Isolated before from the same species [4]. **@**Confirmed by UV *λ*max. <sup>a</sup> Organic acid, <sup>b</sup> phenolic acid, <sup>c</sup> amino acid derivative, <sup>d</sup> flavan-3-ol, <sup>e</sup> phenylpropanoic acid, <sup>f</sup> stilbenoid, <sup>g</sup> flavone, and <sup>h</sup> biflavonoid.

#### *3.3. <sup>1</sup>H-NMR Analysis of L. coronopifolia Extract*

The extract was dissolved in deuterated dimethyl sulfoxide (DMSO-*d6*) and introduced into the proton NMR (500 MHz) experiment. The resonated peaks at different chemical shifts explained the kind of protons present in the chemical structures and repre-

sented the classes of skeletons in the solution. The 2-phenyl chromen-4-one of the flavone structure bearing substituents at positions 5 and 7 of A-ring was observed as the main skeleton. Through the <sup>1</sup>HNMR chart, the downfield protons that appeared as a doublet at δ 7.93 ppm were characterized for H-20 and 60 of B-ring, while the protons resonating at δ 6.92 ppm were assigned for H-30 and 50 of B-ring, with the *ortho*-coupling constant (*J* = 9.0 Hz) suggesting that the B-ring bearing the hydroxyl group was at position 40 , while the appearance of a singlet proton at δ 6.83 ppm was characterized for H-3 of the flavone structure. In addition, the downfield shift observed at δ 6.42 and 6.81 ppm for H-6 and H-8 of the A-ring of the flavone structure with *meta*-coupling (2 Hz) or broad singlet was predicted due to the substitution at position 7. The previous interpretation confirmed the presence of the apigenin derivative in the solution with substitution at position 7. In addition, the ABX system of B-ring was observed along with the chart through the coupling constant of resonated protons as a doublet of doublet (*ortho*- and *meta*-coupled), doublet (*meta-*coupled), and doublet (*ortho*-coupled) at δ 7.46, 7.43, and 6.92 ppm, respectively, were assigned, respectively, for H-60 , H-20 , and H-50 of the B-ring, suggesting that the B-ring was substituted with substituents at positions 30 and 40 . The rest of H-3, H-6, and H-8 proton signals resonated around the mentioned chemical shifts, which confirmed the presence of a luteolin derivative with 7-*O*-substituents. In addition, the resonated singlet signal at δ of 3.83 ppm was assigned to 4'-O-methylated flavone (i.e., the presence of an acacetin or methyl apigenin derivative in the solution). Moreover, the series of signals between δ 3.14 and 3.51 ppm were attributable to a sugar moiety, and the doublet signal with a coupling constant of 7.2 Hz was distinctive for the anomeric proton of sugar with O-*β*-D-linkage. Depending on the previously published data and MS assignment, the major attached sugar for the flavone structure is glucuronide moiety.

#### *3.4. Insecticidal Activity*

Plant extracts are considered a new ecofriendly and efficient alternative means for controlling mosquitoes. The larvicidal activity of the extract was evaluated against the 3rd instar larvae of *C. pipiens.* The fiducial limits were calculated for LC25, LC50, and LC<sup>95</sup> at *p* < 0.05 (Table 2). The extract exhibited considerable larvicidal activity against *C. pipiens* larvae where the LC<sup>50</sup> values after 24, 48, and 72 h of exposure were 52.74, 34.07, and 29.076 µg/mL, respectively. The essential oil from the Egyptian plants exhibited insecticidal activity against the 4th larval instar of *C. pipiens* [42]. Similar activities were reported from essential oils of other *Lavandula* species, among them *L. stoechas* and *L. dentata* [43,44].


**Table 2.** Larvicidal activity of *L. coronopifolia* extract against the 3rd instar larvae of *Culex pipiens* 24, 48, and 72 h post treatment.

\* Fiducial limits; <sup>a</sup> chi square.

