Phytochemistry, Pharmacology and Mode of Action of the Anti-Bacterial Artemisia Plants

Over 70,000 people die of bacterial infections worldwide annually. Antibiotics have been liberally used to treat these diseases and, consequently, antibiotic resistance and drug ineffectiveness has been generated. In this environment, new anti-bacterial compounds are being urgently sought. Around 500 Artemisia species have been identified worldwide. Most species of this genus are aromatic and have multiple functions. Research into the Artemisia plants has expanded rapidly in recent years. Herein, we aim to update and summarize recent information about the phytochemistry, pharmacology and toxicology of the Artemisia plants. A literature search of articles published between 2003 to 2022 in PubMed, Google Scholar, Web of Science databases, and KNApSAcK metabolomics databases revealed that 20 Artemisia species and 75 compounds have been documented to possess anti-bacterial functions and multiple modes of action. We focus and discuss the progress in understanding the chemistry (structure and plant species source), anti-bacterial activities, and possible mechanisms of these phytochemicals. Mechanistic studies show that terpenoids, flavonoids, coumarins and others (miscellaneous group) were able to destroy cell walls and membranes in bacteria and interfere with DNA, proteins, enzymes and so on in bacteria. An overview of new anti-bacterial strategies using plant compounds and extracts is also provided.


Introduction
Since the 1940s, antibiotics have been widely used to treat bacterial infections in humans and animals. However, misuse and abuse of antibiotics have raised public health concerns about antimicrobial resistance (AMR), transmission of antibiotic resistance genes, ineffectiveness of current antibiotics, and the generation of superbugs. Over 700,000 people worldwide die of AMR each year, and by the year 2050, that number is projected to reach 10 million incurring medical expenses of more than 100 trillion USD [1]. There is thus an urgent need for measures to stop the spread of these disease-causing microbes. With COVID-19, combating AMR and preventing the emergence of new emerging drug-resistant organisms has become even more complex since 70% of COVID-infected patients rely on antibiotics for bacterial infections [2]. Therefore, searching for new anti-bacterial remedies is becoming an extremely important unmet need. In the first step, we selected and curated the phytochemicals of the Artemisia plants from the KNApSAcK metabolomics databases in cross-reference with other databases (Pubmed, Google scholar and Web of Science (WOS)). In the second step, we checked 946 compounds with text search into Pubmed, Google scholar, and Web of Science (WOS) using anti-bacterial or antimicrobial key words. As a result, 75 compounds from this genus were identified and classified based on their chemical structures.

Terpenes and Terpenoids
Forty-nine terpenes and terpenoids with anti-bacterial properties found in Artemisia plants are listed in Table 2. They constitute the majority of compounds in Artemisia as described in Section 1.2 (Chemical composition). Terpenes, a simple hydrocarbons structures, while terpenoids (oxygen-containing hydrocarbons) are defined as modified class of terpenes with various functional groups and oxidized methyl groups moved or deleted at various places which classified into alcohols, ethers, aldehydes, phenols ketones, esters, and epoxides that are volatile [30,31]. Terpenes contain ten monoterpenes and seven sesquiterpenes. There are also compounds identified as terpenoids, consisting of sixteen monoterpenoids, twelve sesquiterpenoids, one diterpenoids, and three triterpenoids as listed in Table 2. Table 2. Classification, structure, and anti-bacterial properties of terpenes and terpenoids from the Artemisia species.  (Tables 2-4), terpenes and terpenoids (Table 2), polyphenol (Table 3), and a miscellaneous (other) group (Table 4). The information about the structure, molecular weight, bacteria, anti-bacterial activity, and plant species of the 75 compounds is appended in each Table. Figure 1. Schema delineating the characterization of anti-bacterial compounds from the Artemisia species. In the first step, we selected and curated the phytochemicals of the Artemisia plants from the KNApSAcK metabolomics databases in cross-reference with other databases (Pubmed, Google scholar and Web of Science (WOS)). In the second step, we checked 946 compounds with text search into Pubmed, Google scholar, and Web of Science (WOS) using anti-bacterial or antimicrobial key words. As a result, 75 compounds from this genus were identified and classified based on their chemical structures.

Polyphenols
In Artemisia, there are 13 anti-bacterial compounds, including 11 flavonoids and 2 coumarins, as listed in Table 3.

Polyphenols
In Artemisia, there are 13 anti-bacterial compounds, including 11 flavonoids and 2 coumarins, as listed in Table 3.

Polyphenols
In Artemisia, there are 13 anti-bacterial compounds, including 11 flavonoids and 2 coumarins, as listed in Table 3.

