*Article* **Phytochemical Profiling of** *Lavandula coronopifolia* **Poir. Aerial Parts Extract and Its Larvicidal, Antibacterial, and Antibiofilm Activity against** *Pseudomonas aeruginosa*

**Mahmoud Emam 1,2, Doaa R. Abdel-Haleem <sup>3</sup> , Maha M. Salem <sup>2</sup> , Lina Jamil M. Abdel-Hafez <sup>4</sup> , Rasha R. Abdel Latif <sup>2</sup> , Shaimaa Mahmoud Farag <sup>3</sup> , Mansour Sobeh 5,\* and Mohamed A. El Raey 2,\***


**Abstract:** Infections associated with the emergence of multidrug resistance and mosquito-borne diseases have resulted in serious crises associated with high mortality and left behind a huge socioeconomic burden. The chemical investigation of *Lavandula coronopifolia* aerial parts extract using HPLC–MS/MS led to the tentative identification of 46 compounds belonging to phenolic acids, flavonoids and their glycosides, and biflavonoids. The extract displayed larvicidal activity against *Culex pipiens* larvae (LC<sup>50</sup> = 29.08 µg/mL at 72 h). It significantly inhibited cytochrome P-450 monooxygenase (CYP450), acetylcholinesterase (AChE), and carboxylesterase (CarE) enzymes with the comparable pattern to the control group, which could explain the mode of larvae toxification. The extract also inhibited the biofilm formation of *Pseudomonas aeruginosa* by 17–38% at different Minimum Inhibitory Concentrations (MICs) (0.5–0.125 mg/mL) while the activity was doubled when combined with ciprofloxacin (ratio = 1:1 *v*:*v*). In conclusion, the wild plant, *L. coronopifolia,* can be considered a promising natural source against resistant bacteria and infectious carriers.

**Keywords:** *Lavandula coronopifolia*; *Culex pipiens*; larvicidal; antibiofilm formation; LC-MS/MS; molecular networking

#### **1. Introduction**

Mosquito vector-borne diseases are considered a global problem, which highlights the necessity for new prospects and cost-effective agents for vector control. Around 100 species of mosquitoes transmit viral and bacterial disorders such as malaria, lymphatic filariasis, dengue, and yellow fever, affecting several millions of people worldwide. In 2017, WHO recorded the highest mortality and morbidity due to mosquito-borne disorders that affect human health and economic society. Therefore, the development of novel mosquito repellents and antibacterial agents to overcome the microbial resistance threat is highly demanded. This could also help to avoid disrupting the ecological balance [1–3]. Plant secondary metabolites could furnish safe, efficacious, and multi-mechanistic candidates that might be useful as insecticidal and antibacterial agents.

The genus *Lavandula* (commonly known as lavender) comprises 45 species that are mainly distributed in subtropical and tropical regions [4,5]. Plants of the genus have been used in folk medicine since ancient times to treat pain, headache, migraine, and

**Citation:** Emam, M.; Abdel-Haleem, D.R.; Salem, M.M.; Abdel-Hafez, L.J.M.; Latif, R.R.A.; Farag, S.M.; Sobeh, M.; El Raey, M.A. Phytochemical Profiling of *Lavandula coronopifolia* Poir. Aerial Parts Extract and Its Larvicidal, Antibacterial, and Antibiofilm Activity against *Pseudomonas aeruginosa*. *Molecules* **2021**, *26*, 1710. https://doi.org/ 10.3390/molecules26061710

Academic Editors: Manuela Pintado, Ezequiel Coscueta and María Emilia Brassesco

Received: 20 February 2021 Accepted: 15 March 2021 Published: 19 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

antiepileptic, antidiuretic, antirheumatic, and carminative agents. They become famous for their multiple uses in different pharmaceuticals, aroma, and food products [6,7]. The phytochemistry of the genus is centered on mono- and sesquiterpenoids, together with traces of alkaloids, and phenolic structures [7].

*Lavandula coronopifolia* Poir. (Arabic: Khozama) is a shrublike perennial, growing in the rocky environment and desert plains mainly distributed in subtropical and tropical regions [8]. The first attention to *L. coronopifolia* traces back to 1999, when El-Garf et al. isolated and identified numerous hydroxyl flavones such as hypolaetin, isoscutellarien, and luteolin from its dried aerial parts [4]. Then, several studies reported the presence of polyhydroxyoleanolic acids, polyhydroxyursolic acids and their glycosides, caffeic acid, rosmarinic acid, rutin, quercetin, and hesperidin [9,10].