#### *3.5. Biochemical Activity*

Insects release several detoxifying enzymes such as esterases, oxidases, and reductases to face and detoxify many invader pesticides [45]. To get an insight into the mechanisms involved, we explored the activities of five different enzymes in the 3rd larval instar of *C. pipiens*. The extract, at a concentration of LC<sup>50</sup> and exposure time of 72 h, inhibited cytochrome P-450 monooxygenase, acetylcholinesterase, and carboxylesterase by −9.92%, −19.41%, and −25.47%, respectively, compared to the control group (Figure 3), while the

treated larvae showed elevation of α-esterases and glutathione S-transferase contents by 15.63% and 37.02% compared to the untreated larvae. This could highlight the significant role of glutathione S-transferase and α-esterases in the detoxification mechanism of the extract. Our results come in agreement with those of the Huang et al. study, which reported an increase in the intracellular glutathione content when the larvae were treated with a polyphenolic-rich extract [46–48]. *Molecules* **2021**, *26*, x FOR PEER REVIEW 11 of 16

**Figure 3.** Effects of *L. coronopifolia* extract (L. cor.) on the enzymatic activities of cytochrome P-450 monooxygenase (CYP-450s) (**A**), carboxylesterase (CarE) (**B**), acetylcholinesterase (AchE) (**C**), αesterases (**D**), and glutathione S-transferase (GSTs) (**E**) in the 3rd larval instar of *C. Pipiens*. (**F**); % of changes. Data were represented as mean ± SE. Lowercase letters above the bars indicate significant differences between different treatment groups (Duncan's multiple range test, *p* < 0.01). Error bars indicate 95% confidence intervals. **Figure 3.** Effects of *L. coronopifolia* extract (L. cor.) on the enzymatic activities of cytochrome P-450 monooxygenase (CYP-450s) (**A**), carboxylesterase (CarE) (**B**), acetylcholinesterase (AchE) (**C**), α-esterases (**D**), and glutathione S-transferase (GSTs) (**E**) in the 3rd larval instar of *C. Pipiens*. (**F**); % of changes. Data were represented as mean ± SE. Lowercase letters above the bars indicate significant differences between different treatment groups (Duncan's multiple range test, *p* < 0.01). Error bars indicate 95% confidence intervals.

*3.6. Microbiological Studies* 3.6.1. Antimicrobial Susceptibility, MIC, and MBC The antimicrobial susceptibility screening was performed by the cup diffusion method. The results showed that all tested *P. aeruginosa* were susceptible to the extract compared to the anti-pseudomonal activity of ciprofloxacin (2 mg/mL; Table 3). The extract displayed moderate activities against all the clinical isolates of *P. aeruginosa* and *P.*  As the time of exposure increased, the toxicity of the extract to the 3rd instar larvae increased, followed by a substantial decrease in carboxylesterase (CarE), acetylcholinesterase (AChE), and cytochrome P-450 monooxygenase (CYP450) levels. This suggests the temporary response and neurotoxic effects of the extract. Our results come in agreement with those of the Gershenzon et al. and Salunke et al. studies, which reported the inhibitory effect of natural secondary metabolites for the detoxification enzymes mentioned previously [49–53].

**Extract Ciprofloxacin MIC MBC**

**mg/mL**

(MBC) of the *L. coronopifolia* extract and ciprofloxacin against the tested *P. aeruginosa.*

*P. aeruginosa* **Isolates**

*aeruginosa* ATCC (12924) in the microdilution test (Table 3).

**Table 3.** Antimicrobial susceptibility, minimum inhibitory concentration (MIC), and minimal bactericidal concentration

C1 20 1748 0.3125 1.25 C2 20 40 0.3125 1.25 C3 20 50 0.3125 1.25 C4 28 35 0.3125 1.25

ATCC (12924) 26 22 0.1562 1.25

#### *3.6. Microbiological Studies*

3.6.1. Antimicrobial Susceptibility, MIC, and MBC

The antimicrobial susceptibility screening was performed by the cup diffusion method. The results showed that all tested *P. aeruginosa* were susceptible to the extract compared to the anti-pseudomonal activity of ciprofloxacin (2 mg/mL; Table 3). The extract displayed moderate activities against all the clinical isolates of *P. aeruginosa* and *P. aeruginosa* ATCC (12924) in the microdilution test (Table 3).

