Conyza canadensis from Jordan: Phytochemical Profiling, Antioxidant, and Antimicrobial Activity Evaluation

In this investigation, the chemical composition of the hydro-distilled essential oil (HD-EO), obtained from the fresh aerial parts (inflorescence heads (Inf), leaves (L), and stems (St)) of Conyza canadensis growing wild in Jordan was determined by GC/MS. Additionally, the methanolic extract obtained from the whole aerial parts of C. canadensis (CCM) was examined for its total phenolic content (TPC), total flavonoids content (TFC), DPPH radical scavenging activity, iron chelating activity and was then analyzed with LC-MS/MS for the presence of certain selected phenolic compounds and flavonoids. The GC/MS analysis of CCHD-EOs obtained from the different aerial parts revealed the presence of (2E, 8Z)-matricaria ester as the main component, amounting to 15.4% (Inf), 60.7% (L), and 31.6% (St) of the total content. Oxygenated monoterpenes were the main class of volatile compounds detected in the Inf-CCHD-EO. However, oils obtained from the leaves and stems were rich in polyacetylene derivatives. The evaluation of the CCM extract showed a richness in phenolic content (95.59 ± 0.40 mg GAE/g extract), flavonoids contents (467.0 ± 10.5 mg QE/ g extract), moderate DPPH radical scavenging power (IC50 of 23.75 ± 0.86 µg/mL) and low iron chelating activity (IC50 = 5396.07 ± 15.05 µg/mL). The LC-MS/MS profiling of the CCM extract allowed for the detection of twenty-five phenolic compounds and flavonoids. Results revealed that the CCM extract contained high concentration levels of rosmarinic acid (1441.1 mg/kg plant), in addition to caffeic acid phenethyl ester (231.8 mg/kg plant). An antimicrobial activity assessment of the CCM extract against a set of Gram-positive and Gram-negative bacteria, in addition to two other fungal species including Candida and Cryptococcus, showed significant antibacterial activity of the extract against S. aureus with MIC value of 3.125 µg/mL. The current study is the first phytochemical screening for the essential oil and methanolic extract composition of C. canadensis growing in Jordan, its antioxidant and antimicrobial activity.


Introduction
Conyza genus, a member of the Asteraceae (formerly known as Compositae) family, comprises more than 150 identified species of the flowering plants recognized as weeds in different types of crops and vineyards [1].Despite their wide spread and distribution worldwide, the main origin of this genus is North and South America [2].All species belonging to this genus are characterized by the production of a large number of seeds that can be dispersed by wind over long distances [3].There are only five Conyza species reported in the flora of Jordan.These include Conyza aegyptiaca (L.) Aiiton, Conyza albida Sprigel, Conyza bonariensis (L.) Cronquist, Conyza canadensis (L.) Cronquist, and Conyza stricta [2].
Conyza canadensis L. Cronquist (Synonyms Erigeron canadensis), commonly known as horseweed or Canada fleabane, is an annual or perennial common weed native to North America [4].The plant is recognized by its upstanding hairy long stem, growing up to 1.5 m tall.The plant branches above and overtopping its main axis with terminal cluster inflorescence, consisting of a large number of very small flower heads with a ring of white or pale purple ray florets and a center of yellow disc florets [5].The flowering season extends from spring to autumn.The aerial parts of this weed are widely used in folk medicine to treat headache, dental pain, rheumatism, kidney problems, gastrointestinal disorders, and respiratory tract infections [6], in addition to wounds and skin burns treatments [7].
In this study, hydro-distillation (HD) was used to extract the essential oils (EOs) from the inflorescence heads (Inf), leaves (L), and stems (St) of natural populations of C. canadensis from Jordan.The different CCHD-EOs were then investigated for their chemical composition using the GC/MS technique.Moreover, the methanolic extract (CCM) obtained from the fresh aerial parts of C. canadensis was assayed for its total phenolic content (TPC), total flavonoids content (TFC), in vitro DPPH scavenging potential, and iron chelating activity.Additionally, the presence of a selected set of 14 phenolics compounds and 11 flavonoids was investigated by LC-MS/MS.The antimicrobial activity of CCM was also evaluated against a set of microbes including Gram-positive, Gram-negative bacteria, and fungi.