*L. coronopifolia* showed a plethora of substantial biological activities. These include antioxidant [11], antimicrobial [12], α-glucosidase inhibitory [10], and hepatoprotective [13] activities. These activities were attributed to the presence of flavonoids, especially flavones and their glucuronides, in addition to triterpenes [9,10]. Moreover, *L. coronopifolia* essential oils possessed substantial antibacterial activity against the Gram-negative bacteria and methicillin-resistant *Staphylococcus aureus* bacteria [14].

In this work, we comprehensively characterized the phytoconstituents of the aerial parts of *L. coronopifolia* extract by HPLC–MS/MS and confirmed the existence of possible skeletons quantitatively using <sup>1</sup>H-NMR and molecular networking. We also evaluated the insecticidal activities against *Culex pipiens* larvae and the antibiofilm formation activity against isolates of *Pseudomonas aeruginosa*. We explored several biochemical parameters to investigate the mechanism of the insecticidal activities.

#### **2. Material and Methods**

#### *2.1. Plant Material, Extraction, and Preliminary Qualitative Analysis*

*Lavandula coronopifolia* Poir. was collected from the Western Desert of Egypt in March 2018. A voucher sample was placed at the international herbarium of the National Research Centre (CAIRC) (S.N: 1023). The plant aerial parts (250 g) were crushed into small pieces and then extracted by dipping into 70% MeOH:H2O (*v:v*) at ambient temperature for one week. The solution was filtered (Whatman no. 1), then concentrated till dryness using Rotavapor® (Heizbad Hei-VAP, Heidolph, Germany), yielding 28.45 g, and stored at 4 ◦C for further experiments. The *L. coronopifolia* extract was screened for its phyto-constituents as flavonoids (Shinoda's test), phenolics (FeCl<sup>3</sup> test), ellagitannins (NaNO<sup>2</sup> assay), and gallotannins (KIO<sup>3</sup> test) [15,16]. The proton NMR (Jeol ECA-500 MHz, Japan) experiment using DMSO-*d6* was done for the total extract.

#### *2.2. HPLC-MS/MS*

HPLC-PDA-MS<sup>n</sup> mass spectra was performed through a ThermoFinnigan (Thermo Electron Corporation, Austin, TX, USA) LC system coupled with a mass spectrometer (LCQ-Duo ion trap) having an ESI source (ThermoQuest, Thermo Scientific, Waltham, MA, USA) [15]. The injection process, flow rate, elution solvents, resolution, and negative MS operating parameters were described previously [17]. In brief, a Zorbax Eclipse XDB-C18, rapid resolution, 150 × 4.6 mm, 3.5 µm column was used (Agilent, Santa Clara, CA, USA). A gradient consisting of water and acetonitrile (ACN), each having 0.1% formic acid, was applied, and ACN was increased from 5% to 30% within 60 min and then to 90% within the next 30 min at a flow rate of 1 mL/min and a 1:1 split before the ESI source [17].

#### *2.3. Molecular Networking Workflow Description*

The mgf formatting mass file was uploaded to the online platform of GNPS (http: //gnps.ucsd.edu) (accessed on 27 June 2020). Then the data were filtered as described previously and the visualization of molecular networking (MNW) workflow was carried out using Cytoscape 3.6.1 software [15,18].

#### *2.4. Larvicidal Assay*

#### 2.4.1. Insects

A laboratory susceptible strain of *Culex pipiens* was obtained from the Research and Training Center on Vectors of Diseases (RTC), Ain Shams University. It was colonized in the entomology department insectary at 27 ± 2 ◦C, 75 ± 5% relative humidity (RH), and a 14 h/10 h light/dark photoperiod following standard procedures [19]. The larvae were reared in enamel dishes containing 2000 mL of distilled water. Newly hatched larvae were fed on Tetra-Min, Germany. Adults were reared in (30 × 30 × 30 cm) wooden cages and provided with 10% sucrose solution daily, as well as a pigeon for female blood feeding.

#### 2.4.2. Bioassay

The larvicidal activity was evaluated against the third larval instar of *C. pipiens* under the same controlled laboratory conditions. The bioassay was assessed using the standard method described in [20]. The extract was dissolved in water to prepare the stock solution. Batches of 25 of 3rd instar larvae of *C. pipiens* were transferred by a plastic dropper to small disposable test cups and treated with different concentrations of the extract (10, 25, 50, 100, 150, and 200 µg/mL prepared in distillated water) and control with distillated water only, in a triplicate manner. Mortality was recorded after 24, 48, and 72 h post treatment.

