Anti-Bacterial Artemisia Plants: History
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Contributor:
Khotibul Umam
,
Ching-Shan Feng
,
Greta Yang
,
Ping-Chen Tu
,
Chih-Yu Lin
,
Meng-Ting Yang
,
Tien-Fen Kuo
,
Wen-Chin Yang
,
Hieu Tran Nguyen Minh

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.

  • anti-bacterial activities
  • Artemisia plants
  • compound structure

1. 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.
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 active in suppressing five Gram-positive bacteria (MRSA, S. aureus ATCC 25923, S. lutea ATCC 14579, and B. subtilis ATCC 6633) and a Gram-negative P. aurugi [1]. Furthermore, the next active extract was a methanolic extract of A. absinthium, which inhibited MRSA, S. lutea, B. subtilis, and P. auruginosa. The ethanolic extracts of A. vulgaris, and A. absinthium, later A. annua hexane extract and A. vulgaris ethanolic extract, inhibited only one to three pathogens, while no anti-bacterial activity was observed in A. absinthium and A. vulgaris hexane extracts [1]. The results of these investigations conclude that anti-bacterial activity can differ among various Artemisia species. This difference could be ascribed to the composition of their phytochemicals.
A. annua is the most studied plant in the Artemisia genus. Ivarsen et al. reported that dichloromethane and n-hexane extracts of the aerial parts of A. annua were active against C. perfringens with a MIC value of 185 and 270 µg/mL, respectively [2]. In addition, A. annua extracts have also been reported to inhibit a significant number of pathogens such as E. coli, S. typhi, B. subtilis, S. aureus, and C. perfringens [3][4]. The high anti-bacterial properties of A. annua are due to its primary compounds from essential oil including monoterpenoids group were 30.7% artemisia ketone (18), 15.8% camphor (33), and 18.2% from sesqueiterpenes [5] and also from sabinene (1), linalool (19), camphene (8), α-pinene (9), α-terpineol (24), borneol (31&32), camphor (33), eugenol (75), and coumarins [2]. The terpenoids (monoterpenoids) α-terpineol (24) is also found in A. feddei [6] and A. princeps Pamp [7] with a very low to very high activity against 20 pathogenic bacteria with a MIC value of 30 to 3200 µg/mL [6][7]. Likewise, borneol (31&32), commonly present in A. feddei, A. indica, A. argyi, and A. iwayomogi, showed a MIC value of 2 to 12,800 µg/mL against 44 zoonotic bacteria such as S. aureus, S. typhimurium, S. enteritidis, B. cereus, V. vulnificus, F. nucleatum, P. intermedia, S. pyogenes, E. coli, S. sobrinus, S. epidermidis, S. pyogenes, S. mutans, S. anginosus, S. gordonii, A. actinomycetemcomitans, P. gingivalis, E. faecalis, E. gallinarum, S. sanguinis, S. ratti, S. criceti, P. aeruginosa, K. pneumonia, and S. dyssenteriae [6][8][9][10]. Using a bioactivity-directed fractionation and isolation strategy, β-caryophyllene (12) was identified and this compound inhibited 36 pathogenic and clinical bacteria, particularly S. pyogenes, S. aureus, E. gallinarum, H. influenzae, K. pneumonia, MRSA, methicillin and gentamycin-resistant S. aureus (MGRSA), etc. with a MIC value ranging from 25 to over 12,800 µg/mL [6][11][12]. Furthermore, it is possible that most terpenoids have greater anti-bacterial activity than terpenes. The MIC value of one terpenoids (monoterpenoids), camphor (33), and one triterpenoid, ursolic acid (49), towards different bacteria was 2 to 1280 µg/mL [9] and 32 to 1280 µg/mL [13], respectively. Accordingly, a terpenoids (sesquiterpenoids), artemisinin (44) (MIC = 90 µg/mL), had a higher activity than a monoterpenoid, linalool (19) (MIC 250 µg/mL) against B. cereus, E. coli, S. aureus, and S. typhimurium in A. annua [14]. One monoterpene, myrcene (2), presents in the A. absinthium extract, indicated very low-moderate activities against S. epidermidis, B. subtillis, S. dyssenteriae, and K. pneumonia with a MIC value of 121, 322.11, 325, and 400 µg/mL, respectively [15]. Similarly, α-thujone (29) and β-thujone (30) suppressed S. aureus, S. epidermidis, E. coli, K. pneumoniae, P. aeruginosa, and E. cloacae with MIC values of 90, 100, 350, 650, 750, and 830 µg/mL, respectively [8][16].
Ponticaepoxide (70), (MIC = 100–200 µg/mL), identified from A. annua, had a higher anti-bacterial activity than (+)-threo-(5E)-trideca-1,5-dien-7,9,11-triyne-3,4-diol (71) (MIC = 400–800 µg/mL) [2]. Terpinen-4-ol (23) of A. feddei had a MIC of 50–3200 µg/mL against 15 pathogens [6]. A phenylpropanoid, estragole (73) showed a higher activity towards S. aureus species with a MIC value of 101.6 to128 µg/mL [17] than that (MIC = 200 µg/mL) of eugenol (75), [18]. flavonoids possess low anti-bacterial activities. For instance, chrysosplenol D (55), a major flavonoid found in A. annua extracts, displayed a very low to low anti-bacterial activity against C. perfringens, E. coli, B. subtilis, M. tetragenus, and P. fluorescens with a MIC value of 200 to 500 µg/mL [2]. Moreover, the other flavonoids, casticin (51) showed lower anti-bacterial activity against C. perfringens 200302-1-1-Ba with a MIC value of 800 µg/mL [2]. Moreover, a popular phenolic, rosmarinic acid (RA) (74), showed very low anti-bacterial activity against S. aureus, E. coli, and MRSAcs with a MIC of 500 to 10,000 µg/mL [19][20]. This compound was found in A. absinthium, A. annua, A. alba, and so on [21].
As mentioned in Section 1.2, artemisia ketone (18), germacrene B (16) (MIC = 32–128 µg/mL), and borneol (33&34) (MIC = 32–128 µg/mL) were discovered to exhibit moderate to very high anti-bacterial activities. In A. indica found that the essential oil of artemisia ketone (18), germacrene B (16), and borneol (31&32) were 42.1%, 8.6%, and 6.1% respectively. In contrast, (Z)-chrysanthenyl acetate (27) (MIC value of 128–512 µg/mL) with 4.8% essential oil had very low to moderate anti-bacterial activities against 7 clinical bacteria [8]. Similar to the bacterial efficacy of artemisia ketone (18), sabinene (1), and p-cymene (5) at MICs ranging 32 to >128 µg/mL in the same test [8]. Interestingly, among the terpenoids in A. indica, nerol (20) had high to very high anti-bacterial activity against S. aureus, S. epidermidis, P. aeruginosa, B. subtilis, S. typhi, and K. pneumonia, with MIC values ranging from 32 to over 64 µg/mL [8]. It is obvious that the action of some compounds in killing Gram-negative pathogens is more effective than that in Gram-positive pathogens. In short, α-elemene (11) inhibited the Gram-negative bacteria S. enterica and E. coli with a MIC value of 0.1 µg/mL and 25 µg/mL, respectively [22]. However, the Gram-positive bacteria, S. typhimurium, B. cereus, S. aureus, and L. monocytogenes, had a MIC value ranging from 60 µg/mL to 262.5 µg/mL [14][22]. The same phenomenon was also observed in A. indica and/or A. dracunculus for the low-very high anti-bacterial activities of carveol (26) [14][22], while α-farnesene (13), methyl linolenate (72), diisooctyl phthalate (72) had very low-very high anti-bacterial actions [22]. Camphene (8) [8][12] had low to high anti-bacterial activities.
