Comparative LC–LTQ–MS–MS Analysis of the Leaf Extracts of Lantana camara and Lantana montevidensis Growing in Egypt with Insights into Their Antioxidant, Anti-Inflammatory, and Cytotoxic Activities

Lantana camara L. and Lantana montevidensis Briq. (F. Verbenaceae) are invasive ornamental weeds native to the tropical regions of Africa and America. The leaves of both species have been traditionally used as infusions for treating fever, rheumatism, and cancer. LC–MS–MS-guided profiling of the methanolic extracts of the leaves of L. camara and L. montevidensis growing in Egypt led to the putative identification of 59 compounds belonging to terpenoids, flavonoids, iridoid glycosides, phenolic acids, and their derivatives. The in-vitro antioxidants and anti-inflammatory and anticancer activities of the two extracts were investigated. L. camara and L. montevidensis inhibited DPPH• (IC50 = 34.01 ± 1.32 and 47.43 ± 1.74 µg/mL), ABTS+ (IC50 = 30.73 ± 1.42 and 40.37 ± 1.51 µg/mL), and superoxide anion (IC50 = 1.57 ± 0.19 and 1.31 ± 0.14 μg/mL) free radicals. A potent anti-inflammatory effect was observed for both species through the inhibition of elastase release in fMLF/CB-induced human neutrophils (IC50 = 2.40 ± 0.16 and 1.90 ± 0.07 μg/mL). The extracts showed significant cytotoxic activity against a panel of cancer cell lines with the most potent activity against Caco cells (IC50 = 45.65 ± 1.64 and 40.67 ± 1.52 µg/mL for L. camara and L. montevidensis, respectively). Western blotting supported by FACS analysis revealed that the extracts inhibited cancer cell proliferation, reduced metastasis, and induced apoptosis resulting in cell cycle arrest. This was achieved via increasing mRNA and protein expressions of p53 and GSK-3β as well as decreasing the expression of PI3K, Akt, and cyclin D1.

G1/S specific cyclin D1 is a mitogenic signal sensor whose degradation is regulated by GSK-3β and its gene expression is downregulated due to the negative regulation of GSK-3β on the oncogenic Wnt/β-catenin signaling. Cyclin D1 activity is normally intensified in cancer; therefore, cancers showing overexpression of cyclin D1 are susceptible to GSK-3β activation [25].
In continuation of our work (in finding new sources of pharmacologically active molecules from Egyptian flora [26,27]), we performed a detailed LC-MS-MS metabolome profiling of the polyphenol-rich leaf extracts of L. camara L. and L. montevidensis Briq. A comparative assessment of the extracts' antioxidant potential, their inhibitory effects on neutrophil elastases, as well as their cytotoxic activities on cancer cell proliferation and metastasis supported by mechanistic studies on their effects on PI3K/AkT and GSK-3β/Cyclin D1 signaling pathways, were likewise investigated.

