounds, on the other hand, exhibited weak to moderate antibacterial activity, with IZDs ranging from 6.00 ± 0.300 to 13.50 ± 0.50 mm. The most potent antibacterial activity was observed for compound 6, which showed an IZD of 13.30 ± 0.50 mm against E. coli and 13.50 ± 0.50 mm against P. aeruginosa. This activity was comparable to that of the positive control ceftriaxone, which had IZDs of 14.1 ± 0.3 and 13.8 ± 0.5 mm against E. coli and P. aeruginosa, respectively. The in silico molecular docking analysis against DNA gyrase B revealed that compound 5 showed a higher binding affinity (−6.9 kcal/mol), followed by compound 10 (−6.7 kcal/mol) and compound 12 (−6.3 kcal/mol), whereas ciprofloxacin showed −7.3 kcal/mol. The binding affinities of compounds 5, 11, 10, and 9 were found to be −5.0, −4.3, −4.2, and −4.0 kcal/mol against S. aureus Pyruvate kinase, respectively, whereas ciprofloxacin showed a binding affinity of −4.9 kcal/mol, suggesting that compound 5 had a better binding affinity compared with ciprofloxacin. The effect of extracts of A. abyssinica was evaluated for cytotoxic activity against the breast cancer cell line (MCF-7) by the MTT assay. The extracts induced a decrease in cell viability and exerted a cytotoxic effect at a concentration of 20 μg/mL. The highest percent cell viability was observed for the methanol extract of the stem (92.9%), whereas the least was observed for the methanol extract of the root (34.5%). The result of the latter was significant compared with the positive control. The binding affinities of the isolated compounds were also assessed against human topoisomerase inhibitors IIβ. Results showed that compound 5 showed a binding affinity of −6.0 kcal/mol, followed by 11 (−5.4 kcal/mol), 10 (−5.0 kcal/mol), and 11 (−4.9 kcal/mol). Similar to ciprofloxacin, compounds 4, 5, 6, 9, 10, and 12 comply with Lipinski’s rule of five. Overall, the comprehensive investigation of the chemical constituents and their biological activities reinforces the traditional medicinal applications of A. abyssinica and warrants further exploration of this plant as a source of novel therapeutic agents.

1. Introduction

The genus Artemisia in the family Asteraceae consists of over 500 species geographically distributed all over the world except Antarctica.1 Species of this genus can be perennial, biennial or annual grasses, shrubs or bushes that are generally aromatic, with erect or ascending stems.2Artemisia species have a wide range of uses in folk medicine and have been the subject of numerous chemical and biological studies.3 The genus has been reported to be rich in various secondary metabolites such as flavonoids, lignans, sesquiterpene lactones, coumarins, caffeoylquinic acids, acetylenes, and sterols,4 which possess various biological activities including diuretic,5 antimalarial, antimicrobial, antiviral, anthelmintic, anti-inflammatory, bronchodilator, hypolipidemic, antihypertensive, antioxidant, immunomodulatory, cytotoxic, antitumor, and laxative.6,7

Cancer is a disease characterized by abnormal cell division and proliferation that results from the disruption of molecular signals that control these processes. The prevalence of this disease is rising rapidly in Africa, Asia, and Central and South America, which in fact account for about 70% of cancer deaths in the world. The most commonly diagnosed cancer is female breast cancer, followed by lung cancer.8−10 Previous studies on Artemisia species have shown medicinal properties, such as antibacterial and anticancer effects. Many phytochemicals exert their cytotoxic effects by acting as cell cycle and apoptosis regulators, as well as anti-inflammatory agents.9

Artemisia abyssinica Sch. Bip. ex A. Rich is an aromatic, gray, silky-hairy plant with pale yellow flower-heads and is well-known as a stimulant and an analgesic.11 The plant has been used as an anthelmintic, antispasmodic, antirheumatic, and antibacterial agent.12 The aforementioned uses could be attributed to the presence of various secondary metabolites such as alkaloids, flavonoids, sterols, tannins, anthraquinones, and volatile oils.13 The plant is a popular hedge plant in Ethiopia and is traditionally used for intestinal problems, infectious diseases, and as an antileishmanial.14 The whole herb is employed to alleviate tonsillitis, and a traditional infusion is consumed as a remedy for colds and illnesses in children.15 Despite the tremendous traditional uses of the plant against various arrays of diseases, there are limited previous studies of the chemistry and biological activities of this species. Hence, in this paper, a comprehensive investigation of the chemical constituents, antibacterial activities, and in silico molecular docking analysis of extracts and compounds isolated from A. abyssinica is presented.

2. Materials and Methods

2.1. Plant Material Collection, Authentication, and Preparation

The whole plant of A. abyssinica was collected from Tiyo Woreda, Arsi zone [7°57′N 39°7′E/7.95°N 39.117°E/7.95] southeastern Ethiopia with an elevation of 2430 m16 on July 2020. The specimen was authenticated by Mr. Wege Abebe of Adiss Ababa University, and the voucher specimen was deposited in the National Herbarium, Addis Ababa University (Code: G-003). The plant material was washed with distilled water, air-dried, powdered using a milling machine, and then stored in a polyethylene bag in a refrigerator.

2.2. Extraction

The air-dried and powdered leaves, stems, and roots (each 300 g) of A. abyssinica were extracted successively by maceration using n-hexane (each 1.5 L), ethyl acetate (each 1.5 L), and methanol (each 1.5 L) at room temperature for 72 h. Removal of the solvent at 40 °C under reduced pressure gave 1 g (0.3%) n-hexane, 5.3 g (1.8%) EtOAc, and 11.3 g (3.8%) MeOH extracts for the leaves; 0.8 g (0.27%) n-hexane, 3.5 g (3.5%) EtOAc, and 10.2 g (4.4%) MeOH extract for the stems; and 0.6 g of n-hexane (0.2%), 1.1 g (0.7%) EtOAc, and 5.1 g (1.7%) MeOH extracts for the roots. Analysis using TLC showed that the EtOAc and methanol extracts had similar spots and were hence combined for the isolation of compounds.

2.3. Isolation

The combined extract of the leaves of A. abyssinica (16 g) was adsorbed on silica gel (12 g) and subjected to silica gel column chromatography over silica gel (240 g) eluted by increasing the gradient of EtOAc in n-hexane to afford 230 fractions, 10 mL each. Fractions 1–10 (10%) EtOAc in n-hexane were subjected to silica gel column chromatography to afford 12 subfractions, each 10 mL. Subfractions 8–10 were identified as compound 1 (53 mg). Fractions 17–24 (20% EtOAc in n-hexane) after silica gel column chromatography afforded 8 subfractions, each 10 mL. Subfractions 4–7 were identified as compound 2 (30 mg). Fractions 25–47 (20% EtOAc in n-hexane) were fractionated over silica gel column chromatography to afford 12 subfractions, each 10 mL. Subfractions 5–10 were identified as compound 3 (51 mg). Fractions 81–95 (30% EtOAc in n-hexane) were combined and rechromatographed to afford 6 subfractions, each 10 mL. Subfractions 4–6 were identified as compound 4 (20.6 mg). Fractions 190–199 (90% EtOAc in n-hexane) were subjected to silica gel column chromatography to afford 11 subfractions, each 10 mL. Subfractions 7–11 and subfraction 12 were identified as compounds 5 (33 mg) and 6 (21 mg).

