Metabolomic profiling and anti-infective potential of Zinnia elegans and Gazania rigens (Family Asteraceae).

The present study evaluates the chemical composition of Zinnia elegans and Gazania rigens based on their metabolomic profiles using liquid chromatography coupled with high-resolution mass spectrometry (LC-HR-MS), alongside with the anti-infective activities of their ethanol extracts, as well as, different fractions. A significant difference was observed between the LC-MS profiles of the two plants such as, coumarins, sesquiterpene lactones and phenylethanoids which were characteristic for Z. elegans, while amides and phenolic acid derivatives were characteristic for G. rigens. These results highlight the chemical potential of Z. elegans and G. rigens. Furthermore, the ethyl acetate fraction of Z. elegans showed a significant antimalarial activity with IC50 values of 21.03 and 13.72 µg/mL against Plasmodium falciparum D6 and P. falciparum W2, respectively.

was then re-equilibrated with 10% B for 9 min before the next injection. The total analysis time for each sample was 45 min., where 10 µL were injected and the tray temperature was maintained at 12 o C. High-resolution mass spectrometry was carried out in both positive and negative ESI ionization modes with a spray voltage at 4.5 kV and capillary temperature at 320 o C. The mass range was set from m/z 150-1500.

Metablomic analysis
In MZmine, the raw data was imported by selecting the ProteoWizard-converted positive or negative files in mzML format. The peaks in the samples and blanks were detected using the chromatogram builder. Mass ion peaks were isolated with a centroid detector threshold that was greater than the noise level set to 1.0 × 10 4 and an MS level of one. Following this, the chromatogram builder was used with a minimum time span set to 0.2 min, and the minimum height and m/z tolerance to 1.0 × 10 4 and 0.001 m/z or 5.0 ppm, respectively. Chromatogram deconvolution was then performed to detect the individual peaks. The local minimum search algorithm (chromatographic threshold: 95%, search minimum in RT range: 0.4 min, minimum relative height: 5%, minimum absolute height: 3.0 × 104, the minimum ratio of peak top/edge: 3, and peak duration range: 0.2-5 min) was applied. Isotopes were also identified using the isotopic peaks grouper (m/z tolerance: 0.001 m/z or 5.0 ppm, retention time tolerance: 0.1 absolute (min), maximum charge: 2, and representative isotope: most intense). The chromatographic alignment and gap-filling were then used to reduce inter-batch variation. The peak lists were all aligned using the join aligner parameters set to m/z tolerance: 0.001 m/z or 5.0 ppm, weight for m/z: 20, retention time tolerance: 5.0 relative (%), weight for retention time: 20. The values for the weight of m/z and retention time should be kept the same; this means that both retention time and m/z are given equal importance. Missing peaks were reported using the gap filling peak finder with an intensity tolerance of 25%, m/z tolerance of 0.001 m/z or 5.0 ppm, and retention time tolerance of 0.5 absolute (min). An adduct search was performed for Na-H, K-H, NH 4 , formate, and ACN + H (RT tolerance: 0.2 absolute (min), m/z tolerance: 0.001 m/z or 5.0 ppm, max relative adduct peak height: 30% 0.2 absolute (min), m/z tolerance: 0.001 m/z or 5.0 ppm, and with maximum complex peak height of 50%). Adjust parameters with heuristics element count with all three sub-options to get the isotope pattern filter working with all features with isotope peaks.
Excel macros were written to enable the subtraction of background peaks and to combine positive and negative ionization mode data files generated by MZmine.
Peaks originating from the culture medium were extracted. By applying an algorithm to calculate the intensity of each m/z in both plant extracts and different fractions. The positive and negative ionization mode data sets from each of the respective extracts or fractions were combined with the macro enabling ion peaks that were observed in either or both positive and negative modes to be overlaid for further statistical analysis. The Excel macro was used to dereplicate each m/z ion peak with compounds in the customized database (using RT and m/z threshold of ±3 ppm) which provided details on the putative identities of all metabolites in plant fraction and sequentially sorted the number of the remaining unknowns for each fraction. (El-Sayed et al., 2018).

Evaluation of antimalarial activity
All fractions were investigated for their in vitro antimalarial activity and were evaluated for their ability to inhibit the chloroquinesensitive (D6, Sierra Leone) Plasmodium falciparum protozoan and the chloroquine-resistant (W 2 ) strains. The samples were tested against a suspension of red blood cells infected with P. falciparum. A 200 μL, with 2% parasitemia and 2% hematocrit in RPMI-1640 medium supplemented with 10% human serum and 60 μg/mL amikacin was added to the wells of a 96-well plate containing 10 μL of test samples at 15.87 μg/mL in duplicates and the percentage of inhibition was calculated relative to the negative and positive controls. The samples that showed % inhibition ≥ 50% were further proceeded to the second phase assay. In the second phase assay, the tested samples examined at 47.6, 15.87 and 5.29 μg/mL and the tested concentrations that afforded 50% inhibition of the protozoan relative to positive and negative controls (IC 50 ) against the chloroquinesensitive (D 6 ) and the chloroquine-resistant (W 2 ) strains were reported.
Concomitantly, all samples were tested against the VERO mammalian cell lines as an indicator of general cytotoxicity. The selectivity indices (SI) and the ratio of VERO IC 50 to D 6 or W 2 IC 50 were calculated. The standard antimalarial drug chloroquine (0.079 μg/mL) was used as the positive control and DMSO (0.25%) was used as a vehicle. All IC 50 were calculated using the XLfit curve (Makler and Hinrichs, 1993).