Polyphenols
In Artemisia, there are 13 anti-bacterial compounds, including 11 flavonoids and 2 coumarins, as listed in Table 3

Polyphenols
In Artemisia, there are 13 anti-bacterial compounds, including 11 flavonoids and 2 coumarins, as listed in Table 3. Table 3.

. Miscellaneous Group
This group contains the thirteen compounds that are absent from the first three groups. Their chemical structure, molecular weight, anti-bacterial properties, and plant species are tabulated below.

. Miscellaneous Group
This group contains the thirteen compounds that are absent from the first three groups. Their chemical structure, molecular weight, anti-bacterial properties, and plant species are tabulated below.

. Miscellaneous Group
This group contains the thirteen compounds that are absent from the first three groups. Their chemical structure, molecular weight, anti-bacterial properties, and plant species are tabulated below. A. afra [9] P. intermedia 0.5 [9] A. naeslundii

. Miscellaneous Group
This group contains the thirteen compounds that are absent from the first three groups. Their chemical structure, molecular weight, anti-bacterial properties, and plant species are tabulated below. S. aureus, E. coli 100 µg/5 µL DMSO/disc [46] a SN: serial number, CS clinical strain.

Miscellaneous Group
This group contains the thirteen compounds that are absent from the first three groups. Their chemical structure, molecular weight, anti-bacterial properties, and plant species are tabulated below.

. Miscellaneous Group
This group contains the thirteen compounds that are absent from the first three groups. Their chemical structure, molecular weight, anti-bacterial properties, and plant species are tabulated below.

. Miscellaneous Group
This group contains the thirteen compounds that are absent from the first three groups. Their chemical structure, molecular weight, anti-bacterial properties, and plant species are tabulated below.

Anti-Bacterial Properties of the Artemisia Plants
Since the Artemisia plants have been used medicinally for bacterial and other infections, their extracts and compounds are often assessed in vitro and in animal models against typical harmful zoonotic bacteria such as methicillin-resistant S. aureus (MRSA), E. coli, S. typhimurium, S. aureus, etc. The inhibitory effects of the extracts and compounds of the Artemisia plants on bacteria are generally assessed based on serial dilution, agar plate assay, and/or disc diffusion methods as used in Table 2

Anti-Bacterial Properties of the Artemisia Plants
Since the Artemisia plants have been used medicinally for bacterial and other infections, their extracts and compounds are often assessed in vitro and in animal models against typical harmful zoonotic bacteria such as methicillin-resistant S. aureus (MRSA), E. coli, S. typhimurium, S. aureus, etc. The inhibitory effects of the extracts and compounds of the Artemisia plants on bacteria are generally assessed based on serial dilution, agar plate assay, and/or disc diffusion methods as used in Table 2, Table 3, Table 4. Their minimum inhibitory concentration (MIC) is used if applicable. Table 2, Table 3, Table 4 list the compounds with MIC values against specific bacteria. The anti-bacterial activity of the Artemisia species and compounds can be ranked based on the MIC value [61] as described in Table S1.
Using 0.2 mL of each extract of three different Artemisia species in the agar diffusion method, Poiată et al. discovered that A. annua methanolic and ethanolic extracts are most tochemicals possess a variety of anti-bacterial mechanisms [37,52,55,59,65]. They include destruction of cell wall, membrane, and cytosol, morphological changes, decreased virulence, and interference with DNA, protein, and cell division in bacteria as shown in Figure 2. P. aeruginosa, and A. baumannii A52 cs (MIC = 80 µg/mL) in A. haussknechtii plant [42].
Together, based on the anti-bacterial activity, 64 out of 75 compounds can be categorized into 15 rankings although 11 compounds have so far not been tested for their MIC (Table S2).