GC/MS Analysis
HD-EOs obtained from the different fresh aerial parts of C. canadensis (Inf, L and St) were analyzed for their chemical constituents using GC/MS technique (Figures S1-S3), results are listed in Table 1.The structures of the main constituents detected in the analyzed EOs are shown in Figure 1. Figure 2 reveals the main classes of volatile constituents detected in the analyzed oils.

Total Phenolics Content (TPC), Total Flavonoids Content (TFC), DPPH Radical Scavenging Activity, and Iron Chelating Activity
The CCM extract was assessed for its TPC, TFC, and antioxidant potential using the DPPH radical scavenging method and Fe 2+ chelating activity (Table 2).Figure 3 displays the DPPH scavenging activity percent versus the concentration of CCM and two positive controls (ascorbic acid and α-tocopherol).

Total Phenolics Content (TPC), Total Flavonoids Content (TFC), DPPH Radical Scavenging Activity, and Iron Chelating Activity
The CCM extract was assessed for its TPC, TFC, and antioxidant potential using the DPPH radical scavenging method and Fe 2+ chelating activity (Table 2).Figure 3 displays the DPPH scavenging activity percent versus the concentration of CCM and two positive controls (ascorbic acid and α-tocopherol).

Total Phenolics Content (TPC), Total Flavonoids Content (TFC), DPPH Radical Scavenging Activity, and Iron Chelating Activity
The CCM extract was assessed for its TPC, TFC, and antioxidant potential using the DPPH radical scavenging method and Fe 2+ chelating activity (Table 2).Figure 3 displays the DPPH scavenging activity percent versus the concentration of CCM and two positive controls (ascorbic acid and α-tocopherol).

LC-MS/MS Analysis for Phenolic Compounds and Flavonoids
The CCM extract was also assayed using LC-MS/MS analysis, for the presence of selected 25 authentic compounds, comprising 14 phenolics and 11 flavonoids.The identity and concentration of the detected compounds (mg/kg plant) are shown in Table 3.

LC-MS/MS Analysis for Phenolic Compounds and Flavonoids
The CCM extract was also assayed using LC-MS/MS analysis, for the presence of selected 25 authentic compounds, comprising 14 phenolics and 11 flavonoids.The identity and concentration of the detected compounds (mg/kg plant) are shown in Table 3.

LC-MS/MS Analysis for Phenolic Compounds and Flavonoids
The CCM extract was also assayed using LC-MS/MS analysis, for the presence of selected 25 authentic compounds, comprising 14 phenolics and 11 flavonoids.The identity and concentration of the detected compounds (mg/kg plant) are shown in Table 3.

LC-MS/MS Analysis for Phenolic Compounds and Flavonoids
The CCM extract was also assayed using LC-MS/MS analysis, for the presence of selected 25 authentic compounds, comprising 14 phenolics and 11 flavonoids.The identity and concentration of the detected compounds (mg/kg plant) are shown in Table 3.

LC-MS/MS Analysis for Phenolic Compounds and Flavonoids
The CCM extract was also assayed using LC-MS/MS analysis, for the presence of selected 25 authentic compounds, comprising 14 phenolics and 11 flavonoids.The identity and concentration of the detected compounds (mg/kg plant) are shown in Table 3.

LC-MS/MS Analysis for Phenolic Compounds and Flavonoids
The CCM extract was also assayed using LC-MS/MS analysis, for the presence of selected 25 authentic compounds, comprising 14 phenolics and 11 flavonoids.The identity and concentration of the detected compounds (mg/kg plant) are shown in Table 3.

LC-MS/MS Analysis for Phenolic Compounds and Flavonoids
The CCM extract was also assayed using LC-MS/MS analysis, for the presence of selected 25 authentic compounds, comprising 14 phenolics and 11 flavonoids.The identity and concentration of the detected compounds (mg/kg plant) are shown in Table 3.

Antimicrobial Activity, Minimum Inhibitory Concentration (MIC), and Minimum Bactericidal Concentration (MBC) Determination
The agar-well diffusion method was used to assess the in vitro antifungal and antibacterial activity of CCM against three species of Candida, one species of Cryptococcus, four species of Gram-positive bacteria, and three species of Gram-negative bacteria.Around each well, the clear zone of inhibition (ZOI-mm) was measured.
The lowest concentration of CCM extract that inhibited bacterial growth was determined using the MIC assay, while the lowest concentration that killed 99.9% of the bacterial cells was calculated using the MBC assay.The ZOI, MIC, and MBC values are presented in Table 4.