LC-LTQ-MS-MS Analysis and GNPS-Aided Annotation of L. camara and L. montevidensis Constituents
The metabolomic mass profiles of the two Lantana extracts, L. camara and L. montevidensis, were screened using the Global Natural Product Social Molecular Networking (GNPS) based on tandem mass spectrometry data (Figures 1 and 2) [28][29][30] in the positive ionization mode of Lantana extract samples. The metabolites were represented by nodes in the molecular network, with chemically related metabolites clustered together. The network in Figure 2 was displayed as a pie chart to reflect the relative abundance of each ion in the analyzed Lantana extract samples. The results demonstrated a total of 157 nodes assigned for the parent ions of L. camara demonstrated in Figure 2 as yellow-colored nodes, of which, five parent ions matched five known standards in the GNP library (Table 1) belonging to pentacyclic triterpenes, sesquiterpenes, flavonoids, and amides. On the other side, a total of 153 nodes were assigned for the parent ions of L. montevidensis demonstrated in Figure 2 as blue-colored nodes, of which, four parent ions matched the known standards in the GNP library, namely palmitamide, α-humulene, coprostanone, and carminic acid. The network showed the similarities and variances of metabolites in both extracts and prescribed N1-59 as the identified metabolites (Table 1).  Table 1.  Table 1.   LC-LTQ-MS-MS analysis and molecular networking analysis resulted in the tentative identification of 59 compounds from both Lantana species, including 37 terpenoids, 3 iridoid glycosides, 11 flavonoids/flavonoid glycosides, 11 phenolic acids and their derivatives, among others ( Table 1). The iridoid momordol and copaenol sesquiterpene were identified for the first time from the genus Lantana. The iridoid glycoside, durantoside, was previously identified in the roots of L. viburnoides [40]. Other identified compounds were previously reported from L. camara and L. montevidensis.
Terpenoids are the major metabolites produced by the genus Lantana [40]. Various pentacyclic triterpenoids have been reported in different Lantana species and are known for their wide range of pharmacological activities. Lantadenes A, D, and C, icterogenin, and Plants 2022, 11, 1699 9 of 21 pomonic acid identified in our studied Lantana species showed mass data following the previously reported data. Amyrin and lantabetulic acid were exclusively identified in L. montevidensis.
Flavonoids represented one of the chief constituents in the genus Lantana, particularly flavones and flavonols [40]. Vicenin-2 (m/z 594.96) was tentatively identified in L. camara based on the characteristic fragmentation patterns of C-glycosides by cross-ring cleavage of the glucose moiety and the subsequent formation of the fragment ions [M+H-120] + [59]. The dimethoxy flavone pectolinarigenin (m/z 315.5381) was identified in both L. camara and L. montevidensis. Flavonoid glycosides, such as pectolinarin, camaroside, and lantanoside, were detected in both species. Their identification was based on the molecular ion peaks and the respective sugars lost. A fragment ion at [M+H-162] + indicated the loss of a hexoside moiety, while a fragment ion at [M+H-204] + corresponded to a loss of acetylhexoside.
Simple phenolic acids, such as gallic acid, ferulic acid, and coumaric acid having m/z at 170.5321, 193.3430, and 166.5324, respectively, were detected in both Lantana extracts. Other phenolic acids including cistanoside C, lipedoside A, osmanthuside B, forsythoside A, calceolarioside E, isonuomioside A were also identified. Their mass fragmentation patterns were characteristic, revealing the type of the attached phenolic acid, for example, a loss of 163 Da corresponded to the loss of a caffeoyl moiety, while the loss of 147 Da corresponded to the loss of coumaric acid moiety. Iridoids such as verbascoside (m/z 624.8313, C 29 H 36 O 15 ) were found in both species with fragment ions corresponding to the loss of both a caffeoyl moiety and a dehydrated rhamnose part [60].

Assessment of the Antioxidant Effects of L. camara and L. montevidensis Extracts
The stable DPPH • radical scavenging activity assay was used to evaluate and compare the antioxidant potential of L. camara and L. montevidensis extracts. The abilities of both extracts to scavenge free radicals were assessed by measuring the change in absorbance produced by the decrease of DPPH • radicals ( Figure 3). The results demonstrated the dosedependent radical scavenging capabilities of the two extracts. L. camara demonstrated more potent activity in scavenging DPPH • free radicals with an IC 50 value of 34.01 ± 1.32 µg/mL compared to L. montevidensis, which displayed an IC 50 of 47.43 ± 1.74 µg/mL. The results were comparable to the standard L-ascorbic acid (IC 50 20.3 ± 1.24 µg/mL).