**Table 3.** Antimicrobial susceptibility, minimum inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) of the *L. coronopifolia* extract and ciprofloxacin against the tested *P. aeruginosa.*


#### 3.6.2. Biofilm Formation and Quantification Assay film using the microtiter plate method. Out of the tested isolates, four bacteria showed

Twenty clinical isolates of *Pseudomonas* were examined for their ability to form a biofilm using the microtiter plate method. Out of the tested isolates, four bacteria showed biofilm formation, where the clinical isolate C4 exhibited the strongest biofilm formation, while C1, C2, C3, and *P. aeruginosa* ATCC (12924) showed moderate biofilm formation (Figure 4). biofilm formation, where the clinical isolate C4 exhibited the strongest biofilm formation, while C1, C2, C3, and *P. aeruginosa* ATCC (12924) showed moderate biofilm formation (Figure 4).

**Figure 4.** Results of biofilm formation of *P. aeruginosa*. Isolates were classified as negative (OD ≤ ODc), weak (ODc ≤ OD ≤ 2ODc), moderate (2ODc < OD ≤ 4ODc), and strong biofilm production (4ODc <OD). OD = optical density. <sup>a</sup> Significant compared to control at *p* < 0.05. <sup>b</sup> pared to C1 at *p* < 0.05. <sup>c</sup> Significant compared to C2 at *p* < 0.05. <sup>d</sup> **Figure 4.** Results of biofilm formation of *P. aeruginosa*. Isolates were classified as negative (OD ≤ ODc), weak (ODc ≤ OD ≤ 2ODc), moderate (2ODc < OD ≤ 4ODc), and strong biofilm production (4ODc < OD). OD = optical density. <sup>a</sup> Significant compared to control at *p* < 0.05. <sup>b</sup> Significant compared to C1 at *p* < 0.05. <sup>c</sup> Significant compared to C2 at *p* < 0.05. <sup>d</sup> Significant compared to C3 at *p* < 0.05. <sup>e</sup> Significant compared to C4 at *p* < 0.05.

(1/2, 1/4, 1/8 MICs). The extract inhibited the biofilm formation of *P. aeruginosa* in a dose-

dependent manner and ranged from 17 to 38% (Figure 5). These considerable antibiofilm

properties are similar to that of polyphenol-rich extract from the bark of *Salix tetrasperma*

and the leaves of *Annona glabra* and *Gynura procumbens* [54–56]. A recent study by Koely

et al. described the antibiofilm activities of *Enydra fluctuans* against *P. aeruginosa*. They also

attributed these activities to the presence of several bioactive phenolic compounds, among

them kaempferol, quercetin, and luteolin and their glycosides [57]. In addition, different

crude extracts from *Arbutus unedo* having high phenolic contents demonstrated compara-

Significant compared to C4 at *p* < 0.05.

Significant com-

Significant compared to C3 at *p* <

3.6.3. Biofilm Inhibition Assay

ble antibacterial activity [58].

0.05. <sup>e</sup>

#### 3.6.3. Biofilm Inhibition Assay 3.6.3. Biofilm Inhibition Assay

**C1**

**a**

**C2**

**a**

**C3**

**a**

**C4**

**ac**

**ade**

**P. aeruginosa**

0.05. <sup>e</sup> Significant compared to C4 at *p* < 0.05.

(Figure 4).