CCHD-EOs Data Analysis
The GC/MS analysis of the CCHD-EOs, obtained from the different aerial parts (Inf, L, and St), resulted in the identification of 109 compounds, of which, 64 were detected in the hydro-distilled oil obtained from the inflorescence heads, 56 in the leaves, and 54 in the stems.While the HDEOs of the different parts contained different classes of volatile compounds, all the EOs were characterized by their high polyacetylenes content, mainly the presence of (Z)-lachnophyllum ester and matricaria ester derivatives.In particular, (2E,8Z)-matricaria ester was the major component detected in the different oils in this study.
Previous studies on the essential oil composition of several Conyza species revealed the detection of several volatile organic compounds with a wide spectrum of biological potential such as antibacterial, antioxidant, cytotoxic, anti-inflammatory, analgesic, antiviral, antiproliferative, and insecticidal activities [6,19].Limonene was recognized as a major component detected in the EO of different Conyza species.This compound along, with other monoterpenes, detected in this species are known for their antioxidant, antimicrobial [23], and insecticidal activities [19].Polyacetelenes derivatives, such as (Z)-lachnophyllum ester and matricaria ester isomers, were investigated for their antimicrobial [24] and antileishmanial [25] potentials.Studies on caryophyllene, caryophyllene oxide, and other sesquiterpenes revealed cytotoxic, anticancer, antioxidant, and antimicrobial properties [19].Oxygenated monoterpenes were evaluated for antibacterial, antifungal, and antioxidant activity [26].
The essential oil of C. canadensis from Turkey, Pakistan, and Brazil, was dominated by limonene and polyacetylene derivatives, mainly (Z)-lachnophyllum ester and matricaria ester isomers [8,[10][11][12].In most reports, limonene dominated the EO obtained from whole aerial parts, while polyacetylene derivatives dominated the essential oil obtained from the roots [4,5,9].Noticeably, the CCHD-EO of different aerial parts was rich in (2E,8Z)matricaria ester isomer which was almost absent in EO of the plant from other regions.These results indicate clearly that the variation in the chemical composition of the essential oil was affected not only by the organ being investigated, but also by other environmental and experimental factors.These include soil properties, climate conditions, the time of harvesting, and extraction method.Table 5 summarizes the main variation in the HDEO composition of our study with the previous work.

TPC, TFC, DPPH Scavenging and Iron Chelating Activity for CCM Extract
As could be deduced from the data shown in Table 2, the CCM extract had relatively high DPPH scavenging power (23.75 ± 0.86 µg/mL) as compared to the tested positive controls (ascorbic acid: 1.79 ± 0.12 µg/mL; α-tocopherol: 5.00 ± 0.24 µg/mL).This result is mainly attributed to the high TPC and TFC in this extract (95.59 mg GAE/g extract, and 467 mg QE/g extract, respectively).In addition, the measured chelating effect of the extract on Fe 2+ revealed a low chelating effect with IC 50 5396.07± 15.05 µg/mL compared to EDTA (20.15 ± 0.09 µg/mL).
Few studies reported the TPC and TFC for the methanolic plant extract of C. canadensis.The TPC and the TFC of C. canadensis from Moroccan origin [27] (2.54 µg /mg DM and 19.31 µg/mg DM, respectively) and Turkish origin [28] (71.34 ± 0.53 mg GAE/g extract; 18.91 ± 1.46 mg CA/g extract, respectively) were lower than those detected in our current investigation.Also, the observed DPPH radical scavenging power in our current study was higher than those observed in previous reports [20,27,28].This could be mainly attributed to the high TPC and TFC detected in our study.Further confirmation was obtained upon LC-MS/MS analysis of this extract that revealed the detection of considerable concentration levels of rosmarinic acid, caffeic acid phenethyl ester, and apigenin-7-O-glucoside.
The antioxidant capacity of extracts obtained from Conyza genus was reported.Our research revealed interestingly moderate DPPH radical scavenging power as compared to other species from different geographical area (Table 6).In fact, the comparison of IC 50 values of the different reports reveals the impact of environmental conditions on the chemical composition and antioxidant power.
The results revealed the detection of high concentration levels of rosmarinic acid (1441.1 mg/kg plant extract).Moreover, caffeic acid phenethyl ester was detected in moderate concentration levels (231.8 mg/kg extract).It is worth mentioning that this is the first report for the detection of 3-O-methylquercetin, hesperetin, resveratrol, salvianolic acid, hesperidin, and caffeic acid phenethyl ester in C. canadensis.Among the different flavonoids detected, it was noticed that apigenin-7-O-glucoside and 3-O-methylquercetin were the most abundant (46.13 and 36.64 mg/ kg plant, respectively).Trace amounts of each of chlorogenic acid, rutin, carnosic acid, gallic acid, and luteolin-7-O-glucoside were detected.The detection of high concentration levels of rosmarinic acid and other phenolics and flavonoids supports the observed DPPH radical scavenging power of the CCM extract obtained from C. canadensis from Jordan.
Rosmarinic acid was determined in a considerable concentration in our study.It was absent in the methanolic extract of Moroccan origin [22].These findings further confirm the effect of environmental and climatic conditions on the biosynthetic pathways of plants, consequently leading to a wide spectrum of differences in secondary metabolite composition and bioactivity potentials.