The combined extract of the stem of A. abyssinica (13 g) was adsorbed with the same amount of silica gel and subjected to column chromatography over silica gel (220 g) using n- hexane: EtOAc of increasing polarity afforded 200 fractions, 10 mL each. Fractions 11–30 (20% EtOAc in n-hexane), after silica gel column chromatography, afforded 16 subfractions, each 10 mL, from which subfractions 6–14 and subfractions 15–16 were identified as compounds 7 (20 mg) and 8 (15 mg), respectively. Fractions 141–152, eluted using n-hexane: EtOAc (4:1), were subjected to silica gel column chromatography to afford 20 subfractions, each 10 mL, and subfractions 11–18 yielded compound 9 (22 mg). Fractions F121–F130 (0.5 g) were eluted using 40% EtOAc in n-hexane (6:4) after silica gel column chromatography to afford 15 subfractions, each 10 mL, and subfractions 7–15 were identified as compound 10 (30 mg).

The combined extract of the root of A. abyssinica (6 g) was adsorbed on silica gel (10 g) and subjected to silica gel column chromatography over silica gel (230 g) using n-hexane: EtOAc of increasing polarity to afford 200 fractions, 10 mL each. Fractions 11–20 (20% EtOAc in n-hexane) were fractionated over silica gel column chromatography to afford 8 fractions, each 10 mL, while fractions 3–6 gave compound 11 (35 mg). Fractions 25–30 (20% EtOAc in n-hexane) were rechromatographed over silica gel to afford 9 subfractions, each 10 mL, from which subfractions 4–7 were identified as compound 12 (40 mg).

2.4. In Vitro Biological Activity

2.4.1. Cytotoxicity Assay

MCF-7 breast cancer cell lines were obtained from NCCS (National Center for Cell Science), Pune, India. The cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. Upon reaching confluency, the cells were trypsinized and passaged to be used for further assays. The antiproliferative effect of the extracts of A. abyssinica (20 μg each) was studied on human breast cancer cell lines (MCF-7); doxorubicin was used as a positive control. The cells were grown in T25 culture flasks containing DMEM and L-15 supplemented with 10% FBS and 1% antibiotics (100 μg mL–1 penicillin and 100 μg mL–1 streptomycin). The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Upon reaching confluence, the cells were detached using a Trypsin–EDTA solution and were subcultured at a density of 5000 cells per well. At 50% confluence, the culture medium was aspirated, and the cells were treated with 20 μg of plant extract in DMSO (20 μL) for 24 h at 37 °C in the CO2 incubator. Later, cells were incubated with MTT (4 mg mL–1) for 3 h. The absorbance was measured at 540 nm with a standard microplate reader. The experiments were done in triplicate, and the results were reported as the M ± SD.

2.4.2. Antibacterial Activity

The antibacterial activity of the extracts and isolated compounds was evaluated using a disc diffusion assay.17 Clinical bacterial strains with American standards, including Escherichila coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), and Staphylococcus pyogens (ATCC 19615), were obtained from the Oromia Regional Laboratory and Quality Control, Adama, Ethiopia. McFarland number 0.5 standard was prepared by mixing 9.95 mL 1% H2SO4 in distilled water and 0.05 mL 1% BaCl2 in distilled water to estimate bacterial density.18 The prepared sample was stored in an airtight bottle and used for comparison of bacterial suspension. Extracts and isolated compounds were prepared in DMSO to give 30, 15, 7.5, and 3.75 μg/mL concentrations. Bacteria cell suspensions were adjusted to the 0.5 McFarland turbidity standard to prepare a 1.5 × 108 CFU/mL inoculum. Each bacterial suspension was inoculated on Mueller–Hinton agar plates, and the plates were allowed to dry for 5 min. The sterile filter paper disks (6 mm in diameter) were soaked in 30, 15, 7.5, and 3.75 μg/mL concentrations of the crude extracts and isolated compounds. The extracts and isolated compounds soaked in filter paper disks were placed on the inoculated Mueller–Hinton agar plates, and the analyses were conducted in triplicate. Ciprofloxacin (CPFX, 30 μg/mL) disk was used as the positive control, and DMSO was used as the negative control. The plates were incubated for 18 h at 35 ± 2 °C. After incubation, the zones of inhibition were recorded as the diameter of the growth-free zones measured in mm using an antibiotic zone reader. The experiments were done in triplicate, and the results were reported as the M ± SD.

3. Methodology

3.1. Computational Study

The test compounds were subjected to computational studies to predict their drug-likeness property, viz., Lipinski’s rule of five (ROF),19 Veber rule,20 pharmacokinetics, and drug-likeness using SwissADME,21 PreADMET,22 and in silico cytotoxicity using ProTox-II23 online tools.

3.2. In Silico Molecular Docking

The test compounds’ chemical structures were drawn using ACD/ChemSketch, and Gaussian0924 software was used to optimize the structures by the DFT/B3LYP25 technique, utilizing basis sets of 6-31G(d,p).25 The energy-minimized ligands were then put into the docking procedure. The binding site attributes: center_x = 61.680259; center_y = 28.330852; center_z = 64.290148; and size_x; size_y; size_z = 20. The targets, Escherichia coli DNA gyrase (PDB ID: 6F86), S. aureus Pyruvate kinase (PK) (PDB ID: 3T07), and Human topoisomerase IIβ were retrieved from the Protein Data Bank (https://www.rcsb.org/). For the docking studies, S. aureus PK (PDB ID: 3T07) was used as a target, and CPFX was used as a reference control. Binding site attributes: center_x = −12.446889; center_y = 0.983556; center_z = 2.585889; and size_x; size_y; size_z = 20. Biovia Discovery Studio 2020 was used to prepare the targets.26 The protein complex’s water molecules were removed, and the bound ligand was chosen to determine the characteristics of the binding sites before being removed from the complex.27 Validation of the binding sites was done by redocking the bound ligand with the target protein.28 The target was augmented with polar hydrogen atoms and the necessary charges. Using AutoDoc Vina (MGLTools-1.5.6), the target and ligand were generated in the necessary format (pdbqt) for docking, and docking was performed.27 Hundred conformers and their associated binding energies were generated for each ligand during the docking procedure.29 Using Biovia Discovery Studio 2020, the receptor and ligand interactions were ascertained by selecting the conformation with the lowest binding energy. In the present study, human TOP2β (PDB ID: 3QX3) was used as a target, and EVP was used as a reference control. Binding site attributes: center_x = 33.025762; center_y = 95.765381; center_z = 51.567476; size_x; size_y; size_z = 20.

4. Results and Discussion

Silica gel chromatographic fractionation of various parts of the extracts of A. abyssinica has led to the isolation of 12 compounds (Figure 1). The detailed structural elucidations were presented as follows.

Figure 1.

Figure 1

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Compounds isolated from Artemisia abyssinica.

Compound 1 was obtained as a white solid from the combined ethyl acetate and methanol extracts of the leaves of A. abyssinica. The 1H NMR spectrum (Figure S1) revealed signals in the region δH 1.2 to 0.80. The signal observed at δH = 0.80 is due to methyl protons. The 13C NMR spectrum (Figure S2) showed a signal due to sp3 quaternary oxygenated carbon at δC 77.2 and methyl carbon peaks at δC 32.9, 32.8, and 32.7. The remaining signals at δC 31.9, 29.7, 26.7, 29.6, 29.4, 27.1, and 22.7 were due to methylene carbons, which were supported by the DEPT-135 spectrum (Figure S3). The above spectral data, is in good agreement with previous reports,30 suggesting the compound a diterpene named 3,7,11,15-tetramethylhexadecan-3-ol reported from this species for the first time. The structure of the compound was also confirmed by 1H–1H COSY and HMBC spectra (Figure S4).