**Control**

**0.0**

**0.2**

**0.4**

**OD ± SD ( = 620 nm)**

**0.6**

*Molecules* **2021**, *26*, x FOR PEER REVIEW 12 of 16

3.6.2. Biofilm Formation and Quantification Assay

The biofilm inhibition activity of the extract was evaluated using three concentrations (1/2, 1/4, 1/8 MICs). The extract inhibited the biofilm formation of *P. aeruginosa* in a dosedependent manner and ranged from 17 to 38% (Figure 5). These considerable antibiofilm properties are similar to that of polyphenol-rich extract from the bark of *Salix tetrasperma* and the leaves of *Annona glabra* and *Gynura procumbens* [54–56]. A recent study by Koely et al. described the antibiofilm activities of *Enydra fluctuans* against *P. aeruginosa*. They also attributed these activities to the presence of several bioactive phenolic compounds, among them kaempferol, quercetin, and luteolin and their glycosides [57]. In addition, different crude extracts from *Arbutus unedo* having high phenolic contents demonstrated comparable antibacterial activity [58]. The biofilm inhibition activity of the extract was evaluated using three concentrations (1/2, 1/4, 1/8 MICs). The extract inhibited the biofilm formation of *P. aeruginosa* in a dosedependent manner and ranged from 17 to 38% (Figure 5). These considerable antibiofilm properties are similar to that of polyphenol-rich extract from the bark of *Salix tetrasperma* and the leaves of *Annona glabra* and *Gynura procumbens* [54–56]. A recent study by Koely et al. described the antibiofilm activities of *Enydra fluctuans* against *P. aeruginosa*. They also attributed these activities to the presence of several bioactive phenolic compounds, among them kaempferol, quercetin, and luteolin and their glycosides [57]. In addition, different crude extracts from *Arbutus unedo* having high phenolic contents demonstrated comparable antibacterial activity [58].

**Figure 4.** Results of biofilm formation of *P. aeruginosa*. Isolates were classified as negative (OD ≤ ODc), weak (ODc ≤ OD ≤ 2ODc), moderate (2ODc < OD ≤ 4ODc), and strong biofilm production (4ODc <OD). OD = optical density. <sup>a</sup> Significant compared to control at *p* < 0.05. <sup>b</sup> Significant compared to C1 at *p* < 0.05. <sup>c</sup> Significant compared to C2 at *p* < 0.05. <sup>d</sup> Significant compared to C3 at *p* <

Control C1 C2 C3 C4 P. aeruginosa

Twenty clinical isolates of *Pseudomonas* were examined for their ability to form a biofilm using the microtiter plate method. Out of the tested isolates, four bacteria showed biofilm formation, where the clinical isolate C4 exhibited the strongest biofilm formation, while C1, C2, C3, and *P. aeruginosa* ATCC (12924) showed moderate biofilm formation

**Figure 5.** Biofilm formation inhibition activities of *L. coronopifolia* extract against *P. aeruginosa* isolates.

#### 3.6.4. Synergistic Activities

Antimicrobial resistance (AMR) has increased markedly in the recent years and is causing a major threat to patients' treatment. *P. aeruginosa*, for example, has developed antibiotic resistance, and its increasing dissemination is causing severe infections in hospitals. Combinations of plant extracts with antibiotics represent a novel approach to increase their effectiveness and to overcome AMR. In an attempt to explore the synergistic activities of the extract, we combined it with the reference drug ciprofloxacin in a 1:1 ratio. The combination of ciprofloxacin and the extract substantially potentiated the reduction of biofilm from 24%, 20%, and 19% to 53.5%, 48.9%, and 45.26% at 1/2, 1/4, and 1/8 MICs, respectively (Figure 6). Our findings come in agreement with those of Okansi et al. (2013), who reported a synergy when ciprofloxacin was combined with the methanol extract of *Phyllantus muellerianus* leaves (containing flavonoids) against *P. aeruginosa* [59]. Another study described the synergistic activities against *P. aeruginosa* when zingerone extract was combined with the reference drug ciprofloxacin [60].

Coronavirus disease 2019 (COVID-19) has become the utmost and worst public health crisis of our generation. There are several risk factors associated with COVID-19, among them secondary bacterial infections, which in turn lead to serious negative outcomes and fatal clinical complications. To prevent these negative outcomes and secondary bacterial infections, patients with serious illness are treated with antibiotics. As a result, the use of antibiotics has increased, and this will significantly elevate the antibiotic resistance rates [61]. Plant extracts, with diverse secondary metabolites and several molecular targets, alone or as an adjuvant therapy, would not only boost the overall antimicrobial properties

lates.