Antimicrobial Assay
In this study, the CCM extract was assayed for its in vitro antifungal activity against three species of Candida (Candida albicans, Candida krusei, and Candida glabrata), one species of Cryptococcus (Cryptococcus neoformans (Sanfelice) vuillemin), and for its antibacterial activity against four species of Gram-positive bacteria (Staphylococcus aureus, Staphylococcus hominis, Bacillus cereus, and Streptococcus pyogenes), and three species of Gram-negative bacteria (Salmonella typhi, Escherichia coli, and Pseudomonas aeruginosa).The CCM extract was inactive at a concentration level of 100 ppm against all tested fungal species as compared to the positive control fluconazole.However, the extract revealed strong inhibitory effect against S. aureus with a minimum inhibitory concentration of 3.125 µg/mL (corresponding to MBC value of 6.25 µg/mL).The extract showed no interesting antibacterial activity against the other tested bacterial species at 100 ppm extract concentration.The characteristics of the microorganisms' cell walls can be linked to the effectiveness of CCM extract as an antibacterial agent.For Gram-positive bacteria, teichoic acids make up over 60% of their cell wall [33].In addition, Gram-positive bacteria only have one cell membrane, while Gram-negative bacteria have two: the outer and plasma membranes [34].The outer membrane protects the bacterial cells from potentially hazardous substances by acting as a selective permeability barrier [35].Furthermore, extracellular polymeric substances (EPS) that provide protection against harmful environmental conditions can be produced by Gram-negative bacteria, which could help and explain their resistance to a particular concentration of CCM extract [36].
The considerable antibacterial activity CCM extract against S. aureus may be attributed also to the high content of rosmarinic acid and caffeic acid phenethyl ester.The CCM extract obtained from the plant from Jordan showed higher inhibitory effects against the Grampositive S. aureus (3.125 µg/mL) when compared to the alcoholic extract from Tunisian origin plant (MIC: 5 mg/mL) [20].The extract obtained from the C. canadensis from Turkey showed even lower activity as compared to ours (ZOI: 7.0, 40.0 mm) [21].It is worth noting that the extracts obtained from C. canadensis from Tunisian and Turkish origins both showed moderate antibacterial activity against E. coli [20,21].Again, this variation is mainly attributed to the differences in the chemical composition resulting mainly from the effect of both environmental and climatic changes on the biosynthetic pathways in the plants.
Staphylococcus aureus is one of the main human pathogens that cause a wide variety of clinical illness.It is a leading cause of multiple human infection such as bacteremia, skin and soft tissue infections, pulmonary infections gastroenteritis, meningitis, and urinary tract infections.Treatment strategies are considered devastating due to the appearance of multi-drug resistant strains of species such as MRSA (Methicillin-Resistant Staphylococcus aureus) [37].
Several previous works have reported the antibacterial activity of different Conyza species against S. aureus.The comparison of the results obtained from the previous work with our current findings clearly indicated the significant antibacterial potentials of C. canadensis as compared to other Conyza species (Table 7).This could be attributed to the secondary metabolite composition and its effect on bioactivity.CCM could be a candidate as a plant-based drug for the treatment of infections caused by S. arueses.Table 7 summarizes the antibacterial effect (reported as MIC) observed for different Conyza species against S. arueses.