Compound 2 was obtained as a white solid from the combined ethyl acetate and methanol extracts of the leaves of A. abyssinica. The 1H NMR spectrum (Figure S5) revealed oxymethylene signals at δH 3.98 (2H, t, J = 6.4 Hz), methylene attached to carbonyl at δH 2.22, and terminal methyl protons at δH 0.8 (3H). The 13C NMR spectrum with the aid of the DEPT-135 (Figures S6 and S7) showed diagnostic signals due to ester carbonyl and oxymethylene at δC 174.1 and 64.4, respectively. The signal characteristics of methylene adjacent to carbonyl carbon appeared at δC = 34.4. The above spectral data, along with the literature reported for the same compound,31 suggest that compound 2 is identical with a fatty acid ester named propyl stearate. This was confirmed by the spectral data obtained from 1H–1H COSY and HMBC (Figure S8).

Compound 3 was isolated as a white solid from combined ethyl acetate and methanol extracts of the leaves of A. abyssinica. The 1H NMR spectrum (Figure S9) revealed an sp3 oxygenated methine signal at δH 4.42 (1H, m), oxymethylene at δH 4.20 (2H, t, J = 6.4 Hz), methylene attached to carbonyl at δH 2.44 (2H), and terminal methyl protons at δH 0.81. The 13C NMR spectrum with the aid of the DEPT-135 spectrum (Figures S10 and S11) showed signals at δC 174.1, 63.1, 81.0, 34.4, and 14.1 due to ester carbonyl, oxymethylene, sp3 oxygenated methine, and methylene attached to carbonyl and terminal methyl carbons, respectively. The spectrum also displayed signals due to aliphatic methylenes at δC = 34.4, 32.8, 32.0, 29.7, 29.6, 29.4, and 22.7. Compound 3 is the same as compound 2, except for the presence of a hydroxyl group at C-9. Hence, compound 3 is identified as a fatty acid ester named propyl-9-hydroxyoctadecanoate. The 1H–1H COSY (Figure S12) and HMBC spectra also confirm the assertion.

Compound 4 was isolated as a white solid from the combined ethyl acetate and methanol extracts of the leaves of A. abyssinica. The 1H NMR spectrum of compound 4 (Figure S13) revealed a signal at δH 3.62 (3H, m) for methine and δH 3.60 (2H, m) for methylene protons attached to carbon connected to a hydroxyl group. Signals due to methylene protons were observed in the region between δH 1.3 to 1.6. The signal due to terminal methyl was evident at δH = 0.9 (3H, t). The 13C NMR spectrum with the aid of the DEPT-135 spectrum (Figures S14 and S15) showed peaks at δC 81.7, 72.5, 63.0, and 13.1 due to sp3 quaternary, oxymethylene, and terminal methyl carbons, respectively. Other signals were evident at δC 35.2, 31.7, 29.4, 29.3, 29.2, 29.2, 29.1, 29.0, 28.2, 27.5, and 22.4. The above spectral data, along with the literature report, suggest that compound 4 is a terpene, 3-methyltetradecane-1,3,6-triol. This was further established by the 1H–1H COSY and HMBC correlations of the compound (Figure S16).

Compound 5 was isolated as a brown crystal from the combined ethyl acetate and methanol extracts of the leaves of A. abyssinica. The compound melted at 240–242 °C (lit. 242–245).31 The 1H NMR spectrum of compound 5 (Figure S17) revealed signals due to meta-coupled protons at δH 6.7 (1H, d, J = 2.4 Hz) and 6.4 (1H, d, J = 2.4 Hz) with an AB spin pattern, along with three aromatic protons at δH 7.7 (1H, m), 7.1 (1H, d), and 7.7(1H, m) with an ABX spin pattern attributed to rings A and B of flavonoid skeleton, respectively. Signals at δH = 4.0 (3H, s), 3.9 (3H, s), and 3.8 (3H, s) suggest the presence of three methoxy groups. The 13C NMR spectrum with the aid of the DEPT-135 spectrum (Figures S18 and S19) showed carbonyl carbon at δC 178.7 (C-4), and sp2 oxygenated quaternary carbons at δC 165.9 (C-7), 161.4 (C-5), 156.9 (C-8a), 156.7 (C-2), 149.8 (C-4′), 147.6 (C-5′), and 138.4 (C-3). Signals due to two sp2 aromatic quaternary species were observed at δC 122.4 (C-1′) and 105.4(C-4a), along with five sp2 methine carbons at δC 122.4(C-2′), 115.0(C-3′), 111.5(C-6′), 97.6 (C-6), and 91.8 (C-8). The above spectral data, along with comparison with the literature report, suggest that compound 5 is a flavonoid named quercetin 3,3,4′-trimethyl ether.32 The structure of compound 5 was also confirmed by 2D NMR, including COSY and HMBC (Figure S20).

Compound 6 was isolated as a brown crystalline solid from the combined ethyl acetate and methanol extracts of the leaves of A. abyssinica. The 1H NMR spectrum of compound 6 (Figure S21) revealed the presence of five olefinic protons at δH 6.92(1H, m), 6.09 (1H, m), 5.96 (1H, m), 5.28 (1H, m), and 5.12 (1H, m). The 13C NMR (Figure S22) spectrum demonstrated the presence of nine carbon signals of which the most downfield signal at δC 199.8 is attributed to carbonyl carbon, whereas sp2 methine carbons were evident at δC 145.2, 144.4, 133.2, and 111.3. The latter is due to terminal methylene carbon (Figure S23). Two methylene carbons were observed at δC 44.8 and 29.4, supported by the DEPT-135 spectrum. Signals at δC 26.4 and 25.3 belong to the sp3 methine and methyl carbons, respectively. Based on the above spectral data along with the COSY and HMBC spectra (Figure S24), compound 6 is suggested as 8-methylnona-1,5-dien-4-one6.

Compound 7 was isolated as a white crystalline from the combined ethyl acetate and methanol extracts of the stem of A. abyssinica. The UV–vis spectrum of compound 7 revealed an absorption maxima at 205 nm (Figure S25), suggesting the absence of conjugation in the structure. The 1H NMR spectrum of compound 7 (Figure S26) revealed signals due to olefinic protons at δH 5.72 (2H, s), 5.16 (1H, m), and 5.04 (1H, m). The spectrum displayed a signal due to oxymethine at δH = 3.6 (2H). The proton decoupled 13C NMR spectrum of compound 7 analyzed with the aid of the DEPT-135 spectrum (Figures S27 and S28) showed signals at δC 199.8, 171.82, and 171.8 due to the presence of three carbonyl carbons, with the earlier due to ketone carbonyl. Signals due to the presence of four olefinic carbons were evident at δC 138.1, 129.5, 123.7, and 123.6. The presence of three oxygenated aliphatic carbons was observed at δC 81.1, 77.2, and 63.1. The DEPT-135 spectrum established the latter to be a methylene carbon, while the earlier two to be quaternary carbons. Other signals due to aliphatic carbons were observed in the region between δC 55.9 to 12.2. The COSY and HMBC spectra were also used to confirm the structure of compound 7 (Figure S28). Hence, compound 7 was identified as a steroid, namely, 3-(2-acetoxypropan-2-yl)-6-(2,3,4,5,6,7,8,9,10,13,14,15,16,17-tetradecahydro-10-(hydroxymethyl)-13-methyl-3-oxo-1H-cyclopenta[a]phenanthren-17-yl)heptan-3-yl oleate.