Of note, 1,8-cineole (28) had low-high anti-bacterial activities against 37 bacterial pathogens [6][12][22][23] and this compound is present in A. indica, A. feddei, A. vestica, A. iwayomogi, etc [24]. Moreover, Alpha-pinene (9) had similar inhibition for Gram-negative and Gram-positive bacteria since its MIC had a low to very high activity. Gram-negative bacteria such as K. pneumonia (32 µg/mL), P. aeruginosa (64 µg/mL), E. colics (98 µg/mL), H. influenzae (126 µg/mL), S. typhi (128 µg/mL), S. dyssenteriae (128 µg/mL), and K. pneumoniae (178 µg/mL)) and Gram-positive bacteria (S. aureus (32 µg/mL), B. subtilis (128 µg/mL), S. epidermidis (>128 µg/mL), S. pyogenes (132 µg/mL), MRSAcs (172 µg/mL), S. pneumoniae (172 µg/mL), MRSA (210 µg/mL), MGRSA (256 µg/mL), and S. aureus (172 µg/mL)) [8][23]. As mentioned above, it also was similar condition for the anti-bacterial activity presented by β-pinene (10) and α-thujone (29) in A. indica which had a low to very high activity for Gram-negative and Gram-positive bacteria [8][23][25]. In addition, grandisol (21) and piperitone (22) had better results in A. vestica against 6 pathogens [23].
In parallel, dichloromethane extract of A. integrifolia was identified as the most active fraction. Six compounds were identified and their anti-bacterial activities for E. coli, B. cereus, S. aureus, and Y. enterocolitica are listed as follows in descending order: integracid (73) (MIC = 60–120 µg/mL) > zerumbone (37) (MIC = 90–230 µg/mL) > (+)-(S)-ar-turmerone (35) (MIC = 100–260 µg/mL) = α-curcumene (14) > (+)-(S)-dihydro-ar-turmerone (36) (MIC = 120–160 µg/mL) > dihydro-ar-curcumene (15) (MIC = 120–4000 µg/mL) [26]. Furthermore, a crude extract of A. afra inhibited Gram-positive bacteria (A. naeslundii, A. israelii, and S. mutans), and Gram-negative bacteria (P. intermedia, P. gingivalis, and A. actinomycetemcomitans) with a MIC value of 25 to 1600 µg/mL [27]. The highly active anti-bacterial action of the A. afra extract was attributable to six phytochemicals, including phytol (46), α-amyrin (47), and betulinic acid (48), acacetin (50), 12α,4α-dihydroxybishopsolicepolide (39), and scopoletin (61). All six compounds had very high anti-bacterial potential [27]. Especially, the three most active compounds, betulinic acid (48), acacetin (50), and scopoletin (61) which exerted an excellent antimicrobial activity with a MIC value of 0.3 to >1 µg/mL against the above mentioned pathogens [27]. Similarly, the sesquiterpenoid vulgarone B (34) found in A. iwayomogi species had a very high anti-bacterial activity against S. typhimurium, S. aureus, and S. enteritidis with a MIC value of 0.5 to >2 µg/mL [10].
The main compound in A. herba-alba oils, sesquiterpene germacrene D (17), was found against six bacteria, B. subtillis, S. aureus, P. vulgaris, S. dyssenteriae, K. pneumonia, and S. typhi, with MIC values varying from 30.31 to 90.15 µg/mL [15]. Mohamed et al. reported that six of seven secondary metabolites isolated from the organic extract of A. herba-alba aerial parts showed anti-bacterial activities. Using disc diffusion assays [28], two novel compounds, 1,3,8-trihydroxyeudesm-4-en-7α,11βH-12,6α-olide (40) and 3α,8β-dihydroxygermacr-4(15),9(10)-dien-7β,11αH,12,6α-olide (41) suppressed B. subtilis and S. aureus growth at 25 to 50 µg per disc and B. subtilis, S. aureus, and E. coli growth at 50 to 100 µg per disc, respectively. Similarly, the other compounds, 1β,8α-dihydroxy-11α,13-dihydrobalchanin (42), 11-epiartapshin (43), and 5-β-D-glucopyranosyloxy-7-methoxy-6H-benzopyran-2-one (62) could also inhibit the three pathogenic strains at 25 to 100 µg per disc, except that benzoic acid p-(β-D-glucopyranosyloxy)-methyl ester (65) at dose of 25 and 50 µg could kill S. aureus and B. subtilis, respectively [28]. In order to study the action of the A. capillaris extract on clinically drug-resistant bacteria, limonene (6&7), from this extract were shown to have very low to very high anti-bacterial activities against E. colics, H. influenzae, MRSAcs, S. pyogenes, K. pneumoniae, S. pneumoniae, MRSA, MGRSA, and L. monocytogenes with a MIC value of 20 to 332 µg/mL [11][29]. In addition, capillin (64), an aromatic ketone or ynone from A. capillaris, had moderate to high activities with a MIC value of 64 to 165 µg/mL inhibiting the listed pathogens [11].
Zhang et al. screened five fractions of aqueous extract of A. argyi leaf to find the most active fraction against S. aureus. As a result, its chloroform fraction showed a very low anti-bacterial activity with a MIC value of 3000 µg/mL [30]. One flavonoid, jaceosidin (59), was detected among 24 phytochemicals from the chloroform fraction using LC-MS [30]. In addition, this compound was also found in A. californica and it had a MIC value of 3.3 µg/mL for E. coli ΔtolC, E. coli ΔtolC (fabl), and that of 33 µg/mL for E. coli WT [31]. Screening of these pathogens showed that jaceidin (60) from A. californica had very high anti-bacterial activity with a MIC value of 18 to 36 µg/mL [31]. However, chrysosplenol B (54) had very low to low anti-bacterial activity with a MIC value of 200 to 500 µg/mL [31]. More anti-bacterial compounds have been discovered in A. argyi species. Carveol (26), a monoterpenoid in the terpenoid group, had very high anti-bacterial activity towards S. enterica, S. aureus, E. coli ATCC25922, S. typhimurium, E. coli ATCC8739, L. monocytogenes, and B. cereus with a MIC value of 0.1, 15, 25, 30, 60, 100, and 120 µg/mL, respectively [14][22].
Two isomers β-ocimene (3&4) were major compounds of A. dracunculus; (E)-β-ocimene (3) had a lower MIC value of 130 to 650 for B. subtillis, S. epidermidis, P. vulgaris, S. dyssenteriaem, and K. pneumonia than (Z)-β-ocimene (4) with a MIC value of 130 to 600 [15]. Several other active compounds were purified from other Artemisia species. The A. douglasiana extract inhibited standard H. pylori and six clinical strains with a MIC value of 60 to 120 µg/mL. Consistently, its active phytochemical, dehydroleucodine had a MIC value of 1 to 8 µg/mL [32]. Moreover, five active compounds isolated from the A. rupestris extract, chrysosplenetin (52), chrysoeriol (53), penduletin (56), artemetin (57), and pachypodol (58) showed synergistic effects with norfloxacin, ciprofloxacin, and/or oxacillin against S. aureus isolates [33]. Two novel compounds ((Z)-2-(hexa-2,4-diyn-1-ylidene)-1,6-dioxaspiro(4.5)dec-3-ene (68) and (E)-2-(hexa-2,4-diyn-1-ylidene)-1,6-dioxaspiro(4.5)dec-3-ene (69)) were isolated from the acetone extract of A. pallens which had outstanding anti-bacterial activity against Gram-negative and Gram-positive infections such as B. subtilis, S. aureus, P. aeruginosa, and E. coli with a MIC value of 2.6–80.6 ug/mL, 4.7–10 µg/mL, 2.5–21.8 ug/mL, and 1.6–2.7 µg/mL, respectively [34]. In addition, thymol (25) was found to be one moderately active compound against S. aureus and K. pneumoniae K38cs (MIC = 60 µg/mL), and E. coli, P. aeruginosa, and A. baumannii A52cs (MIC = 80 µg/mL) in A. haussknechtii plant [35].