Plant Material
The aerial parts of the plant were collected from the Al Mansour neighborhood, Al-Jubeiha, Amman governorate, Jordan, during the autumn of 2023.The taxonomic identity of the plant was confirmed by Prof. Dr. Hala I. Al-Jaber, Department of Chemistry, Faculty of Science, Al-Balqa Applied University, Al-Salt, Jordan.A voucher specimen (No: Ast/Cc/2023) was deposited at the herbarium of the Faculty of Science (Natural Products Laboratory Herbarium), Al-Balqa Applied University, Al-Salt, Jordan.

Hydro-Distillation and Extraction of Essential Oils
Essential oils were extracted from fresh aerial parts of the of C. canadensis (inflorescence heads, leaves, and stems) according to the procedure described in the literature [40,41].Briefly, a weighed sample of the specified fresh organ (Inf: 42.0 g, leaves: 86.21 g, stems: 107.0 g) was coarsely powdered and then subjected to hydro-distillation for 3 h in a Clevenger-type apparatus.The obtained essential oil (HDEO) from each organ was extracted (twice) with GC-grade n-hexane, dried using anhydrous Na 2 SO 4 , and then stored in an amber glass vials at 4 • C until analysis was performed.(% yield of the CCHD-EO: Inf: 0.38%, L: 1.71%, St: 0.023%).

GC-MS Analysis
GC/MS analysis was performed according to the procedure previously described in the literature [42,43].The analysis was performed on Shimadzu QP2020 GC-MS equipped with GC-2010 Plus (Shimadzu Corporation, Kyoto, Japan) with split-splitless mode, utilizing a DB-5MS fused silica column (5% phenyl, 95% polydimethylsiloxane, 30 m × 0.25 mm, 25 µm film thickness).For the best component separation, a linear temperature program was used.Briefly, the oven temperature was set to 50 • C for 1 min, the temperature increasing from 50 • C to 280 • C, at a heating rate of 7 • C/min; then held at 280 • C for 10 min.The total run time was 44 min.The injector temperature was 260 • C with a split ratio of 20:1; an injection volume of 1 µL; a carrier gas: helium (flow rate 1.50 mL/min); and a flow control mode: pressure, 88.3 kPa.MS source temperature/detector temperature: 240 • C; interface temperature: 250 • C; ionization energy (EI): 70 eV; scan range 35-500 amu; scan speed 1666.
The solvent cut was 3 min, while these data were acquired in 4.5 min.These data were collected using Windows based Lab-Solution GC-MS version 4.45SP1 Software.The mass spectra of isolated components were compared to those reported in ADAMS-2007 and NIST 2017 mass spectrometry libraries.To confirm the identified compound, a comparison performed between the reported values and relative retention indices (RI) with reference to n-alkanes (C 8 -C 30 ) in addition to these data published in the literature [25,44,45].

Preparation of the Alcoholic Extract
The whole and fresh aerial parts of C. canadensis (20 g sample) were soaked in methanol (200 mL) at room temperature for 24 h as described in literature [42,46].The procedure was repeated three times.The obtained extracts were combined and the solvent was then evaporated under reduced pressure at 55 • C. The obtained methanol extract (CCM yield: 7.63%) was then used for TPC, TFC, LC-MS/MS profiling, antioxidant activity evaluation, and bioactivity screening.

Total Phenolic Content (TPC) and Total Flavonoids Content (TFC)
The TPC and TFC were determined according the methods described in the literature [47] with slight modification.The Folin-Ciocalteu method was used to determine TPC.A 2.5 mL of Folin-Ciocalteu reagent (2N diluted ten folds) and 2 mL of Na 2 CO 3 solution (75 g/L) were added to 0.5 mL of CCM (500 µg/mL).After incubating the solution for 1 h at room temperature, the absorbance of the resulting solution was measured at 765 nm.Methanol was used as a blank reference.Measurements were performed on Infinite M. Plex microplate reader (Tecan, Männedorf, Switzerland).The TPC of the CCM extract is reported as mg/g gallic acid equivalent.All measurements were performed in triplicates.
Briefly, the TFC was determined by diluting of 1.0 mL sample of extract (500 µg/mL) with 4.0 mL distilled water into a 10 mL volumetric flask and then 0.30 mL of the NaNO 2 solution was added.After 5 min, 0.30 mL of the AlCl 3 solution (10% w/v) was added to the mixture.The solution was incubated for 6 min, and then 2.0 mL of the 1.0 M NaOH solution was introduced and the final volume of the solution was adjusted to 10.0 mL with distilled water.After another 15 min, the absorbance of the resulting solution was measured at 510 nm using methanol as a blank.Measurements were performed on Infinite M. Plex microplate reader (Tecan, Männedorf, Switzerland).The TFC content in the plant extracts was determined and expressed in mg quercetin/g dry extract.