Compound 8 was isolated as a white solid from the methanol extracts of the stem of A. abyssinica. The UV–vis of compound showed λmax at 207 nm (Figure S49) confirming the absence of a conjugated chromophore. The 1H NMR spectrum of compound 8 (Figure S30) revealed signals due to five olefinic protons at δH 6.36 (1H, m), 5.89 (2H, m), and 5.40 (2H, m). The diagnostic signal due to proton on oxygenated carbons was at δH 4.45 (1H, m) and 4.42 (1H, m). The triplet signal at δH 2.36 is due to protons on carbon adjacent to carbonyl carbon. The intense peak at δH 1.12 is evidence of the presence of many overlapping methylenes. The signal at δH = 0.89 is assigned to terminal methyl protons. The 13C NMR (Figures S31 and S32) spectrum of compound 8 showed a signal at δC 179.4 due to ester carbonyl carbon. Characteristic signals due to olefinic carbons were observed at δC 144.1, 133.1, 111.5, and 120.5, where the latter is due to terminal olefinic carbon. The spectrum also demonstrated the presence of oxygenated carbons at δC values of 74.3 and 66.0. The spectrum also showed signals due to aliphatic carbons in the region between δC 33.9 to 14.1. Hence, compound 8 is identified as a (E)-3-(tetrahydro-5-vinylfuran-2-yl)-4-hydroxybut-1-enyl octadecanoate.

Compound 9 was isolated as a pale yellow crystalline solid from the methanol extracts of the stem of A. abyssinica, which melted at 201–203 °C (Lit. 201).33 The UV–vis spectrum showed λmax at 346 nm (Figure S33), which is diagnostic for the presence of conjugation. The 1H NMR spectrum of compound 9 (Figure S34) revealed the presence of two ortho-coupled protons at δH values of 7.81 (1H, d, J = 9.2 Hz) and 6.15 (1H, d, J = 9.5 Hz). Two singlet aromatic signals were observed at δH 7.07 (1H) and 6.72 (1H). The proton-decoupled 13C NMR spectrum of compound 9 analyzed with the aid of DEPT-135 spectrum (Figures S35 and S36) showed ten carbon resonances with the most downfield signal at δC 162.6 due to α,β conjugated carbonyl carbon. The presence of three oxygenated aromatic carbons is apparent at δC 151.1, 149.5, and 145.7. The spectrum displayed signals due to olefinic carbons at δC 111.7 and 145.6 due to carbons α and β being adjacent to the carbonyl carbon. The remaining signals at δC 111.3, 109.5, and 102.9 are due to aromatic methine carbons. Hence, compound 9 is identified as 5-hydroxy-6-methoxycoumarin.34,35

Compound 10 was obtained as a white solid from the methanol extracts of the stem of A. abyssinica. The UV–vis spectrum demonstrated absorption maxima at λmax of 348 nm (Figure S37). The 1H NMR spectrum of compound 10 (Figure S38) revealed signals due to ortho-coupled protons at δH = 7.89 (1H, d, J = 9.6 Hz) and 6.20 (1H, d, J = 9.6 Hz). Two singlet signals were evident at δH 7.15 (1H, s) and 6.77 (1H, s). The signal at δH 3.97 (3H, s) is due to methoxy protons, while that at δH 1.98 (3H) is due to methyl protons. The 13C NMR spectrum of compound 10 analyzed with the aid of the DEPT-135 spectrum (Figures S39 and S40) showed resonances of 13 carbons. The signals at δC 161.9, 151.3, 149.7, 145.7, 145.3, 111.9, 111.1, 109.7, and 103.1 establish the skeleton of the compound as a coumarin. Furthermore, the spectrum had four additional signals at δH 66.5, 65.2, 56.3, and 20.9 due to oxygenated methine, oxygenated methylene, and methoxy and methyl groups, respectively. Hence, compound 10 is identified as 6-(2-hydroxypropoxy)-7-methoxy-2H-chromen-2-one.

Compound 11 was isolated as a white crystalline solid from the combined extract of the root of A. abyssinica. The compound displayed an absorption maxima at 206 nm (Figure S41). The 1H NMR spectrum of compound 11 (Figure S42) revealed four signals in the olefinic regions at δH 5.75 (2H, s), 5.16 (1H, dd, J = 8.0 and 12.0 Hz), and 5.04 (1H, dd, J = 8.0 and 12.0 Hz). The signal due to the oxymethine proton is evident at δH 4.20 (1H, m). The proton decoupled 13C NMR (Figures S43 and S44) spectrum of compound 11 displayed diagnostic signals due to the ketone carbonyl at δC 199.7. The presence of an ester carbonyl was also observed at δC 171.8. Four olefinic signals were observed at δC 138.1, 129.5, 123.8, and 123.7. The spectrum displayed a signal due to oxymethine carbon at δC 77.2. Characteristic signals of the steroid skeleton were observed in the region δC 56.0 to 12.3. Hence, compound 11 is a steroid identified as (9E)-17-(5-ethyl-6-methyl-4-oxoheptan-2-yl)-2,3,4,5,6,7,8,9,10,13,14,15,16,17-tetradecahydro-10,13-dimethyl-1H cyclopenta[a]phenanthren-3-yl-octadec-9-enoate.36

Compound 12 was obtained as a white crystalline solid melted at 317–319 °C. The UV–vis spectrum showed λmax 210 and 275 nm (Figure S45). The 1H NMR spectrum of compound 12 (Figure S46) revealed signals due to aromatic protons at δH 7.5 (2H, m) and 7.7 (2H, m). A signal due to oxymethylene was observed at δH 4.20 (4H, t). The 13C NMR (Figures S47 and 48) spectrum of compound 12 showed a signal due to ester carbonyl at δC 167.6. Other aromatic carbons were observed at δC 132.2, 130.9, and 128.8. The signal at δC = 61.6 is due to oxymethylene carbon. Hence, compound 12 is identified as o-dipropyl phthalate.37

4.1. Biological Activity of the Extracts and Isolated Compounds

4.1.1. Antibacterial Activity

The in vitro antibacterial activity of the extracts and isolated compounds was measured using a disc diffusion assay. The results were expressed in terms of inhibition zone diameter (IZD) and are presented in Table 1.