#### 2.4.3. Preparation of Samples for Biochemical Assay

The 3rd larval instar of *C. pipiens* was treated by LC<sup>50</sup> values, and then the insects were prepared as described by Amin et al. [21]. The whole bodies of larvae were homogenized in distilled water (50 mg/1 mL). The homogenates were centrifuged at 8000 r.p.m. for 15 min at 4 ◦C. The supernatants were used for biochemical analyses. Acetylcholinesterase (AChE) and carboxylesterase assays were measured according to the method described by Simpson et al. [22], using acetylcholine bromide (AchBr) and methyl n butyrate (MeB) as substrates, respectively. Alpha esterase (α-esterase) activity was determined according to Van Asperen [23] using α-naphthyl acetate as substrate. Glutathione S-transferase (GST) catalyzes the conjugation of reduced glutathione (GSH) with 1-chloro 2,4-dinitrobenzene (CDNB) via the –SH group of glutathione. The conjugate S-(2,4-dinitro-phenyl)-L-glutathione could be detected as described by the method of Habig et al. [24]. Cytochrome P-450 monooxygenase activity was determined using *p*-nitroanisole o-demthylation according to the method of Hansen and Hodgson [25] with slight modifications.

#### *2.5. Microbiological Assay*

#### 2.5.1. Sample Collection and Identification of Isolated Bacteria

Clinical isolates of *Pseudomonas aeruginosa* were collected from burn wounds, otitis media, and urine as previously described [26] and were kept for scientific research. The isolates were grown on Tryptic soya agar (TSA) (DifcoTM, Strasbourg, France) for 24 h at 37 ◦C, then one single colony of each isolate was inoculated into 2 mL Tryptic soya broth (TSB) (DifcoTM, France) with overnight incubation at 37 ◦C. The samples were cultivated on Cetrimide agar media, after which the isolated species were identified by morphology (pale yellow colonies on MacConkey and green exopigment on Cetrimide agar), Gram staining (Gram-negative bacilli), and biochemical reactions (oxidase-positive). The Microbact™ Gram-negative system was implemented in compliance with the manufacturer's protocol (Oxoid, Hampshire, UK). A standard strain of *P. aeruginosa* (ATCC 12924) was kindly provided by NAMRU as a frozen cultural broth containing 40% glycerol.

#### 2.5.2. The Antimicrobial Susceptibility Testing

The antimicrobial susceptibility testing was done using cup agar diffusion method [27]. The bacterial cultures were adjusted to an optical density (OD) of 0.5 at 600 nm, TSA plates were covered with 100 µL of each bacterial isolate, and 5 mm pores were filled with 100 µL of extract dissolved in sterile water at 2.5 mg/mL. The plates were incubated for 24 h at 37 ◦C. The zone of inhibitions was measured in mm.

#### 2.5.3. Minimum Inhibitory Concentration (MIC)

MIC is used as the gold standard method for detecting the sensitivity of the organisms to antimicrobial agents [28]. The final concentrations of the extract ranging from 2.5 to 0.0195 mg/mL were prepared and then added to test tubes containing 1 mL of sterile TSB media. The bacterial suspensions with OD 0.5 at 600 nm were diluted 1:100 (≈106 CFU/mL), and then 50 µL of inoculums were added to each tube. The tubes were incubated at 37 ◦C ± 2 ◦C for 24 h. A tube containing TSB broth without extract was taken as control. The MIC was defined as the lowest concentration of the tested extract that restricted the visible growth of tested strains compared to the blank [29].

#### 2.5.4. Minimal Bactericidal Concentration (MBC)

The minimal bactericidal concentration (MBC) was determined by the Petri dish sowing method [30]. This procedure was dependent on the procedures for the determination of MIC. After the incubation period for the determination of the MIC, an aliquot of 0.1 mL was taken from each of the test tubes that were not showing growth, and then was inoculated into a TSA agar plate. The plates were then incubated at a temperature of 37 ± 2 ◦C for 24 h. After this period, the presence of bacterial colonies was observed in each plate. The MBC was defined as the lowest concentration of the plant extract that was able to prevent microbial growth in a culture medium (formation of bacterial colonies). The bioassays were performed in duplicate with three repetitions for each bacterial isolate.