DPPH Free Radical Scavenging Activity
The free radical scavenging activity of CCM extract was determined by the 1,1diphenyl-2-picryl-hydrazil (DPPH) according to the procedure described in the literature with minor modification [48].Briefly, a volume of 1.0 mL from the prepared standard solutions at different concentrations (5-500 µg/mL) was added to 1.0 mL of the freshly prepared methanolic DPPH solution.After 30 min of incubation, the absorbance of the different solutions were measured at 517 nm.Ascorbic acid and α-tocopherol were used as positive control.A standard curve was prepared using different concentrations of the DPPH.
IC 50 values of extract and standard were determined from the plot of scavenging activity against the compound's concentrations, which were defined as the total antioxidant necessary to decrease the initial DPPH radical concentration by 50%.Experiments were carried out in triplicates.Measurements were performed on Infinite M. Plex microplate reader (Tecan, Männedorf, Switzerland).

Iron Chelating Activity
The chelating effect on ferrous ions by CCM was estimated by the method described by Sudan et al. with slight modifications [49].Briefly, to 250 µL of the extract sample, 750 µL of methanol was added.Then, 50 µL of the 2 mM FeCl 2 solution was added.The reaction was initiated by the addition of 100 µL of the 5 mM ferrozine into the mixture, which was then left at room temperature for 10 min.The absorbance of the mixture was determined at 562 nm.

LC-MS/MS Analysis of the CCM
The analysis of flavonoids and phenolic compounds was performed using a SciEx UPLC (Exion-UPLC, Framingham, MA, USA) equipped with the LC-ESI-MS/MS-4500-QTRAP system (AB Sciex Instrument, Framingham, MA, USA), utilizing Analyst 1.7 software for data analysis.As described in the literature [50], a chromatographic separation was conducted at 30 ± 1 • C using a Phenomenex column (Torrance, CA, USA, 3.0 × 50 mm, 5 µm).A gradient elution consisted of a mobile phase A (5 mM ammonium format in water: methanol (95:5; v/v)) and mobile phase B (methanol, 1 mM formic acid).The following ratio of mobile phase B was applied during gradient program (% B, min): 5-90% B (0.00-8.00 min), 90-90% (8.00-12.00min), 90-5% (12.01-15.00min).The solvent flow rate was 0.35 mL/min and the injection volume was 5 µL.MS/MS analysis was performed in positive and negative ion mode.Nitrogen gas was applied at a pressure of 60 psi as the nebulizing and drying gas.The mass spectra were obtained over an m/z range of 100-900 amu.

Antifungal and Antibacterial Activity
The CLSI (Clinical and Laboratory Standards Institute) agar well diffusion method was used to evaluate the antibacterial activity of CCM [51].On SDA, fungi were cultivated, and the mixture was incubated for 72 h at 25 • C. In the meanwhile, bacterial strains were grown on MHA and incubated for 24 h at 37 • C. Test strains of fungus (2 × 10 4 CFU/mLcolony forming unit) and bacteria (~1 × 10 7 CFU/mL) that matched a 0.5 McFarland were produced into a workable solution.In summary, 100 µliters of the suspension were applied individually to an SDA/MHA plate, and the suspension was then uniformly distributed throughout the agar's surface.Next, using a 6 mm punching tool, wells were punched in each petri dish, and 100 µL of CC extract was added to each well.Fluconazole (10 µg/mL) and Ciprofloxacin (5 µg/mL) were used as a positive control for fungal and bacterial strains, respectively.The zone of inhibition around each well was measured in mm using a caliber.The assay was repeated and carried out in triplicate for each test isolate in a similar manner.