Table 1. Antibacterial Activity of Constituents of A. abyssinicaa.
    zone of inhibition (mm)
samples concentration (μg/mL) E. coli P. aerugisnosa S. aureus S. pyogens
CAALM 30 9.30 ± 0.20 8.00 ± 0.20 8.50 ± 0.20 8.10 ± 0.20
  15 8.80 ± 0.30 7.20 ± 0.30 7.90 ± 0.30 7.90 ± 0.30
  7.5 7.30 ± 0.10 7.00 ± 0.10 7.30 ± 0.10 7.30 ± 0.10
  3.25 6.90 ± 0.20 6.90 ± 0.20 6.70 ± 0.20 6.90 ± 0.20
  1.625 6.50 ± 0.40 6.40 ± 0.40 6.60 ± 0.40 6.50 ± 0.40
CAASM 30 7.40 ± 0.40 7.80 ± 0.20 8.10 ± 0.20 7.40 ± 0.30
  15 7.00 ± 0.30 7.40 ± 0.30 7.10 ± 0.10 7.00 ± 0.30
  7.5 6.80 ± 0.10 6.60 ± 0.20 6.90 ± 0.10 6.90 ± 0.20
  3.25 6.50 ± 0.10 6.30 ± 0.20 6.70 ± 0.30 6.50 ± 0.20
  1.625 6.30 ± 0.40 6.10 ± 0.30 6.40 ± 0.40 6.30 ± 0.50
CAARM 30 8.80 ± 0.20 9.00 ± 0.20 7.30 ± 0.20 7.10 ± 0.20
  15 8.60 ± 0.30 7.20 ± 0.30 6.80 ± 0.30 7.00 ± 0.30
  7.5 7.30 ± 0.10 7.00 ± 0.20 6.60 ± 0.10 6.80 ± 0.10
  3.25 6.90 ± 0.20 6.60 ± 0.30 6.60 ± 0.20 6.90 ± 0.40
  1.625 6.50 ± 0.40 6.40 ± 0.50 6.50 ± 0.40 6.10 ± 0.40
1 30 6.50 ± 0.20 8.50 ± 0.80 6.40 ± 0.20 7.00 ± 0.20
  15 6.40 ± 0.30 6.30 ± 0.20 6.40 ± 0.20 6.90 ± 0.30
  7.5 6.30 ± 0.20 6.50 ± 0.40 6.30 ± 0.20 6.80 ± 0.30
  3.25 6.20 ± 0.30 6.30 ± 0.10 ±0.10 6.30 ± 0.20
  1.625 6.10 ± 0.20 6.10 ± 0.10 6.10 ± 0.20 6.10 ± 0.30
2 30 7.20 ± 0.30 8.40 ± 0.30 6.60 ± 0.20 8.80 ± 0.60
  15 6.60 ± 0.30 6.80 ± 0.50 6.50 ± 0.10 7.00 ± 0.50
  7.5 6.70 ± 0.20 6.70 ± 0.40 6.30 ± 0.10 6.80 ± 0.50
  3.25 6.40 ± 0.40 ±0.10 6.20 ± 0.10 6.20 ± 0.20
  1.625 6.20 ± 0.20 6.10 ± 0.10 6.10 ± 0.20 6.10 ± 0.20
3 30 11.5 ± 0.30 10.1 ± 0.50 7.0 ± 0.30 10.5 ± 0.50
  15 11.1 ± 0.30 8.3 ± 0.50 6.9 ± 0.30 8.4 ± 0.50
  7.5 9.9 ± 0.40 7.3 ± 0.30 6.6 ± 0.10 7.0 ± 0.60
  3.25 7.7 ± 0.30 6.6 ± 0.20 6.2 ± 0.20 6.2 ± 0.30
  1.625 6.8 ± 0.20 6.3 ± 0.20 6.1 ± 0.20 6.1 ± 0.20
4 30 12.1 ± 0.30 12.9 ± 0.90 12.7 ± 0.90 10.8 ± 1.10
  15 11.1 ± 0.10 10.9 ± 0.90 11.6 ± 0.70 8.0 ± 0.80
  7.5 10.3 ± 0.20 10.0 ± 0.90 9.3 ± 0.70 7.4 ± 0.80
  3.25 8.1 ± 0.30 8.3 ± 0.40 8.1 ± 0.60 6.1 ± 0.20
  1.625 6.8 ± 0.30 7.8 ± 0.20 8.0 ± 0.20 6.0 ± 0.20
5 30 7.8 ± 0.30 8.7 ± 0.30 6.6 ± 0.40 7.9 ± 0.40
  15 7.6 ± 0.40 7.5 ± 0.20 6.5 ± 0.10 7.5 ± 0.20
  7.5 7.5 ± 0.30 7.2 ± 0.40 6.4 ± 0.40 6.9 ± 0.20
  3.25 6.4 ± 0.30 7.3 ± 0.60 6.2 ± 0.30 6.5 ± 0.10
  1.625 6.2 ± 0.30 6.8 ± 0.50 6.1 ± 0.30 6.3 ± 0.30
6 30 13.3 ± 0.50 13.5 ± 0.50 7.9 ± 0.30 11.4 ± 0.40
  15 11.3 ± 0.40 11.6 ± 0.40 7.1 ± 0.50 8.5 ± 0.40
  7.5 9.9 ± 0.60 9 ± 0.50 6.6 ± 0.40 7.7 ± 0.20
  3.25 7.1 ± 0.30 7.0 ± 0.20 6.5 ± 0.60 6.7 ± 0.30
  1.625 6.8 ± 0.40 6.8 ± 0.40 6.3 ± 0.30 6.5 ± 0.30
7 30 8.2 ± 0.10 8.2 ± 0.60 6.9 ± 0.30 6.7 ± 0.20
  15 7.6 ± 0.30 6.6 ± 0.10 6.6 ± 0.30 6.4 ± 0.20
  7.5 7.1 ± 0.30 7.5 ± 0.90 6.6 ± 0.30 6.4 ± 0.30
  3.25 6.2 ± 0.20 6.7 ± 0.50 6.2 ± 0.20 6.2 ± 0.10
  1.625 6.0 ± 0.30 6.4 ± 0.30 6.1 ± 0.30 6.1 ± 0.30
8 30 6.8 ± 0.70 9.2 ± 0.50 8.1 ± 0.40 7.1 ± 0.50
  15 6.7 ± 1.00 8.6 ± 0.40 7.4 ± 0.10 6.8 ± 1.00
  7.5 6.6 ± 0.30 8.2 ± 0.80 6.9 ± 0.20 6.7 ± 0.30
  3.2 6.3 ± 0.50 7.5 ± 0.40 6.6 ± 0.30 6.4 ± 0.20
  1.625 6.1 ± 0.30 6.6 ± 0.30 6.3 ± 0.30 6.2 ± 0.10
9 30 8.1 ± 0.20 8.1 ± 0.30 7.5 ± 0.30 8.3 ± 0.30
  15 7.9 ± 0.30 7.6 ± 0.60 7.0 ± 0.50 7.4 ± 0.80
  7.5 7.3 ± 0.10 7.1 ± 0.80 6.8 ± 0.70 7.1 ± 1.00
  3.25 6.9 ± 0.20 7.0 ± 0.50 6.5 ± 0.50 7.0 ± 1.00
  1.625 8.1 ± 0.20 8.1 ± 0.30 7.5 ± 0.30 8.3 ± 0.30
10 30 8.3 ± 0.30 7.1 ± 0.50 8.2 ± 0.50 9.1 ± 0.60
  15 7.7 ± 0.80 6.8 ± 0.30 7.6 ± 0.40 9.0 ± 0.50
  7.5 7.4 ± 1.00 6.5 ± 0.30 7.3 ± 0.50 8.0 ± 0.40
  3.25 6.9 ± 1.00 6.4 ± 0.50 7.0 ± 0.50 7.2 ± 0.30
  1.625 6.5 ± 0.20 6.3 ± 0.30 6.6 ± 0.20 7.0 ± 0.30
11 30 9.5 ± 0.20 8.1 ± 0.20 8.1 ± 0.40 8.1 ± 0.30
  15 9.3 ± 0.30 7.5 ± 0.40 7.9 ± 0.30 8.0 ± 0.20
  7.5 7.5 ± 0.20 7.1 ± 0.10 7.3 ± 0.10 7.6 ± 0.31
  3.25 6.8 ± 0.20 6.8 ± 0.50 6.9 ± 0.20 7.0 ± 0.30
  1.625 6.3 ± 0.50 6.5 ± 0.40 6.5 ± 0.40 6.9 ± 0.50
12 30 8.3 ± 0.30 8.1 ± 0.20 8.1 ± 0.20 8.1 ± 0.20
  15 7.6 ± 0.30 7.2 ± 0.30 7.9 ± 0.30 7.6 ± 0.30
  7.5 7.1 ± 0.20 6.9 ± 0.10 7.3 ± 0.10 7.3 ± 0.10
  3.25 6.8 ± 0.20 6.7 ± 0.20 6.9 ± 0.20 7.0 ± 0.20
  1.625 6.5 ± 0.50 6.5 ± 0.40 6.0 ± 0.40 6.5 ± 0.20
ceftriaxone 30 14.1 ± 0.30 13.8 ± 0.50 14.4 ± 0.90 16.2 ± 0.60
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a

CAALM (A. abyssinica leaves methanol extract), CAASM (A. abyssinica stem methanol extract), and CAARM (A. abyssinica root methanol extract).