ABTS + Assay
The ABTS + cation radical scavenging activity was measured using the decolorization assay at various concentrations of L. camara and L. montevidensis. The results showed that the ABTS + cation radical scavenging activity of both extracts was concentration-dependent, similar to the DPPH assay ( Figure 3). The IC50 scavenging capability exhibited by L. camara and L. montevidensis values were 30.73 ± 1.42 and 40.37 ± 1.51 µg/mL, respectively, compared with the standard L-ascorbic acid showing IC50 of 15.7 ± 1.21 µg/mL. Based on these findings, it can be concluded that both extracts exhibited a high radical scavenging

ABTS + Assay
The ABTS + cation radical scavenging activity was measured using the decolorization assay at various concentrations of L. camara and L. montevidensis. The results showed that the ABTS + cation radical scavenging activity of both extracts was concentration-dependent, similar to the DPPH assay ( Figure 3). The IC 50 scavenging capability exhibited by L. camara and L. montevidensis values were 30.73 ± 1.42 and 40.37 ± 1.51 µg/mL, respectively, compared with the standard L-ascorbic acid showing IC 50 of 15.7 ± 1.21 µg/mL. Based on these findings, it can be concluded that both extracts exhibited a high radical scavenging capacity by reducing oxidative stress.
2.3. In Vitro Assessment of the Anti-Inflammatory Effects of L. camara and L. montevidensis The methanol extracts of L. camara and L. montevidensis were investigated for their antiinflammatory effects through the inhibition of superoxide anion generation and elastase release in fMLF/CB-induced human neutrophils. The LDH assay was likewise performed to assess the safety and/or toxicity of the tested extracts. LDH is a stable enzyme, present in all cell types, rapidly released into the cell culture medium upon the damage of the plasma membrane. The LDH assay is commonly used for the determination of cell death and cytotoxicity [61]. The results of the LDH analysis indicated the nontoxic features of both Lantana extracts at the tested dose of 10 µg/mL with cell viability exceeding 95% (Table 2). Therefore, both samples did not affect the growth of human neutrophils at 10 µg/mL. Lantana extracts showed a dose-dependent inhibition of superoxide anion generation and elastase release in fMLF/CB-induced human neutrophils (Tables 3 and 4). The extract of L. montevidensis demonstrated slightly more potent inhibition of superoxide anion with an IC 50 of 1.31 ± 0.14 µg/mL compared to L. camara (IC 50 of 1.57 ± 0.19 µg/mL). Similarly, L. montevidensis methanolic extract showed marked inhibition of elastase release (IC 50 of 1.90 ± 0.07 µg/mL) compared to L. camara extract (IC 50 of 2.40 ± 0.16 µg/mL) in fMLF/CB-induced human neutrophils. Both extracts at 10 µg/mL almost completely (~100%) attenuated the activation of human neutrophils, which play role in different stages of cancer and viral infections. Our results support previous studies reporting the antiinflammatory activities of L. camara and L. montevidensis in different models [62,63]. This activity was attributed to their phytoconstituents, such as pectolinarigenin, and rutin, among others, which showed anti-inflammatory activity in previous reports [51,[64][65][66].

Alterations in Morphological Features of Treated Cancer Cells
Cytotoxic agents frequently alter cell morphology, resulting in abnormal morphological changes, elevated cellular debris, and reduction in cell number. In the current study, detectable morphological features of apoptosis were observed in Caco cells treated with L. camara and L. montevidensis extracts, including cellular shrinkage, reduction in cell number, detachment of the cells, cell rounding, and condensation of the cytoplasm. However, the morphology of the untreated cells appeared normal and confluent ( Figure 5). Results were expressed as mean ± SE, (n = 5).

Alterations in Morphological Features of Treated Cancer Cells
Cytotoxic agents frequently alter cell morphology, resulting in abnormal morphological changes, elevated cellular debris, and reduction in cell number. In the current study, detectable morphological features of apoptosis were observed in Caco cells treated with L. camara and L. montevidensis extracts, including cellular shrinkage, reduction in cell number, detachment of the cells, cell rounding, and condensation of the cytoplasm. However, the morphology of the untreated cells appeared normal and confluent ( Figure 5). cancer cell lines. Cells were treated with various concentrations of L. camara, L. montevidensis, and tamoxifen for 48 h and cell viability was plotted against drugs concentration to calculate the IC50. Results were expressed as mean ± SE, (n = 5).