Assay for MIC and MBC
The minimal inhibitory concentration (MIC) of C. canadensis against S. aureus was determined by using the method of microdilution [52].The bacterial strain was inoculated in Muller Hinton broth at 37 • C for 24 h.The turbidity of obtained cultures was adjusted to 0.5 Mcfarland.One hundred µL from diluted cultures were poured into a 96 well microtiter plate.Then, 100 µL of CCM extract stock solution was added to the first well followed by two-fold serial dilution to obtain different CCM concentrations (100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.195, 0.0975, and 0.04875 µg/mL).Then, the 96 well plate was incubated at 37 • C for 24 h.Visual examination of the incubated plate was performed by turbidity detection and changes observation.The optical density (OD) was measured at 600 nm using 96 well reader (Thermo-Scientific Multiskan SKY, Waltham, MA, USA).A control positive well was used, which had the tested culture and a negative control one that contained only sterile broth medium.The MIC defined as the least CCM concentration that inhibited bacterial growth after 24 h of incubation.A 50 µL from all wells that showed no visible growth or turbidity were cultivated on Tryptone Soya Agar (TSA), (Biolab, Hungary) and incubated at 37 • C for 24 h.The MBC (minimal bactericidal concentration) is known as the least CCM concentration that can prevent bacterial growth.

Conclusions
The increased widespread of C. canadensis, known also as horseweed, over the cultivated yards intensifies the interest to search for the weed biological activity and pharmaceutical application.We here report the phytochemical evaluation of the volatile composition and the alcoholic extract of C. canadensis from Jordan.Our current study revealed qualitative and quantitative variations in the composition of the HDEO of C. canadensis as compared to other previous studies.The detecting 2E,8Z-Matricaria ester isomer is reported at high concentration levels, which was almost absent in previous studies.Despite these changes, some similarities were observed.The richness of the CCHD-EOs in limonene and polyacetyelene derivatives encourages the future assessment of the oil for herbicidal and fungicidal activities.Moreover, the plant from Jordanian origin was found to be rich in rosmarinic acid as evidenced from its content in the CCM extract (1441.125 mg/kg plant).This is the first report for the detection and quantitation of 3-O-methylquercetin, hesperetin, resveratrol, salvianolic acid, hesperidin, and caffeic acid phenethyl ester in C. canadensis.Additionally, CCM also showed moderate antioxidant activity and significant antibacterial activity against S. aureus was detected.

Figure 1 .
Figure 1.The structures of the main constituents detected in the CCHD-EO obtained from fresh aerial parts of C. canadensis from Jordan.

Figure 1 . 19 Figure 2 .
Figure 1.The structures of the main constituents detected in the CCHD-EO obtained from fresh aerial parts of C. canadensis from Jordan.Molecules 2024, 29, x FOR PEER REVIEW 6 of 19

Figure 2 .
Figure 2. Different classes of the volatile compounds detected in CCHD-EOs obtained from the fresh aerial parts of C. canadensis from Jordan.

Table 1 .
GC/MS analysis of the HDEO obtained from different parts of fresh C. canadensis grown in Jordan.

Table 2 .
The total phenolic content (mg gallic acid/g extract), total flavonoids (mg quercetin/g extract), and DPPH radical scavenging activity in methanolic extract of C. canadensis.(Values expressed are means ± SD of three parallel measurements).

50 (µg/mL) Iron Chelating Activity IC 50 (µg/mL)
Different classes of the volatile compounds detected in CCHD-EOs obtained from the fresh aerial parts of C. canadensis from Jordan.

Table 3 .
LC-MS/MS data for the phenolic and flavonoid compounds detected in the CCM from Jordan and their concentrations.

Table 3 .
LC-MS/MS data for the phenolic and flavonoid compounds detected in the CCM from Jordan and their concentrations.

Table 3 .
LC-MS/MS data for the phenolic and flavonoid compounds detected in the CCM from Jordan and their concentrations.

Table 3 .
LC-MS/MS data for the phenolic and flavonoid compounds detected in the CCM from Jordan and their concentrations.

Table 3 .
LC-MS/MS data for the phenolic and flavonoid compounds detected in the CCM from Jordan and their concentrations.

Table 3 .
LC-MS/MS data for the phenolic and flavonoid compounds detected in the CCM from Jordan and their concentrations.

Table 3 .
LC-MS/MS data for the phenolic and flavonoid compounds detected in the CCM from Jordan and their concentrations.

Table 4 .
The ZOI-mm, MIC, and MBC of CCM against S. aureus.

Table 5 .
The major components of essential oils from C. canadensis from different locations.

Table 6 .
The DPPH scavenging activity of Conyza spp.from different origins.