Targeting Cell Membrane/Cell Wall
The hydrophobicity of solutions must pass a cell membrane and the membrane's structure dictates a cell membrane's permeability [66]. A large volume of literature demonstrates that the Artemisia essential oils are able to damage the cytoplasmic membrane in bacteria and cause their loss of vital chemicals and cell death [67]. The anti-bacterial action of the essential oils can be ascribed to multiple mechanisms since there are different potential targets in bacteria. As described in Section 1.2. "Chemical compositions", Artemisia plants are rich in terpenes with potent anti-bacterial properties. Terpenes and terpenoids are partitioned into the layer of essential oils. Terpenes and terpenoids have the capability to increase membrane permeability by inserting through the phospholipidic bilayer in bacteria and, consequently, the leaking cell membrane causes the loss of cellular contents and cell lethality in bacteria [40]. For example, limonene (6&7) inhibited L. monocytogenes and other food-borne pathogens due to the interruption of bacterial cell integrity and wall structure observed using scanning electron microscopy (SEM) [34]. Limonene (6&7) enhanced the conductivity and induced nucleic acid and protein leakage that results in damaging cell membrane permeability and cell membrane rupture based on conductivity and PI staining measurements [34]. Furthermore, (+)-limonene (6) could lower inner pH values. pH 4.0 is more effective in eliminating E. coli BJ4. The E. coli MC4100 lptD4213 mutant with enhanced outer membrane permeability showed higher sensitivity to (+)limonene (6) at pH 4.0. Furthermore, reflectance infrared microspectroscopy revealed that β-sheet proteins played a crucial part in the mechanism of (+)-limonene (6). E. coli BJ4 s resistance to (+)-limonene was not altered by rpoS deletion, sub-lethal heat, acid shock, nor both of these conditions [68].
In order to inactivate E. coli O157:H7 in fruit juices or preserved foods [68], a synergistic combination procedure with heat was created based on the investigation of the mechanism of inactivation by (+)-limonene (6). Active compounds such as α-elemene (11), carveol (26), α-farnesene (13), methyl linolenate (72), diisooctyl phthalate (66), camphene (8), and 1,8-cineole (28) showed lower anti-bacterial activities on Gram-negative pathogens than Gram-positive cells. This may be due to the fact that the Gram-positive bacteria have a thicker layer of peptidoglycan that makes compounds more difficult to pass through and leads to imparting rigidity to the bacteria [69]. Yang et al. reported that methanolic crude extract of A. indica and its active major compounds, including carveol (26), 1,8-cineole (28), α-elemene (11), methyl linolenate (72), α-farnesene (13), and diisooctyl phthalate (66) could damage or kill E. coli, S. enterica, and L. monocytogenes as evidenced via membrane destruction based on the PI staining method [6]. Their transmission electron microscope (TEM) data showed that the A. indica crude extract caused bacterial emptiness while its most active compound, carveol (26) also led to severe bacterial membrane defects including membrane poring, wrinkling, emptiness, and membrane discontinuities [6]. Previous study showed that there were also changes in cell morphological under treatment with carveol (26) in S. aureus, E. coli, and S. typhimurium as shown by SEM with irregularly sized cells and the presence of debris in E. coli suggesting that cell division disruption or cellular membrane malfunction may have occurred [40].
Hydoxyl groups are highly reactive in some terpenoids (monoterpenoids) such as thymol (25), carveol (26), terpineol (24); as well as eugenol (75) in the other group (Table 4). The hydrogen bonds indicates for the active sites which targeting the enzymes, causing protein inactivation and cell membrane rupture or malfunction in bacteria [40,70,71]. For instance, a strong anti-bacterial compound, thymol (25) (Table S2) could inhibit Gramnegative and Gram-positive bacteria, including E. coli, A. baumannii A52 cs , S. aureus, P. aeruginosa, and K. pneumoniae K38 cs [42]. The SEM data demonstrated that the mode of action of thymol (25) with the hydroxyl group at a distinct position on the phenolic ring involved the membrane dysfunction in S. typhimurium [72] and disturbance of membrane integrity and impaired permeability that lead to leakage of membrane potential, protons, K + ions, and ATP in E. coli [73]. Eugenol (75) and terpineol (24) also caused cell death through disrupting cellular membrane and function as the cell membrane was entirely destroyed and surrounded by cell debris in S. typhimurium [40]. Although a sesquiterpenoid, vulgarone B (34), did not cause leakage of a significant cytoplasmic component, the SEM data indicated that it indeed altered cell morphology of S. aureus which became bloated and, crushed and aggregated at 8 h and 24 h post treatment, respectively. This led to 99.62% cell death in S. aureus indicating that vulgarone B (34) had strong bacterial effects on S. aureus without significantly breaking the cell membrane [44].
RA (74), a compound that grouped as miscellaneous showed high MIC values against S. aureus, E. coli, and MRSA as aforementioned, which might be explained by its inefficient penetration capability into bacterial cell walls. Interestingly, RA was found to have synergy, particularly in the log phase of bacterial growth, with antibiotics including amoxicillin, vancomycin, and ofloxacin against S. aureus and exclusively with vancomycin against MRSA [59]. In a different study, this acid induced membrane damage, resulting in impaired cell wall and membrane in S. aureus and leakage of bacterial contents and ions [74]. Antimicrobial activity of phenolic compounds was mainly attributed to inactivating cellular enzymes, which depends on the speed of penetration into the cell or its ability to alter membrane permeability [75].