Alterations in Morphological Features of Treated Cancer Cells
Cytotoxic agents frequently alter cell morphology, resulting in abnormal morphological changes, elevated cellular debris, and reduction in cell number. In the current study, detectable morphological features of apoptosis were observed in Caco cells treated with L. camara and L. montevidensis extracts, including cellular shrinkage, reduction in cell number, detachment of the cells, cell rounding, and condensation of the cytoplasm. However, the morphology of the untreated cells appeared normal and confluent ( Figure 5).

Analysis of the Cell Cycle
Cell cycle arrest occurs when the PI3K/Akt protein kinases are inhibited. The activation of GSK-3β and the blockade of the cyclin D1 signaling pathway reduces the proliferation and metastasis of the Caco cancer cell line. When compared with the untreated cells,

Analysis of the Cell Cycle
Cell cycle arrest occurs when the PI3K/Akt protein kinases are inhibited. The activation of GSK-3β and the blockade of the cyclin D1 signaling pathway reduces the proliferation and metastasis of the Caco cancer cell line. When compared with the untreated cells, both extracts increased the percentage of cells in the sub-G 0 /G 1 phase (the phase at which the cells wait before entering the cell cycle to duplicate) in Caco cells at the IC 50 level. When the number of cells in this phase rises, the cell cycle stops, and division as well as DNA replication are impossible. Figure 6 showed that at the IC 50 concentrations, the two Lantana extracts caused cell cycle arrest at rates of 19.2% and 17.2% in the sub-G 0 /G 1 phase, respectively, compared to the untreated Caco cells (3.4%). These findings showed that Lantana extracts could inhibit the PI3K/AkT and GSK-3β/cyclin D1 signaling pathways, as well as trigger apoptosis by arresting the cell cycle in the sub-G 0 /G 1 phase [67,68]. When the number of cells in this phase rises, the cell cycle stops, and division as well as DNA replication are impossible. Figure 6 showed that at the IC50 concentrations, the two Lantana extracts caused cell cycle arrest at rates of 19.2% and 17.2% in the sub-G0/G1 phase, respectively, compared to the untreated Caco cells (3.4%). These findings showed that Lantana extracts could inhibit the PI3K/AkT and GSK-3β/cyclin D1 signaling pathways, as well as trigger apoptosis by arresting the cell cycle in the sub-G0/G1 phase [67,68].

qRT-PCR Assessment
The mRNA expressions of the p53 (apoptotic markers), PI3K, and GSK-3β (proliferative markers) genes in the Caco cell line were measured using qRT-PCR. The expressions of p53 and GSK-3β were significantly (p < 0.0001) enhanced in cells treated with both Lantana extracts compared to the untreated cells. Similarly, PI3K gene expression was downregulated in Lantana-treated cells as compared with the untreated cells (Figure 7). Therefore, Lantana extracts had the potential to limit cancer cell proliferation and metastasis causing cell cycle arrest and apoptosis, which was clarified by the overexpression of p53 and GSK-3β as well as the downregulation of the PI3K gene [69].

qRT-PCR Assessment
The mRNA expressions of the p53 (apoptotic markers), PI3K, and GSK-3β (proliferative markers) genes in the Caco cell line were measured using qRT-PCR. The expressions of p53 and GSK-3β were significantly (p < 0.0001) enhanced in cells treated with both Lantana extracts compared to the untreated cells. Similarly, PI3K gene expression was downregulated in Lantana-treated cells as compared with the untreated cells (Figure 7). Therefore, Lantana extracts had the potential to limit cancer cell proliferation and metastasis causing cell cycle arrest and apoptosis, which was clarified by the overexpression of p53 and GSK-3β as well as the downregulation of the PI3K gene [69].