2. Mechanisms of Action of Artemisia Plants and Their Compounds

Artemisia plants and their phytochemicals are well known for their antimicrobial properties [24][26][27][28]. More and more studies report that Artemisia plants and their phytochemicals possess a variety of anti-bacterial mechanisms [20][23][31][34][36]. 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 1.
Bioengineering 10 00633 g002 550
Figure 1. Anti-bacterial mechanisms of the Artemisia compounds. Four likely mechanisms by which the Artemisia phytochemicals inhibit pathogenic bacteria are proposed in different molecular targets and compartments of bacteria. (1) Artemisia essential oils and 9 constituents target bacterial cell membrane and/or cell wall via changes in membrane structure and function, membrane permeability and rupture of bacterial membrane and/or cell wall, eventually leading to cause the release of vital chemicals and cell death; (2) (+)-Limonene (6&7), vulgarone B (34) and thymol (25) can interfere with DNA structure and function, and, in turn, induce SOS response, and oxidative stress (i.e., over-production of reactive oxygen species (ROS)). Consequently, aberrant ROS kills bacteria; Limonene (6&7), (+)-α-pinene (9), thymol (25), chrysosplenetin (52), chrysoeriol (53), penduletin (56), RA (74), jaceosidin (59), (Z)-2-(Hexa-2,4-diyn-1-ylidene)-1,6-dioxaspiro(4.5)dec-3-ene (68), and (E)-2-(Hexa-2,4-diyn-1-ylidene)-1,6-dioxaspiro(4.5)dec-3-ene (69) can target proteins and enzymes via altering protein functions, denaturing proteins and reducing enzyme activities. Consequently, oxidative stress, heat shock stress, and expressional inhibition of virulent factors are induced in bacteria; and (4) four compounds target the other mechanisms. Thymol (25) and eugenol (75) can target biofilm pathways via reduction and inactivation of biofilm, leading to loss of bacterial virulence and survival. Estragole (73) and artesunate (45) inhibit the efflux pumps and, as a result, accumulate antibiotics within bacteria.

2.1. 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 [37]. 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 [38]. “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 [39]. 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) [29]. 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 [29].
In order to inactivate E. coli O157:H7 in fruit juices or preserved foods [40], 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 [41]. 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 [22]. 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 [22]. 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 [39].
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 [39][42][43]. For instance, a strong anti-bacterial compound, thymol (25) (Table S2) could inhibit Gram-negative and Gram-positive bacteria, including E. coli, A. baumannii A52cs, S. aureus, P. aeruginosa, and K. pneumoniae K38cs [35]. 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 [44] and disturbance of membrane integrity and impaired permeability that lead to leakage of membrane potential, protons, K+ ions, and ATP in E. coli [45]. 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 [39]. 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 [10].
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 [20]. 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 [46]. 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 [47].

2.2. 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 [10]. 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 [10]. 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 [48]. 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 [45].