#### 2.5.5. Biofilm Formation Assay and Quantification

The biofilms were assayed as described in [31,32] using sterile 96-well microtiter plates, each well containing 180 µL TSB broth and 20 µL of bacterial suspension with OD 0.5 at 600 nm. After 24 h incubation at appropriate conditions, all the planktonic cells were removed, and the biofilms were gently washed twice with phosphate buffer saline (PBS) to remove any free-floating bacteria. The biofilm cells formed in each well were stained with 200 µL crystal violet (0.1% *w*/*v*) and incubated at room temperature (28 ◦C) for 10 min. The stain was removed and washed with distilled water for 30–60 s. After 5 min of air drying, the biofilms were solubilized by 200 µL of 98% ethanol, then the optical densities of stained adherent biofilms were measured at 620 nm using a microplate reader. The evaluation of biofilm production was categorized according to the criteria of Stepanovi´c et al. as follows: OD ≤ ODc: not a biofilm producer (non-adherent); ODc < OD ≤ 2ODc: a weak biofilm producer (weakly adherent); 2ODc < OD ≤ 4ODc: a moderate biofilm producer (moderately adherent); 4ODc < OD: a strong biofilm producer (strongly adherent). ODc and OD were defined as the mean OD of the blank wells and wells with biofilm, respectively [33].

#### 2.5.6. Biofilm Inhibition Assay

The ability of the extract to inhibit the biofilms of the clinical isolates of *P. aeruginosa* was evaluated according to Stepanovi´c et al. [34] with some modifications. Microbial biofilms were developed in a round-bottom 96-well microtiter plate. Each clinical isolate was inoculated into each well of the 96-well microtiter plate. The extract was added to each well at 1/2, 1/4, and 1/8 MICs and incubated for 24 h at 37 ◦C. After the incubation period, non-adherent cells were detached by dipping each sample three times in sterile PBS. The samples were fixed for one hour, and the biofilms were stained with 0.1% solution of crystal violet in H2O. After staining, the samples were washed with distilled H2O (DW). The measurable biofilm production was achieved by adding 125 µL of 30% acetic acid to de-stain the samples. Afterwards, the OD at 620 nm was detected using the microplate reader. The percentage (%) of inhibition formula is as follows:

$$\% \text{Inhibition} = \frac{\text{Abs control } - \text{Abs sample}}{\text{Abs control}} \times 100$$

#### 2.5.7. Combination of the Extract with Ciprofloxacin

The ability of ciprofloxacin/extract (1:1) to inhibit the biofilm of the strong biofilm isolate of *P. aeruginosa* was evaluated according to Stepanovi´c et al. [34] with some modifications. Microbial biofilms were developed in a round-bottom 96-well microtiter plate. The clinical isolate C4 was inoculated into each well of the 96-well microtiter plate, and ciprofloxacin/extract (1:1) was added to each well at 1/2, 1/4, and 1/8 MICs and incubated for 24 h at 37 ◦C. After incubation (24 h), non-adherent cells were detached by dipping each sample three times in sterile PBS. The samples were fixed for 1 h, and the biofilms were stained with 0.1% solution of crystal violet in H2O. After staining, the samples were washed with DW (distilled H2O). The quantitative analysis of biofilm production was achieved by adding 125 µL of 30% acetic acid to de-stain the samples. Afterwards, the OD at 620 nm was measured using the microplate reader. The percentage (%) of inhibition formula is as follows:

$$\% \text{Inhibition} = \frac{\text{Abs control } - \text{Abs sample}}{\text{Abs control}} \times 100$$

#### *2.6. Statistical Analysis*

The biofilm formation inhibiting activities of different concentrations of the extract were compared by two-way ANOVA (Bonferroni post hoc tests). *p* < 0.05 was used to detect the significance of differences. The obtained larvicidal data were analyzed using a statistics package (LDP-line) for goodness of fit (chi square test) and to detect LC<sup>50</sup> and LC<sup>95</sup> values with corresponding 95% confidence limits (CL), slope, correlation coefficient and standard error. The results of biochemical determinations were investigated by one-way analysis of variance (ANOVA) using Costat statistical software (Cohort software, Berkeley). When the ANOVA statistics were significant (*p* < 0.01), the means were compared by the Duncan's multiple range test [35]. GraphPad Prism 5.0 software (GraphPad Prism Software Inc., San Diego, CA, USA) was used to draw most of the figures.

#### **3. Results and Discussion**

#### *3.1. Phytochemical Screening and LC-MS/MS Profile of L. coronopifolia*

The preliminary phytochemical screening of *L. coronopifolia* extract revealed the presence of dihydroxy phenolics and flavonoids and/or their glycosides, as well as the absence of ellagic and gallotannins moieties. The mass spectrometry analysis (full scan and product ion scan mode) provided structural information of 46 metabolites, including organic and phenolic acids, flavonoids and their glycosides, and bioflavonoids (Table 1 and Figure 1).