qRT-PCR Assessment
The mRNA expressions of the p53 (apoptotic markers), PI3K, and GSK-3β (proliferative markers) genes in the Caco cell line were measured using qRT-PCR. The expressions of p53 and GSK-3β were significantly (p < 0.0001) enhanced in cells treated with both Lantana extracts compared to the untreated cells. Similarly, PI3K gene expression was downregulated in Lantana-treated cells as compared with the untreated cells (Figure 7). Therefore, Lantana extracts had the potential to limit cancer cell proliferation and metastasis causing cell cycle arrest and apoptosis, which was clarified by the overexpression of p53 and GSK-3β as well as the downregulation of the PI3K gene [69].

Immunoblotting Assay
Compared to the untreated cells, both Lantana extracts resulted in a significant decrease in Akt protein kinase (Figures S1-S3) and cyclin D1 (Figures S4-S6) in Caco cells (Figure 8). These findings revealed that the extracts suppressed PI3K resulting in Akt inhibition via dephosphorylation. The dephosphorylation of Akt activates p53, which subsequently arrests the cell cycle. It likewise stimulated GSK-3β, which inhibited cyclin D1, leading to reduced cellular proliferation, angiogenesis, and metastasis.

Immunoblotting Assay
Compared to the untreated cells, both Lantana extracts resulted in a significant decrease in Akt protein kinase (Figures S1-S3) and cyclin D1 (Figures S4-S6) in Caco cells (Figure 8). These findings revealed that the extracts suppressed PI3K resulting in Akt inhibition via dephosphorylation. The dephosphorylation of Akt activates p53, which subsequently arrests the cell cycle. It likewise stimulated GSK-3β, which inhibited cyclin D1, leading to reduced cellular proliferation, angiogenesis, and metastasis. Results were expressed as mean ± SE, (n = 3). *** p < 0.001 and **** p < 0.0001 is considered significant compared to the Caco control untreated cells. Bands were relatively expressed to β-actin protein (internal control) (Figures S7 and S8) by western blot analysis.

Plant Collection and Extraction
Fresh young leaves of L. camara L. (Syn. Camara vulgaris Benth.) and L. montevidensis (Spreng.) Briq. (Verbenaceae) were collected from South Valley University Garden and Aswan Botanical Garden, Aswan, Egypt, respectively, in March 2020. The woody shrubs of L. camara L. were grown in sandy soil, irrigated by groundwater wells, and supplied by Figure 8. Western blot analysis of L. camara and L. montevidensis extracts in Caco cells. Results were expressed as mean ± SE, (n = 3). *** p < 0.001 and **** p < 0.0001 is considered significant compared to the Caco control untreated cells. Bands were relatively expressed to β-actin protein (internal control) ( Figures S7 and S8) by western blot analysis.

Plant Collection and Extraction
Fresh young leaves of L. camara L. (Syn. Camara vulgaris Benth.) and L. montevidensis (Spreng.) Briq. (Verbenaceae) were collected from South Valley University Garden and Aswan Botanical Garden, Aswan, Egypt, respectively, in March 2020. The woody shrubs of L. camara L. were grown in sandy soil, irrigated by groundwater wells, and supplied by natural fertilizers. Nevertheless, L. montevidensis woody shrubs were grown in loam soil, irrigated by river Nile fresh water, and supplied by natural fertilizers. Photos of the collected fresh leaves are demonstrated in Figure 9. Authentication was achieved by Therese Labib, Consultant at El-Orman Botanic Garden, Giza, Egypt. Voucher specimens were deposited at the herbarium at the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Egypt (PHG-V-LC-244), and (PHG-V-LM-245). The dried plant materials (500 g each) were extracted by successive maceration (3X-2 L each) in methanol (Al-Brouj, Giza, Egypt). The macerate was filtered, evaporated, and concentrated in vacuo at 45 • C to yield 2.17 g and 2.34 g of dark brown extracts of L. camara and L. montevidensis, respectively. The methanol extracts were subsequently defatted using n-hexane (Al-brouj, Giza, Egypt), evaporated, freeze-dried, then stored in amber-colored bottles for further chemical analysis and biological investigations.