The methanol extracts of leaves, stems, and roots tested at a concentration of 30 μg/mL showed weak antibacterial activity against the four bacterial pathogens (S. pyogens, S. aureus, P. aeruginosa, and E. coli) with an IDZ range of 6.1 ± 0.3 to 9.30 ± 0.20 mm. The result obtained herein for the extract is inferior compared with the literature reports.38 However, the antibacterial activity reported for the essential oil by Bibiso et al., was found to be in good agreement with our findings.39 All of the isolated compounds displayed weak antibacterial activity against the tested microorganisms, except for compounds 3, 4, and 6, which showed moderate activity. The best activity was observed for compound 6 against Gram-negative bacteria (E. coli, IZD = 13.3 ± 0.5 and 11.3 ± 0.4 mm and P. aeruginosa, IZD = 13.5 ± 0.5 and 11.6 ± 0.4 mm), respectively, compared to ceftriaxone (IZD = 14.1 ± 0.3, 13.8 ± 0.5, 14.4 ± 0.9, and 16.2 ± 0.6, respectively) at concentration of 30 μg/mL. Compounds 4 and 6 displayed IZD of 12.7 ± 0.9 mm and 11.4 ± 0.4 mm against S. aureus and S. pyogens, respectively, at 30 μg/mL. Hence, the activity displayed by the leaves extract of A. abyssinica could be attributed to the presence of compounds 3, 4, and 6 in the extract. Literature reports revealed that the leaves of many Artemisia species exhibited modest antibacterial activity.40

4.1.2. Cytotoxic Activity

Hence, the effect of extracts of A. abyssinica was evaluated on breast cancer (MCF-7) cells by the MTT assay, which induced a decrease of cell viability and exerted a cytotoxic effect at a concentration of 20 μg/mL. The percent cell viability of the n-hexane and methanol extracts of the leaves of A. abyssinica was 54.9 and 16.3%, respectively. The n-hexane, EtOAc, and methanol extracts of the stems displayed percent cell viability of 68.6, 74.0, and 92.9%, respectively. Likewise, the EtOAc and methanol root extracts showed percent cell viability of 66 and 34.5%, respectively (Figure 2). Generally, the cytotoxicity activities of the extracts could be attributed to the presence of flavonoids, triterpenes, aromatic compounds, and phenolic constituents in the extracts, showing their activity against the tested cancer cell.

Figure 2.

Figure 2

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Cell viability test for the A. abyssinica extract. CAAL hexane: A. abyssinicca leaves hexane extract; CAAL methanol: A. abyssinicca leaves methanol extract; CAAR methanol: A. abyssinicca root methanol extract; CAAR ethyl acetate: A. abyssinicca root ethyl acetate extract; CAAS hexane: A. abyssinicca stem hexane extract; CAAS ethyl acetate: A. abyssinicca stem ethyl acetate extract; and CAAS methanol: A. abyssinicca stem methanol extract.

4.1.2.1. Lipinski’s and Veber Rules

Lipinski’s ROF is one of the most effective tools for predicting new chemical entities’ (NCE) drug-likeness.19 The results are presented in Table 2. Similar to the standard drug, CPFX (hydrochloride), the test compounds 4, 5, 6, 9, 10, and 12 comply with the ROF. Whereas all other test compounds showed one or two violations due to their high molecular weight (M.Wt. > 500) and/or lipophilic character (cLogP > 5). Veber’s rule,20 which specifies that the number of rotatable bonds should be ≤ 10 and the TPSA should be ≤140 Å2 or ≤12 total hydrogen bonds, is a commonly acknowledged technique for predicting the oral bioavailability of NCE. Like CPFX, the test compounds 5, 6, 9, 10, and 12 comply with Veber’s rule. Whereas, the other test compounds showed one violation due to more number of rotatable bonds (>10) as these compounds had open-chain structures. Based on the above two rules, compounds 5, 6, 9, 10, and 12 appeared to be good oral drug candidates.

Table 2. Drug-Likeness Predictions of the Test and Standard Compounds Calculated by SwissADMEa.
C.no formula Mol. Wt. (g/mol) NHD NHA LogP (cLogP) Lipinski’s ROF violation NRB TPSA (Å2) Veber’s rule violation
1 C20H42O 298.55 1 1 6.32 1 13 20.23 1
2 C21H42O2 326.56 0 2 6.98 1 19 26.3 1
3 C21H42O3 342.56 1 3 6.03 1 19 46.53 1
4 C15H32O3 260.41 3 3 3.17 0 12 60.69 1
5 C18H16O7 344.32 2 7 2.54 0 4 98.36 0
6 C10H16O 152.23 0 1 2.66 0 5 17.07 0
7 C49H82O6 767.17 1 6 10.75 2 26 89.9 1
8 C28H50O4 450.69 1 4 7.24 1 22 55.76 1
9 C10H8O4 192.17 1 4 1.51 0 1 59.67 0
10 C13H14O5 250.25 1 5 1.78 0 4 68.9 0
11 C47H80O3 693.14 0 3 12.01 2 23 43.37 1
12 C14H18O4 250.29 0 4 3.05 0 8 52.6 0
CPFX C17H18FN3O3 331.34 2 6 1.1 0 3 74.57 0
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a

NHD = number of hydrogen donor, NHA = number of hydrogen acceptor, NRB = number of rotatable bonds, and TPSA = total polar surface area.

4.1.2.2. ADME Studies

The reference drug, CPFX, and the test compounds exhibited high GI absorption except 1, 2, 7, 8, and 11 (Table 3). Skin permeability is a measure of how quickly a substance permeates the stratum corneum (Kp). This value is frequently used to emphasize the significance of skin absorption and to quantify the movement of molecules in the outermost layer of the epidermal skin. Lesser the log Kp value, the lower the cutaneous permeability of the molecule.41 In this study, compared to CPFX, the test compounds showed a higher log Kp value (Table 3), so these compounds might have better skin permeation than CPFX. Increased P-glycoprotein (P-gp) expression in the intestine can limit the absorption of medicines that are P-gp substrates. As a result, bioavailability is diminished, and therapeutic plasma concentrations are not achieved.42 In the present study, 1, 4, 7, 8, and 11 and CPFX were predicted to be P-gp substrates.

Table 3. ADME Predictions of the Test Compounds Computed by SwissADME and PreADMETa.
  absorption
 distribution
 metabolism
 excretion
  GI ABS log Kp cm/s P-gp substrate BBB permeability (logBB) log VD (L/kg) CYP3A4 inhibitor CYP2D6 inhibitor CYP2C9 inhibitor CYP2C19 inhibitor CYP1A2 inhibitor TC (log mL/min/kg) OCT2 substrate
1 low –2.36 yes 0.769 0.261 no no yes no no 1.588 no
2 low –1.65 no 0.815 0.293 no no no no yes 1.989 no
3 high –3.09 no –0.552 –0.06 yes no no no yes 2.008 no
4 high –5.39 yes –0.2 –0.235 no yes no no no 1.763 no
5 high –5.84 no –0.724 –0.044 yes yes yes no yes 0.764 no
6 high –5.28 no 0.675 0.182 no no no no no 0.459 no
7 low –1.28 yes –0.939 –1.293 yes no no no no 0.722 no
8 low –2.42 yes –0.995 –0.067 no no no no no 1.875 no
9 high –6.49 no –0.309 –0.014 no no no no yes 0.768 no
10 high –6.69 no –0.456 –0.229 no no no no yes 0.876 no
11 low 0.71 yes –0.617 –0.854 no no no no no 0.748 no
12 high –5.51 no 0.008 –0.101 no no no yes yes 0.837 no
CPFX high –9.09 yes –0.425 –0.17 no no no no no 0.633 no
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a

GI = gastro-intestinal, ABS = absorption, BBB = blood–brain barrier, P-gp = P-glycoprotein, and CYP = cytochrome-P.