2.3. 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 ΔkatG729::kan, especially JW3933-3 ΔoxyR749::kan, indicating that hydrogen peroxide played an important role. In addition, both terpenes could induce heat shock to damage the cells [48]. 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 [29]. 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, Ca2+-ATPase) [29]. 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 [29]. 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 [29] 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.
Through hydrophilic and hydrophobic interactions, thymol (25) could interact with membrane bound or periplasmic proteins [49]. For example, thymol (25) upregulated the levels of OmpA, OmpX, GlnH, and FabI that are related to the synthesis of outer membrane proteins [45]. 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.
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 [45].
RA (74) was shown to completely suppress the expression of surface membrane proteins (MSCRAMM’s), 40 to 90 kDa, main factors that cause infections. They are covalently anchored to RA, making them prime targets for antibiotics since they mediate the initial host-bacterial interactions and resistance in MRSA leading to suppression of virulence factors [20]. Other compounds, chrysosplenetin (52), chrysoeriol (53), and penduletin (56) were found to bind protein NorA, a cytoplasmic membrane containing a multidrug efflux protein and reassemble proteoliposomes [50]. This binding could change the morphology and function of NorA [33]. To develop novel antibiotics that generally target fatty acid biosynthesis and FAS II enzyme enoyl reductase in bacteria is thought to be a promising strategy [51][52]. Thus, jaceosidin (59) was examined and found to inhibit FabI in vitro, but no signal was found in vivo against the enzyme in E. coli [31]. Both spiro compounds (Z)-2-(Hexa-2,4-diyn-1-ylidene)-1,6-dioxaspiro(4.5)dec-3-ene (68), and (E)-2-(Hexa-2,4-diyn-1-ylidene)-1,6-dioxaspiro(4.5)dec-3-ene (69) inactivated Gram-positive (B. subtilis and S. aureus) and Gram-negative (P. aeruginosa and E. coli) bacteria, perhaps since these compounds could bind to the active site of the DNA gyrase B, an important bacterial enzyme that catalyzed the negative supercoiling of double-stranded closed-circular DNA in an ATP-dependent manner [53] using molecular docking [34].

2.4. Others

There were also several case studies of Artemisia species acting as inhibitors of both bacterial growth and biofilm formation. For instance, 24 h treatment with essential oils of A. herba-alba, A. campestris, and A. absinthium which major component were 32.07% β-pinene (10), 39.21% chamazulene, and 29.39% α-thujone (29), at 620 µg/mL could reduce biofilm formation by up to 45% for E. coli E2346/69 and 70% for E. coli K-12 [54]. In particular, eugenol (75) at 200 µg/mL destroyed the cell membrane of carbapenem-resistant Klebsiella pneumoniae (CRKP), as characterized by decreased intracellular ATP concentration, reduced intracellular pH and cell membrane hyperpolarization, coupled with enhanced membrane permeability, and led to changes in bacterial cell structure and intracellular component leakage. Additionally, eugenol (75) inhibited biofilm formation and inactivated biofilm in CRKP by using different EM techniques. This happened due to the fact that eugenol (75) strongly inhibited biofilm-associated gene expression (pgaA, luxS, wbbM, and wzm genes of CRKP-12), upregulated mrkA mRNA, and inactivated CRKP cells growing in biofilms [18]. In the case of thymol (25), it was also reported to inhibit biofilm formation against different carbapenemase-producing Gram-negative bacilli [49][55][56].
Estragole (73) has been proposed to be a possible inhibitor of the efflux pump against S. aureus 1199B and S. aureus K2068 species. This inhibition is supported by data indicating that estragole (73) treatment enhanced the action of norfloxacin and ethidium bromide against S. aureus 1199B and lowered the MIC of ethidium bromide against S. aureus K2068 [57]. Artesunate (45) has been shown to increase the antibiotic properties of β-lactams against MRSA by binding penicillin-binding protein 2a (PBP2a) and downregulating MecA expression, which was upregulated by oxacillin. In addition to reducing bacterial load, artesunate (45) was found to protect mice from MRSA WHO-2 (WHO-2) when combined with oxacillin. In mouse peritoneal macrophages stimulated with heat-killed WHO-2, artesunate (45) inhibited the expression level of TLR2 and Nod2, two key players in the inflammasome, suggesting artesunate (45) is a candidate drug for MRSA sepsis [58]. The data obtained from confocal microscopy and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that artesunate (45) increased the accumulation of antibiotics (daunorubicin and oxacillin) in MRSA, suggesting that artesunate (45) could affect the efflux pumps of antibiotics. Consistently, artesunate (45) inhibited the gene expression of NorA, NorB, and NorC but not MepA, SepA, and MdeA, in the efflux pumps [59].

This entry is adapted from the peer-reviewed paper 10.3390/bioengineering10060633

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