LC-LTQ-MS-MS Analysis of L. camara and L. montevidensis Extracts
The defatted methanol extract was analyzed using LC-MS-MS. A Shimadzu LC-10 HPLC with a Grace Vydac Everest Narrowbore C-18 column (100 mm × 2.1 mm i.d., 5 µm, 300 Å). LC-MS, connected to an LTQ Linear Ion Trap MS (Thermo Finnigan, San Jose, CA) was utilized with a mass range of 100-2000 m/z. A 2 µL sample was injected using an autosampler. A 35 min method was used as follows: 5 min isocratic run using 5% acetonitrile (Acn) and 0.05% formic acid (FA), then a gradient was run for 25 min until 95% AcN 0.05% FA. Finally, there was 5 min of conditioning the column with 5% AcN and 0.05% FA. The data were processed and analyzed using foundation 3.1_Xcalibur_3.1.6610. Furthermore, the raw data files were converted to mzXML format using MSConvert from the ProteoWizard suite [70]. The molecular network was created using the Global Natural Products Social Molecular Networking (GNPS) online workflow [28,29]. The spectra in the network were then searched against the GNPS spectral libraries and published data. The DPPH • assay was used to examine the free radical scavenging capacity of the two extracts according to the method published by Burits and Bucar [71] with certain

LC-LTQ-MS-MS Analysis of L. camara and L. montevidensis Extracts
The defatted methanol extract was analyzed using LC-MS-MS. A Shimadzu LC-10 HPLC with a Grace Vydac Everest Narrowbore C-18 column (100 mm × 2.1 mm i.d., 5 µm, 300 Å). LC-MS, connected to an LTQ Linear Ion Trap MS (Thermo Finnigan, San Jose, CA) was utilized with a mass range of 100-2000 m/z. A 2 µL sample was injected using an autosampler. A 35 min method was used as follows: 5 min isocratic run using 5% acetonitrile (Acn) and 0.05% formic acid (FA), then a gradient was run for 25 min until 95% AcN 0.05% FA. Finally, there was 5 min of conditioning the column with 5% AcN and 0.05% FA. The data were processed and analyzed using foundation 3.1_Xcalibur_3.1.6610. Furthermore, the raw data files were converted to mzXML format using MSConvert from the ProteoWizard suite [70]. The molecular network was created using the Global Natural Products Social Molecular Networking (GNPS) online workflow [28,29]. The spectra in the network were then searched against the GNPS spectral libraries and published data.

DPPH • Free Radical Scavenging
The DPPH • assay was used to examine the free radical scavenging capacity of the two extracts according to the method published by Burits and Bucar [71] with certain modifications. Briefly, various concentrations of LC and LM (1.56-100 µg/mL) were added and mixed gently with 975 µL of (0.003 g%) DPPH • in methanol. The reaction mixture absorbance (A) was measured at 515 nm using a Jenway 6305 UV/Vis spectrophotometer after 1 h of dark incubation at room temperature. A reaction without the extract was carried out as a control. As a positive control, L-ascorbic acid (20-100 µg/mL) was utilized, and the DPPH • radical scavenging activity (%) was measured using the equation: DPPH radical scavenging activity % = A control − A sample A control × 100