Blood–brain barrier (BBB) permeability is one of the important parameters that molecules exhibit their action at CNS.43 It has been suggested that molecules with a logBB value more than 0.3 can easily penetrate the BBB, whereas those with a logBB value less than −1 are not well distributed throughout the brain.44 Among the test and standard compounds, only 1, 2, and 6 showed logBB > 0.3 (Table 3). Therefore, these molecules might have readily crossed the BBB and act on the CNS. The volume of distribution (VD) is the theoretical volume that would be required for a drug’s whole dosage to be evenly dispersed to produce a concentration identical to that of blood plasma. If logVD < −0.15, it is considered to be low; if logVD > 0.45, it is considered to be high.43 Compounds 4, 7, 10, and 11 and CPFX might have less VD, since they showed logVD < −0.15, whereas, other compounds might have moderate VD, since these compounds showed logVD between −0.15 and 0.45 (Table 3).

About 60% of prescribed drugs are metabolized by CYP enzymes, with CYP3A4 accounting for about half of this metabolism, followed by CYP2D6, CYP2C9, and CYP2C19.45 The CYP3A4 enzyme was inhibited by compounds 3, 5, and 7, suggesting that this enzyme may not have metabolized these compounds. Whereas, CYP2C19 was inhibited only by 12, and CYP2D6 was inhibited by compounds 4 and 5. Six of the test compounds inhibited CYP1A2 and only 12 inhibited CYP2C19 (Table 3). The primary transporter for cation influx in renal epithelial cells is organic cation transporter 2 (OCT2). It is involved in the initial stage of renal elimination, which involves drug molecules being taken up from the blood and entering the proximal tubule cell through the basolateral membrane. According to the study’s predictions, none of the compounds were OCT2 substrates.

4.1.2.3. Boiled-Egg Model

The compounds’ cLogP and TPSA values were plotted to estimate access to the BBB and human intestinal absorption (HIA) (Figure 3). The egg-shaped plot has been divided into 3 parts, including a white area (HIA), a yellow area (BBB access), and a gray area (no HIA or BBB access). In this prediction, compounds 3 and 5 are in the white area, so these compounds could be absorbed through the intestine, whereas 4, 6, 9, 10, and 12 are in the yellow area, suggesting that these two compounds may pass through the BBB. Compounds 1, 2, and 8 are in the gray area, which indicates no HIA or BBB access (Daina 2016). Compounds 7 and 11 were out of range in this study. Additionally, this model predicted whether those substances are P-gp (PGP) substrates. Blue dots (PGP+) indicate molecules that are substrates of the PGP CNS efflux transporter and could be effluated from the CNS, whereas red dots (PGP−) indicate substances that are not PGP substrates that could cross and act on the CNS.46 In this study, compounds 1, 4, and 8 had blue dots, suggesting that these compounds are PGP substrates and eventually not acting on the CNS.47

Figure 3.

Figure 3

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Boiled-egg model for predicting GIT absorption and brain access.

4.1.2.4. Toxicity Studies

On a scale of 1 to 6, the degree of toxicity is expressed, with a higher number denoting a lower level of toxicity. This assessment is based on the LD50 (mg/kg) value, which represents the dose that kill 50% of the test animals. Among the compounds studied, compounds 4, 7, and 12 were predicted to be less or not toxic as they fall under class 6. Compounds 2, 3, 5, 6, and 10 showed a toxic classification of 5, while compounds 1, 8, and 11 exhibited a toxic classification of 4, similar to that of CPFX. Compound 9 appeared to be more toxic, with a toxic classification of 3. Except for compound 1, none of the tested compounds revealed hepatotoxicity (liver toxicity). All of the compounds also appeared to have no mutagenicity and no cytotoxicity. Except for compounds 2, 3, 4, 6, 8, and 12, the other compounds exhibited immunotoxicity (the ability to disrupt the immune system). Only compounds 2, 9, and 12 showed carcinogenicity (Table 4).

Table 4. Test Compounds Toxicity Prediction Based on Findings by Pro-Tox II.
      organ toxicity
sample LD50 (mg/kg) toxicity class hepatotoxicity carcinogenicity immunotoxicity mutagenicity cytotoxicity
1 1190 4 yes no yes no no
2 5000 5 no yes no no no
3 5000 5 no no no no no
4 20,000 6 no no no no no
5 5000 5 no no yes no no
6 4300 5 no no no no no
7 11,210 6 no no yes no no
8 1330 4 no no no no no
9 280 3 no yes yes no no
10 3800 5 no no yes no no
11 1000 4 no no yes no no
12 10,000 6 No yes no no no
CPFX 2000 4 No no no yes no
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4.1.2.5. Molecular Docking with DNA Gyrase

DNA gyrase is necessary for the topology and integrity of bacterial DNA during transcription and replication. It is currently regarded as one of the main targets and is present in the majority of pathogenic bacteria in clinical settings.48,49 Well-known DNA gyrase inhibitors, fluoroquinolones, target the enzymes A subunit.

In this study, E. coli DNA gyrase (PDB ID: 6F86) was used as a target, and a fluoroquinoline class of antibacterials, CPFX, was used as a reference control for docking with the target. Binding affinity and interactions with different amino acids are presented in Table 5, and 3D and 2D binding interactions are depicted in Supporting Information; Figure S1. Among the test compounds, compound 5 showed the higher binding affinity (−6.9 kcal/mol), followed by compound 10 (−6.7 kcal/mol) and compound 12 (−6.3 kcal/mol), whereas CPFX showed −7.3 kcal/mol. CPFX formed hydrogen bonding interactions with Ser121, Val120, Val97, and Leu98. Similarly, some of the test compounds (compounds 1, 2, 3, 6, 7, 8, and 11) showed H-bonding interactions with one or two of these amino acids, whereas other test compounds showed interactions with Thr165 and/or Asn46. CPFX showed residual interactions with Ile94 and Gly119, whereas most of the test compounds showed interactions with Ile94 and Ile78 along with other amino acids. Only CPFX showed van der Waals interactions, whereas the test compounds did not show van der Waals interactions with any amino acids.

Table 5. Binding Affinity and Interaction with the Target E. coli DNA Gyrase (PDB ID: 6F86).
      residual amino acid interactions
compd affinity (kcal/mol) H-bond hydrophobic/π-cation/π-anion/ π-alkyl interactions van der Waals interactions
1 –4.8 Ser121, Val97 Ile94, Ile78  
2 –4.4 Ser121, Val120 Ile94, Ile78  
3 –4.7 Ser121, Val120, Asp49 Ile94, Ile78, Ala47  
4 –5.5 Thr165, Asn46 Ile94, Ile78, Pro79  
5 –6.9 Thr165, Asn46, Arg76 Ile94, Pro79, Glu50  
6 –5.4 Asn46 Ile94, Pro79, Val71, Val43, Ala47  
7 –5.3 Ser121, Val120, Asn46 Ile94, Pro79  
8 –5.4 Ser121, Asn46, Leu98a Ile94, Ile78  
9 –5.8 Thr165, Asn46 Ile94, Ile78, Ala47  
10 –6.7 Asn46, Glu50 Ile78, Ile94, Pro79, Arg76  
11 –5.2 Val120, Ser121 Ile78, Ile94, Ala90, Val93  
12 –6.3 Asn46, Pro79a Ile78, Ile94, Glu50  
CPFX –7.3 Ser121, Val120, Val97, Leu98 Ile94, Gly119 Glu50, Ile78, Pro79
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a

Carbon hydrogen bond. The binding interactions (3D and 2D) of the isolated compounds and CPFX against E. coli DNA gyrase are presented as Supporting Information (Figure S49).