Targeting DNA
DNA agarose gel analysis revealed that 8 to 24 h incubation of S. aureus with vulgarone B at 1000 µg/mL induced DNA breakage [44]. In addition, vulgarone B (34) was found to cause a single DNA nick and change in DNA mobility shift, indicating a mutual interaction or DNA breakage [44]. While the (−)-limonene (7) has been shown to be more active than (+)-limonene (6). The (−)-limonene (7) damaged DNA, which induced SOS response, membrane impairment and release of heat shock proteins (HSPs) as evidenced by the induction of PkatG and PsoxS promoters in E. coli models via formation of reactive oxygen species. At high concentrations, (−)-limonene (7) causes irreversible degrading processes in both S. aureus (Gram-positive) and E. coli (Gram-negative) for 24 h. This phenomenon was observed to be weaker in E. coli when treated with α-pinene (9) compared to inducing only heat shock [76]. Thymol (25) repressed the hilA gene, which encodes a gene activator for the virulence of S. typhimurium, increased DNA thermal stability, and inhibited transcription by downregulating the DNA-binding protein H-NS [73].

Target Protein and Enzymes
Alpha-pinene (9) at 2.72 µg/mL only partially inhibited protein refolding in E. coli with a HSP IbpB mutation. However, (−)-limonene (7) (1.36 µg/mL) competed with IbpB to bind to hydrophobic sites of DnaKJE chaperone and, thus, inhibited the DnaKJE -ClpB bichaperone-dependent refolding function of heat-inactivated bacterial luciferase in E. coli WT and mutant ∆ibpB strains. Furthermore, (−)-limonene (7) (0.136 µg/mL) induced the overproduction of hydrogen peroxide and superoxide anion radicals, eventually, leading to DNA and protein damage in E. coli as detected by inducible specific lux-biosensors. The induction of oxidative stress in the first minute of (−)-limonene (7) could be related to the synthesis of reactive oxygen species (ROS), which has been reported in several antibiotic cases. Moreover, a significant increase in the inhibitory effect of (−)-limonene (7) was observed without catalase and peroxidase enzymes in bacterial strains JW3914-1 ∆katG729::kan, especially JW3933-3 ∆oxyR749::kan, indicating that hydrogen peroxide played an important role. In addition, both terpenes could induce heat shock to damage the cells [76]. Limonene (6&7) declined the activity of electron transfer chain (ETC), composed of complexes I to V, located on the plasma membrane of L. monocytogene since it significantly downregulated the protein level of complexes III, IV, and V, and some protein units in complexes I and II following 24-h treatment using ESI MS/MS [34]. Limonene inhibited the activity of the ETC complex and ATPase in L. monocytogenes, resulting in a decrease in ATP content and intracellular ATPase activity (Na + K + -ATPase, Ca 2+ -ATPase) [34]. Limonene might inhibit respiration by blocking the electron transmission from NADH to coenzyme Q, which might explain why ATP synthesis is blocked by limonene (6&7).
The increased permeability of the cell membrane was shown to delay the capacity to produce essential compounds for growth and reproduction and, eventually, cell death in bacteria. In light of the aforementioned findings, limonene might decrease enzyme activity, limit respiration, and mess with the ATP balance in L. monocytogenes [34]. Particularly, the expression of the complex I subunit (Unigene11357 CK 0A, CL1094.Contig4 CK 0A, CL1528.Contig4 CK 0A, and CL4703.Contig1 CK 0A), in charge of acquiring two electrons from NADH and transferring them to coenzyme Q via ferritin, was markedly increased, indicating that more electrons from NADH would be transported into the ETC of the L. monocytogenes [34] by limonene (6&7). Accordingly, limonene (6&7) blocked the ETC and accumulated electrons in the cytochrome (CL594.Contig2 CK 0A), one subunit of the complex III, and the cytochrome oxidase subunit (Unigene2340 CK 0A, Unigene7527 CK 0A, CL3277.Contig1 CK 0A) of the complex IV. A considerable downregulation of the majority of ATP synthase subunits in complex V further implied that ATP synthesis was inhibited, which was in good agreement with the drop in ATP content. Such treatment also caused considerable downregulation of the Unigene6313 CK 0A subunit of complex V's V-type proton ATPase. The V-type proton ATPase hydrolyzed ATP to produce an electrochemical gradient across the membrane in addition to controlling pH within and outside of bacteria [34]. Normal bacteria experienced necrosis and apoptosis when exposed to the high levels of extracellular H + produced by the milieu created by a high level of V-ATPase [77].