3.6.4. Synergistic Activities

but can also work as modifying/modulating agents. This will effectively reduce the use of antibiotics and, therefore, reduce the risk of developing antibiotic resistance [62]. study described the synergistic activities against *P. aeruginosa* when zingerone extract was combined with the reference drug ciprofloxacin [60].

**Figure 5.** Biofilm formation inhibition activities of *L. coronopifolia* extract against *P. aeruginosa* iso-

Antimicrobial resistance (AMR) has increased markedly in the recent years and is causing a major threat to patients' treatment. *P. aeruginosa*, for example, has developed antibiotic resistance, and its increasing dissemination is causing severe infections in hospitals. Combinations of plant extracts with antibiotics represent a novel approach to increase their effectiveness and to overcome AMR. In an attempt to explore the synergistic activities of the extract, we combined it with the reference drug ciprofloxacin in a 1:1 ratio. The combination of ciprofloxacin and the extract substantially potentiated the reduction of biofilm from 24%, 20%, and 19% to 53.5%, 48.9%, and 45.26% at 1/2, 1/4, and 1/8 MICs, respectively (Figure 6). Our findings come in agreement with those of Okansi et al. (2013), who reported a synergy when ciprofloxacin was combined with the methanol extract of *Phyllantus muellerianus* leaves (containing flavonoids) against *P. aeruginosa* [59]. Another

*Molecules* **2021**, *26*, x FOR PEER REVIEW 13 of 16

**Figure 6.** Biofilm formation activity of *L. coronopifolia* extract alone and in combination with ciprofloxacin on C4 isolate. **Figure 6.** Biofilm formation activity of *L. coronopifolia* extract alone and in combination with ciprofloxacin on C4 isolate.

#### Coronavirus disease 2019 (COVID-19) has become the utmost and worst public **4. Conclusions**

health crisis of our generation. There are several risk factors associated with COVID-19, among them secondary bacterial infections, which in turn lead to serious negative outcomes and fatal clinical complications. To prevent these negative outcomes and secondary bacterial infections, patients with serious illness are treated with antibiotics. As a result, the use of antibiotics has increased, and this will significantly elevate the antibiotic resistance rates [61]. Plant extracts, with diverse secondary metabolites and several molecular targets, alone or as an adjuvant therapy, would not only boost the overall antimicrobial properties but can also work as modifying/modulating agents. This will effectively In this study, the LC-MS profiling of *L. coronopifolia* extract revealed 46 secondary metabolites. The extract displayed promising insecticidal activities against 3rd instar larvae of *C. pipiens*. The larvae showed a defensive mechanism by increasing the activities of detoxification enzymes of GSTs and α-esterases, while the toxification of *C. pipiens* was significantly observed through the reduction of CarE, AChE, and CYP450 activities. Moreover, the extract demonstrated promising antibiofilm formation against *P. aeruginosa* alone and when combined with the reference drug ciprofloxacin. To sum up, the wild plant, *L. coronopifolia,* exploits a substantial natural source to control disease carriers and manage resistant bacteria infections. Further studies are needed to evaluate the effects of *L. coronopifolia* extract on the life cycle of *C. pipiens* larvae and to explain the regulatory mechanisms of toxification.

**Author Contributions:** Conceptualization, M.E., M.M.S., R.R.A.L., M.A.E.R., M.S., L.J.M.A.-H., D.R.A.-H. and S.M.F.; formal analysis, M.E., D.R.A.-H., L.J.M.A.-H., M.S. and M.A.E.R.; methodology, M.E., D.R.A.-H., M.M.S., L.J.M.A.-H., R.R.A.L., S.M.F., M.S. and M.A.E.R.; validation, D.R.A.-H., L.J.M.A.-H., M.E., and M.S.; visualization, M.E., and M.S.; Writing—original draft, M.E., D.R.A.-H., M.M.S., L.J.M.A.-H., R.R.A.L., S.M.F. and M.A.E.R.; Writing—review and editing, M.S. and M.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data are included at the manuscript.

**Conflicts of Interest:** There is no conflict of interest.

**Sample Availability:** Samples of the extract are available from the authors.

#### **References**


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