4.1.2.6. Molecular Docking with S. aureus PK

PK is an essential enzyme found in staphylococci that controls the bacteria’s growth, antibiotic resistance, and ability to create biofilms.50 Moreover, it was discovered to differ structurally from human homologues, making it a potential target for new antimicrobial drugs.51 Binding affinity and interactions with different amino acids are presented in Table 6, and the 2D and 3D binding interactions are presented as Supporting Information. Among all the compounds, compound 5 showed the highest binding affinity (−5.0 kcal/mol), followed by compound 11 (−4.3 kcal/mol), compound 10 (−4.2 kcal/mol), and compound 9 (−4.0 kcal/mol), whereas CPFX showed binding affinity of −4.9 kcal/mol. CPFX forms H-bonding interactions with Ser362 and Thr366. All the test compounds showed H-bonding interactions with one or two of these amino acids. CPFX showed residual amino acid interactions only with His365. Similarly, the test compounds showed interactions with His365 along with other amino acids, whereas van der Waals interactions were not observed for the standard and test compounds. The binding interactions (3D and 2D) of test compounds and CPFX against S. aureus PK are presented as Supporting Information (Figure S50).

Table 6. Binding Affinity and Interaction with the Target S. aureus PK (PDB ID: 3T07).
      residual amino acid interactions
sample affinity (kcal/mol) H-bond hydrophobic/π-cation/π-anion/ π-alkyl interactions van der Waals interactions
1 –2.8 Ser362 Ile361, Ala358  
2 –2.9 Ser362 His365, Ile361, Ala358  
3 –3.4 Thr366, Asn369 His365, Ile361, Ala358, Leu370  
4 –3.6 Ser362, Thr366, Asn369 Ile361  
5 –5.0 Ser362, Thr366 His365, Ile361  
6 –3.1 Asn369 His365, Ile361, Leu361  
7 –3.8 Thr366, Asn369 His365, Ile361  
8 –3.3 Ser362, Thr366 His365, Leu370  
9 –4.0 Thr366, Asn369 His365  
10 –4.2 Thr366, Asn369 His365, Leu370  
11 –4.3 Thr366, Asn369, His365 His365, Leu368, Leu344, Lys341  
12 –3.8 Thr366, Asn369 His365, Leu370  
CPFX –4.9 Ser-362, Thr-366 His365  
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4.1.2.7. Molecular Docking with Human Topoisomerase IIβ

Topoisomerase II (TOP2) catalyzes the relaxing and unwinding of double-stranded DNA, which is essential for DNA replication, transcription, and repair.52 Recent studies showed that TOP2β is an important target for many anticancer agents, including etoposide (EVP). Binding affinity and interactions with different amino acids are presented in Table 7, and the 2D and 3D binding interactions are presented as Supporting Information. EVP showed a binding affinity of −7.5 kcal/mol, whereas all of the test compounds showed a lesser binding affinity. Compound 5 showed a binding affinity of −6.0 kcal/mol, followed by compound 11 (−5.4 kcal/mol), compound 10 (−5.0 kcal/mol), and compound 11 (−4.9 kcal/mol). EVP showed H-bonding interactions with His775, Lys-505, Asp-561, and Arg-503, while the test compounds showed interactions with one or more of these amino acids. EVP showed residual amino acid interactions with Arg-503, His-775, and Glu-522, whereas most of the test compounds showed interactions with different amino acids. Only EVP showed van der Walls interactions, whereas the test compounds did not show van der Walls interactions with any amino acids. The binding interactions (3D and 2D) of test compounds and EVP against human topoisomerase IIβ are included as Supporting Information (Figure S51).

Table 7. Binding Affinity and Interaction with the Target Human TOP2β (PDB ID: 3QX3).
      residual amino acid interactions
sample affinity (kcal/mol) H-bond hydrophobic/π-cation/π-anion/π-alkyl interactions van der Waals interactions
1 –3.8 His775 Lys505, Ala779  
2 –3.7 His775, Gly776 Lys505, Ala779  
3 –3.5 His775 Lys505, Ala779, Met782  
4 –3.7 His775, Asp561, Arg729, Lys505    
5 –6.0 His775 Asp561, Glu477, Gly478  
6 –3.4 His775 Lys505  
7 –4.3 His775 Lys505, Ala779  
8 –4.0 His775, Gly776    
9 –4.9 His775, Lys505 Ala779, Asp561  
10 –5.0 His775 Lys505, Glu477, Asp557, Asp559  
11 –5.4 Lys505, Arg729 His775, Ala779, Arg503  
12 –4.4 His775, Gly776 Ala779, His775, Asp561  
EVP –7.5 His-775, Lys-505, Asp-561, Arg-503 Arg-503, His-775, Glu-522 Gly776, Ala779, Arg729, Asp559, His774, Glu477
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5. Conclusions

Column chromatographic fractionation of the extracts of A. abyssinica has led to the isolation of 12 compounds, of which compounds 1, 3, 4, 5, and 7-11 are new to the genus Artemisia. All the crude extracts displayed weak antibacterial activity with IZD ranges of 6.1 ± 0.3 to 9.30 ± 0.20 mm at 30 μg/mL. Compound 6 had the best antibacterial activity against E. coli (IZD = 13.30 ± 0.50 mm) and P. aeruginosa (IZD = 13.50 ± 0.50 mm) compared to ceftriaxone (IZD = 14.1 ± 0.3 and 13.8 ± 0.5 against E. coli and P. aeruginosa, respectively). Therefore, it can be concluded that the activity displayed by the leaves extract of A. abyssinica could be attributed to the presence of compounds 3, 4, and 6 in the extract. The in silico molecular docking analysis against DNA gyrase B revealed that compound 5 showed a higher binding affinity (−6.9 kcal/mol), which is comparable with that of CPFX (−7.3 kcal/mol). Similarly, compound 5 had a better result compared with CPFX (−4.9 kcal/mol) against S. aureus PK with a binding affinity of −5.0 kcal/mol. The binding affinities of the isolated compounds against human topoisomerase inhibitors IIβ revealed that compound 5 showed a binding affinity of −6.0 kcal/mol, followed by 11 (−5.4 kcal/mol), 10 (−5.0 kcal/mol), and 11 (−4.9 kcal/mol). Similar to CPFX, compounds 4, 5, 6, 9, 10, and 12 comply with Lipinski’s ROF. The effect of the extracts of A. abyssinica was evaluated against breast cancer (MCF-7) cell lines using the MTT assay. The extracts induced a decrease in cell viability and exerted a cytotoxic effect at a concentration of 20 μg/mL. Therefore, the cytotoxicity, antibacterial activities, and in silico molecular docking analysis displayed by the constituents validate the traditional uses of the plant against microbial infections and cancer.

Acknowledgments

D.T. acknowledges Adama Science and Technology University for sponsorship.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c01096.

  • 1H NMR, 13C NMR, DEPT, and UV-vis spectrum of compounds 1–12 and binding interactions (3D and 2D) of test and standard compounds with E. coli DNA, S. aureus PK, and with human topoisomerase IIβ (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao4c01096_si_001.pdf (5.9MB, pdf)

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