ABTS + Radical Scavenging Activity
The ABTS cation radical (ABTS + ) scavenging activity of the two extracts was measured according to Re et al. [72]. The ABTS solution (14 mM) reacted with the potassium persulfate solution (4.9 mM) for 16 h in the dark. ABTS + cation radicals were produced. The ABTS + solution was diluted with distilled water to achieve an absorbance of 0.734 at 734 nm before use. Then, 975 µL of ABTS + solution was added to 25 µL of the two Lantana extracts containing different concentrations (from 1.56 to 100 µg/mL). Absorbance was measured at 734 nm after 4 min of dark incubation and compared to the control. Ascorbic acid (20-100 µg/mL) was employed as a positive control and the ABTS + cation radical scavenging activity (%) was estimated using the following equation: 3.4. In Vitro Assessment of the Anti-Inflammatory Activity of L. camara and L. montevidensis

Preparation of Human Neutrophils
Blood was collected from healthy human donors (20-35 years old) using a protocol conducted according to the guidelines of the Declaration of Helsinki, and approved by the institutional review board at Chang Gung Memorial Hospital (IRB no. 201902217A3). Informed consent was obtained from all subjects involved in the study. Neutrophils were isolated as previously described [73]. In brief, the blood samples were processed by dextran sedimentation and Ficoll-Hypaque centrifugation, followed by hypotonic lysis of contaminating red blood cells [74]. The segregated neutrophils were suspended and stored in pH 7.4 Ca 2+ -free Hank's balanced salt solution (HBSS) at 4 • C before the experiments. Next, the Wright-Giemsa stain was applied to confirm the purity of the suspension of neutrophils. Finally, the cellular viability of >98% was confirmed by the trypan blue exclusion method.

Lactate Dehydrogenase (LDH) Assay
The damage or toxicity to the cells may be expressed as LDH release because the cell membrane loses its integrity, and LDH stored in the cytoplasm is released outside the cell. The cell viability assay based on LDH release was performed to ensure the safety of the extracts on human neutrophils [75]. In brief, human neutrophils (6 × 10 5 cells/mL) were preheated at 37 • C for 5 min in 1 mM CaCl 2 and were incubated with the tested extracts for 15 min. Total LDH released from the cells was incubated with 0.1% of Triton X-100 for 30 min to completely cause cell lysis. Cells were centrifuged at 4 • C for 200× g for 8 min, an LDH reagent was added to the supernatant, and the mixture was incubated in the dark at room temperature for 30 min. The absorbance was then measured at 492 nm, and the LDH release was calculated and compared to the total LDH release set as 0%; untreated cells were set as 100%.

Measurement of Superoxide Generation
Ferricytochrome c was used to evaluate the superoxide release in human neutrophils [76]. The method was described in a previous study [77]. Human neutrophils (6 × 10 5 cells/mL) were incubated in HBSS containing ferricytochrome c (0.6 mg/mL) and CaCl 2 (1 mM). The mixture was equilibrated at 37 • C for 5 min and then was incubated with the tested samples or DMSO (control) for 5 min. Cells were primed by cytochalasin B (CB, 1 µg/mL) and were activated with formyl-methionyl-leucyl-phenylalanine (fMLF, 100 nM) for 10 min. The absorbance was constantly monitored at 550 nm using a double-beam, six-cell positioned spectrophotometer Hitachi U-3010 with constant stirring (Hitachi Inc., Tokyo, Japan). Calculations were based on the differences in absorbance in the presence or absence of superoxide dismutase (SOD, 100 U/mL) divided by the extinction coefficient for ferricytochrome c reduced form (ε = 21.1/mM/10 mm). LY294002 was used as the positive control.

Measurement of Elastase Release
Elastase release was measured by degranulation of azurophilic granules in human neutrophils [78]. Human neutrophils (6 × 10 5 cells/mL) were equilibrated with elastase substrate MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 µM) in HBSS supplemented with CaCl 2 (1 mM) at 37 • C for 2 min and then were incubated with samples or DMSO (control) for 5 min. Human neutrophils were activated by 100 nM fMLF and 0.5 µg/mL CB and the changes in the absorbance at 405 nm were continuously monitored by a spectrometer (Hitachi U-3010, Tokyo, Japan) to record the elastase release. The results were expressed as the percent of elastase release in the fMLF/CB-activated drug-free control system. LY294002 was used as the positive control.