Through hydrophilic and hydrophobic interactions, thymol (25) could interact with membrane bound or periplasmic proteins [78]. For example, thymol (25) upregulated the levels of OmpA, OmpX, GlnH, and FabI that are related to the synthesis of outer membrane proteins [73]. The build-up of misfolded outer membrane proteins as well as the increase of gene expression in outer membrane protein production was observed in S. enterica after exposing it to thymol (25) at sub-lethal concentrations. Moreover, thymol (25) affected the citrate metabolic pathway, which eventually affected ATP synthesis. Thymol (25) has been shown to alter various pathways of cell metabolism and impair the metabolic pathway. For example, it downregulated the protein PtsH involved in the phosphotransferase system and the sugar transport system, upregulated the proteins Enolase (Eno) and 2,3bisphosphoglycerate-independent phosphoglyceromutase (iPGM), and downregulated the ATP synthase α-subunit, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase dPGM and glyceraldehyde-3-phosphate dehydrogenase A (AtpA, GpmA and GapA) and the dPGM involved in energy metabolism.
The citrate breakdown route involved the enzymes S-adenosylmethionine synthetase and the autonomus glyacyl radical cofactors (MetK and GrcA). In addition, they proposed that downregulation of S-adenosylmethionine synthase. The autonomus glyacyl radical cofactors (MetK and GrcA) implicated in the citrate degradation pathway would suppress the activation of the pyruvate formate lyase, resulting in a blockage of the pathway and a downregulation of AckA, and that a build-up of citrate would result in CitE overexpression. Since acetate kinase was involved in the production of ATP, it is possible that thymol plays a significant role in the dysfunction of this metabolic process [73]. Thymol (25) inhibited protein biosynthesis by upregulating the 30S ribosomal protein S1 (RpsA), which was involved in translation initiation and elongation processes, and down-regulating the 50S ribosomal protein L7/L12 (RplL), the binding site for several factors involved in protein synthesis and translation accuracy. According to these results, thymol (25) might regulate cell wall synthesis that might be linked to cell division with central metabolism.
The limitations and challenges faced in the development of new anti-bacterial compounds from Artemisia plants include: (1) Artemisia plant-derived phytocompounds generally have low anti-bacterial potency; (2) anti-bacterial mechanisms of action of the compounds are not clear; (3) anti-bacterial compounds with high potency need more labor and time to be identified; and (4) total synthesis of the anti-bacterial compounds such as terpenoids is challenging [87,88]. Additionally, Artemisia plants have been used to treat bacterial infections in humans from ancient time. So far, only artemisinin from A.  3) anti-bacterial compounds with high potency need more labor and time to be identified; and (4) total synthesis of the anti-bacterial compounds such as terpenoids is challenging [87,88]. Additionally, Artemisia plants have been used to treat bacterial infections in humans from ancient time. So far, only artemisinin from A. annua has been developed as a prescription drug against malaria. Some gaps in development of anti-bacterial drugs from the Artemisia plants include the identification, safety, efficacy, and synthesis of active compounds from this genus. Currently, components of the essential oils form this genus was successfully identified and they had a MIC of 0.1 µg/mL against certain bacteria but not the other. This suggests other potential anti-bacterial compounds need to be characterized, which may reflect the gaps from current findings to drug discovery [89,90].

Conclusions
The genus Artemisia comprises over 500 species. These plants are annual or perennial aromatic herbs and subshrubs with greenish to yellowish leaves, white or yellow flowers, and small black seeds. Artemisia species are a remarkable source of foods and medicines and their culinary and medicinal functions can be attributed to their rich phytochemicals. Despite significant advances in phytochemical and biological research of Artemisia plants over recent years, comprehensive and critical reviews of this genus are fragmented or limited. The present review updated and summarized information about the chemistry, antibacterial properties, and mechanisms of action of the Artemisia plants and phytocompounds. Seventy-five compounds present in twenty Artemisia species were extensively dis-cussed with regard their chemical structure, anti-bacterial activity and mechanism and structureand-activity relationship. Generally speaking, compounds of the Artemisia plants inhibit bacteria more potently than their crude extracts. However, the structure of these compounds also affects this bacterial inhibition as well as their modes of action. Caution should be taken in the use of the Artemisia plants and phytochemicals for bacterial infections in humans and animals.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/bioengineering10060633/s1, Table S1: Degree of anti-bacterial activity in different compounds based on the MIC value against pathogens; Table S2: Ranking of the anti-bacterial activity of the 75 compounds based on the MIC value against pathogens.