Evaluation of antileishmanial activity
The anti-leishmanial activity of the samples was screened against Leishmania donovani, a fly-borne protozoan that causes visceral leishmaniasis. The promastigotes were grown in RPMI 1640 medium supplemented with 10 % fetal calf serum (GibcoChem. Co.) at 26 ο C. A three-day-old culture was diluted to 5 x 10 5 promastigotes/mL. Drug dilutions were prepared directly in cell suspension in 96-well plates. Plates were incubated at 26 ο C for 48 h, and growth of Leishmania promastigotes was determined by the Alamar Blue TM assay. Standard fluorescence was measured on a Fluostar Galaxy plate reader (BMG Lab Technologies) at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.
Amphotericin B and Alpha-difluoromethylornithine were used as standard antileishmanial agents. IC 50 values were computed from the dose-response curve (Ma et al., 2004).

Evaluation of antitrypanosomal activity
Blood stage forms of Trypanosoma brucei were grown in IMDM medium supplemented with 10 % fetal bovine serum. The assays were set up in a clear 96 well microplates. A two days old culture of T. brucei in the exponential phase was diluted with IMDM to 5000 parasites/mL. For primary screening (Single concentration of 20 μg/mL in duplicates) samples dilutions (1 mg/mL) were prepared from the stock samples (20 mg/mL) in IMDM medium. Each well received 4 μl of the diluted sample and 196 μl of the culture volume (total culture volume 200 μl). The plates were incubated at 37 °C in 5 % CO 2 for 48 h. Alamar blue (10 μl) (ABD Serotec, catalog number BUF012B) was added to each well and the plates were further incubated overnight. Standard fluorescence was measured on a Fluostar Galaxy fluorometer (BMG LabTechnologies) at 544 nm excitation, 590 nm emission. α-difluoromethyl ornithine (DFMO) was tested as standard. The samples that have shown more than 90 % inhibition of T. brucei growth in primary screening were subjected to secondary screening for dose-response analysis (Jain et al., 2016).

Evaluation of antimicrobial activity
All organisms used for the biological evaluation were obtained from the American  intracellulare, A. fumigatus) using the Polarstar Galaxy plate reader (BMG LabTechnologies, Germany) prior to and after incubation. Percentage growth was plotted versus test concentration to afford the IC 50 (Samoylenko et al., 2009).

Results and discussion
The metabolomic analysis was performed according to Elsayed et al. 2018 Metabolomic profiling of Z. elegans and G. rigens revealed that the presence of various classes of metabolites in extracts and different fractions of both plants.
Coumarins, sesquiterpene lactones and phenylethanoids were detected in Z. elegans, whereas amides and phenolic acid derivatives were characteristic for G. rigens ( Figure 1). Identification of compounds was established using METLIN database (Table S1 and S2).
The LC-MS profiling of Z. elegans showed the mass ion peak at m/z 163.039 [M+H] + in agreement with the molecular formula C 9 H 6 O 3 which was dereplicated as umbelliferone, which was isolated formerly from Diplostephium foliosissimum (Morikawa et al. 2011). Furthermore, the mass ion peak at m/z 177.054 [M-H]for the predicted molecular formula C 9 H 6 O 4 was identified as esculetin that was isolated previously from Ligularia sagittal ) . The mass ion peak at m/z 245.117 [M-H]for the predicted molecular formula C 15 H 18 O 3 was identified as zaluzanin C that was isolated from Z. acerosa (Romo et al. 1971) Additionally, two phenolic acid derivatives were detected in G. rigens, of which one mass ion peak at m/z 515.119 [M-H]in agreement with the molecular formula C 25 H 24 O 12 was characterized as 3,5-di-O-caffeoylquinic acid that was formerly isolated from the same plant and G. longiscapa (Desoukey et al. 2016) (Oyugi et al. 2011).
Moreover, the results of the antimalarial activity of the examined samples revealed that the ethyl acetate fraction of Z. elegans exhibited a significant antimalarial activity of a 63% inhibition, followed by the petroleum ether fraction of the same plant, ethyl acetate fraction of G. rigens and the total ethanol extract of roots of Z. elegans (39, 28 and 25%, respectively). All the other tested extracts and fractions showed weaker activity (Table S3). In the secondary phase assay, the ethyl acetate fraction of Z. elegans showed an IC 50 value of 21.03 and 13.72 µg/mL against P. falciparum D 6 and P. falciparum W 2 , respectively. Furthermore, no cytotoxic activity against the VERO mammalian cell lines was observed up to the maximum dose tested 47.6 μg/mL, indicating the safety of the above mentioned plants (Table S4).
Additionally, the antileishmanial activity of the tested samples was determined against  (Table S5).