Cell Morphology Study
Briefly, 1 × 10 5 of the Caco cell line was seeded in a 6-well plate, incubated for 24 h, then treated with Lantana extracts at their IC 50 concentrations. After 48 h of incubation, morphological alterations of the treated and untreated cells were evaluated and captured using an inverted light microscope (Olympus, Tokyo, Japan).

Cell Cycle Examination
Flow cytometry was used to analyze cell cycle phases using an Accuri C6 flow cytometer (Becton Dickinson BD, Franklin Lakes, NJ, USA) on Caco cells 1 × 10 5 that were trypsinized, centrifuged at 5000 rpm at 4 • C, washed with cold phosphate buffer saline (PBS), and fixed with cold absolute ethanol, as described by Noser et al. and Darzynkiewicz et al. [80,81].

Quantitative Real-Time PCR (qRT-PCR)
The Caco 1 × 10 5 control and treated cells were trypsinized, centrifuged at 4500 rpm at 4 • C, and washed with PBS. The pelleted cells were subjected to RNA extraction and transcription to cDNA as described by Kvastad et al. [82]. The expressions of p53, GSK-3β, and PI3K mRNA were measured using Applied qPCR Biosystems (Foster City, CA, USA) on treated and control cells according to Livak and Schmittgen [83]. The primer sequences were designed using primer 3 plus as shown in Table 5.

Western Blot Analysis
The method of Mruk and Cheng was used for immunoblotting assay [84]. Proteins were separated from Caco control and treated cells using cold RIPA lysis buffer and were quantified using Bradford [85]. Equal amounts of proteins (20 mg) were separated and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking the membrane, the primary antibodies, phospho-AkT (ab81283) and cyclin D1 (ab134175), were added and incubated with it. Then, the primary antibodies were removed, carefully washed several times, and incubated with the secondary antibody horseradish peroxidase (HRP) (ab205718). The bands were visualized using enhanced chemiluminescence (ECL) detection kit (Promega, Madison, WI, USA). A gel documentation system (Geldoc-it, UVP, Cambridge, UK), was applied for data analysis using TotalLab analysis software, Newcastle upon Tyne, England, www.totallab.com, (Ver.1.0.1).

Statistical Analysis
Results are expressed as mean ± SEM value of at least three independent measurements unless otherwise specified. The 50% inhibitory concentration (IC 50 ) was calculated from the dose-response curve obtained by plotting the percentage of inhibition versus concentrations (linear function, Microsoft Office, Redmond, WA, U.S). Statistical analysis was performed by Student's t-test (Sigma Plot, Systat software, Systat Software Inc., San Jose, CA, USA, anti-inflammatory assay). Values with * p < 0.05, ** p < 0.01, *** p < 0.001 were considered statistically significant.

Conclusions
LC-MS-MS-guided metabolic profiling of L. camara and L. montevidensis extracts resulted in the tentative identification of 59 compounds belonging to different phytochemical classes including pentacyclic triterpenes, flavonoids, and phenolic acids. In vitro studies revealed that Lantana species displayed potent radical scavenging and anti-inflammatory activities through the inhibition of elastase release in fMLF/CB-induced human neutrophils. The extracts, despite their encouraging safety profile on normal human cells, exhibited potent cytotoxic effects on a wide array of cancer cell lines, especially against Caco cells. Lantana extracts induced apoptosis and triggered cell cycle arrest. They inhibited the proliferation and metastasis of cancer cells by downregulating the PI3K/AkT signaling cascade and reducing cyclin D1 levels via the activation of GSK-3β. Our findings imply that L. camara and L. montevidensis crude extracts could be valuable sources for further research as potential anticancer agents.