#### *3.2. Molecular Networking (MNW) of L. coronopifolia Aerial Parts' Metabolite Perception*

The symmetrical chemical entities were facilitated to be visualized through the molecular networking between the identical fragments (*m/z*) of definite metabolites. Through the network, each node was characterized with the parent mass [M–H]− and the contiguous arrows (edges) connected between the similar nodes. The network was built for the negative ionization (–ve) mode using the GNPS 2 platform (Figure 2). The (–ve) network involved 148 nodes, 71 self-looped (individual) nodes, and 92 connected components. The designed networks facilitated the visual examination of the different compound families and analogues and assisted in isomer differentiation.

In the negative network, five clusters, A, B, C, D, and E, were mentioned and annotated as apigenin derivatives, which were grouped as methylated flavones, *O*-glycosidic flavones, *C*-glycosidic flavones, and/or biflavones. In addition, the other self-looped nodes asterisked within the network were denoted as phenolic-*O*-glycosides, N-acetylamino acid, flavan-3-ol, phenolic acids, and biflavones.

**Organic acids:** Quinic acid was determined with an [M-H]– ion at *m/z* 191 fragmented to *m/z* 111, and 173, whereas malic acid was characterized with an [M-H]– ion at *m/z* 133 and fragmented to *m/z* 115, 89, and 71. **Phenolic acids:** These structures were tentatively identified as eucomic acid, syringic acid-4-*O*- hexoside, sinapic acid 3-*O*-glucoside, and dihydrosinapic acid hexoside. They showed negative molecular ions at *m/z* 239, 359, 375, 385, and 387, respectively. These structures were tentatively identified depending on the main fragments that are shown in Table 1. **Flavonoids and their glycosides:** Most of the aglycones were found to be apigenin derivatives and their *O*- and/or *C*-linkage of mono and/or diglycosides. The MS of *C*-glycosides was characterized through the main fragmentations by the loss of different masses of 60, 90, 120, and 240 Daltons [36]. A molecular ion [M-H]– at *m/z* 289 and yielding a main fragment at *m/z* 245 was identified as catechin. **Biflavonoids:** Several signals that gave parent ions [M–H]− of *m/z* 551, 555, 565, 579, 581, and 609 were fragmented into specific fragments that characterized bioflavonoid derivatives [37]. Their identification, retention times, molecular weights, and fragmentation pattern are shown in Table 1. *Molecules* **2021**, *26*, x FOR PEER REVIEW 6 of 16

**Figure 1.** Base peak chromatogram of *L. coronopifolia* Poir. aerial parts' extract. **Figure 1.** Base peak chromatogram of *L. coronopifolia* Poir. aerial parts' extract.

*3.2. Molecular Networking (MNW) of L. coronopifolia Aerial Parts' Metabolite Perception*

The symmetrical chemical entities were facilitated to be visualized through the molecular networking between the identical fragments (*m/z*) of definite metabolites. Through the network, each node was characterized with the parent mass [M–H]<sup>−</sup> and the contiguous arrows (edges) connected between the similar nodes. The network was built for the negative ionization (–ve) mode using the GNPS 2 platform (Figure 2). The (–ve) network involved 148 nodes, 71 self-looped (individual) nodes, and 92 connected components. The designed networks facilitated the visual examination of the different compound families

In the negative network, five clusters, A, B, C, D, and E, were mentioned and anno-

flavones, *C*-glycosidic flavones, and/or biflavones. In addition, the other self-looped nodes asterisked within the network were denoted as phenolic-*O*-glycosides, N-acetylamino

and analogues and assisted in isomer differentiation.

acid, flavan-3-ol, phenolic acids, and biflavones.

**Figure 2.** Complete molecular networking (MNW) generated using MS/MS data in (–ve) negative mode from *L. coronopifolia* aerial parts' extract. Nodes are labeled with parent mass. **Figure 2.** Complete molecular networking (MNW) generated using MS/MS data in (–ve) negative mode from *L. coronopifolia* aerial parts' extract. Nodes are labeled with parent mass.

**Organic acids:** Quinic acid was determined with an [M-H]–

to *m/z* 111, and 173, whereas malic acid was characterized with an [M-H]–

and fragmented to *m/z* 115, 89, and 71. **Phenolic acids:** These structures were tentatively identified as eucomic acid, syringic acid-4-*O*- hexoside, sinapic acid 3-*O*-glucoside, and dihydrosinapic acid hexoside. They showed negative molecular ions at *m/z* 239, 359, 375,

ion at *m/z* 191 fragmented

ion at *m/z* 133


**Table 1.** Chemical composition of *L. coronopifolia* Poir. aerial parts' extract using LC-MS/MS.
