Evaluation of Zamia floridana A. DC. Leaves and Its Isolated Secondary Metabolites as Natural Anti-Toxoplasma and Anti-Cancer Agents Using In Vitro and In Silico Studies

Toxoplasmosis and cancer are life-threatening diseases with worldwide distribution. However, currently used chemosynthetic treatments are not devoid of their own intrinsic problems. Natural metabolites are gaining attention due to their lower side effects. In this study, we investigated for the first time Zamia floridana leaves extract and its different fractions for their toxoplasmocidal activity, using Virulent RH Toxoplasma gondii, and cytotoxic activity against MCF-7 and HCT-116 cancer cell lines using MTT assay. The n-butanol fraction was the most potent fraction against T. gondii with an EC50 of 7.16 ± 0.4 µg/mL compared to cotrimoxazole (4.18 ± 0.3 µg/mL). In addition, the n-BuOH fraction showed a significant cytotoxicity against MCF-7 and HCT-116 with IC50 of 12.33 ± 1.1 and 17.88 ± 1.4 µg/mL, respectively, compared to doxorubicin (4.17 ± 0.2 and 5.23 ± 0.3 µg/mL, respectively), with higher safety index against normal cell line (WISH). Therefore, the n-BuOH fraction was investigated for its phytochemicals using extensive chromatographic techniques, which led to the isolation of six compounds that were fully characterized using different spectroscopic techniques. Three biflavonoids (1, 2 and 4) in addition to two phenolic acid derivatives (3 and 5) and a flavonoid glycoside (6) were isolated. Compounds (1, 3, 5 and 6) were reported for the first time from Z. floridana. In silico docking studies for toxoplasmocidal and cytotoxic effects of these compounds revealed that compounds (1, 2, 4 and 6) have promising inhibition potential of either thymidylate synthase-dihydrofolate reductase (TS-DHFR) or cyclin dependent kinase 2 (CDK2) target proteins. This study is considered the first report of chemical and biological investigation of Z. floridana leaves.


Introduction
Gymnospermous plants have been documented since 300 BC [1]. The order of cycadales is one of the largest groups of living gymnosperms. It is commonly referred to as the cycads. Cycadaceae and Zamiaceae are the most important families in this order due to their large number of species and wide range of biological activities such as Cycas revoluta, that has cytotoxic and antioxidant activities and Cycas rumphii, which was previously reported as a natural source for toxoplasmocidal and cytotoxic agents [2,3]. In addition, various biological effects have been reported for different species of Zamiaceae such as the antimicrobial effect of Dioon spinulosum and the antileishmanial activity of Zamai lindenii [4,5]. The genus Zamia was found to exert a wide range of significant biological effects due to their high content of biflavonoids, flavonoids, lignans, phenolic acids, fatty acids, sterols and amino acids [6][7][8][9]. Zamia floridana A. DC. is one of many Zamia species that belong to

Plant Material
Leaves of Z. floridana A. DC. were collected from El-Abd Garden at 68 kilos from desert Cairo-Alexandria Road in July 2018. It was kindly provided and identified by researcher Rabea Sharawy Agronomist and palm researcher. A voucher sample (No. PGG-013) was deposited at the herbarium of Faculty of Pharmacy, Tanta University, Egypt.

Extraction and Isolation
The extraction, fractionation and isolation steps are shown in the supplementary material ( Figures S1 and S2). The plant material was dried in the shade, reduced to powder, and stored in tightly closed containers. The plant powder (3.4 kg) was extracted with methanol by cold maceration till exhaustion. The total methanol extract was evaporated under reduced pressure at 40 • C to yield a green residue (181.25 g). Methanol extract residue (161.32 g) was suspended in 50% aqueous methanol (750 mL) and successively fractionated with petroleum ether (40-60 • C), chloroform (CHCl 3 ), ethyl acetate (EtOAc), and n-butanol (n-BuOH) to yield 23.72 g, 4.28 g, 5.83 g and 42.10 g, respectively.
The n-BuOH fraction (42.10 g) was suspended in a deionized water and applied to Diaion HP-20 column (Φ 5 cm × 28 cm, 200 g). The column was first eluted with (4 L) of deionized water followed by (2 L) 100% MeOH. The methanol fraction was concentrated to give a brown residue (3.3 g) to be used for biological screening and chromatography separation. A silica gel column (Φ 2 cm × 48 cm, 82 g) was used to isolate the components using a gradient elution, starting with 100% CHCl 3 and the polarity was increased using MeOH. Fractions (10 mL) were collected and similar fractions on TLC were combined to afford five groups of fractions (F1 to F5). F1 eluted with CHCl 3 :MeOH (95:5) gave a yellow colored residue (172.2 mg) which was chromatographed further on a silica gel column (Φ 1 cm × 17 cm, 6 g) using a gradient elution of CHCl 3 and MeOH to obtain two subfractions F1-1 to F1-2. F1-1 eluted with CHCl 3 :MeOH (97:3) gave a yellow colored residue (84.1 mg) was re-chromatographed on a Sephadex LH-20 column (Φ 1.5 cm × 25 cm, 20 g) using MeOH (HPLC grade) to give compound (1) (9.1 mg).

Toxoplasmocidal Activity
A virulent RH strain of T. gondii was supplied by the Medical Parasitology Department of the Faculty of Medicine (Alexandria University, Egypt) for this experiment. According to the method reported by Kavitha et al., 2012, different concentrations of the total methanol extract of Z. floridana leaves and its different fractions were tested for toxoplasmocidal activity [16]. The mean effective concentration (EC 50 ) was calculated as µg/mL and compared to that of cotrimoxazole as a positive control drug.
Using MTT assay method, the cytotoxicity assay was carried out in accordance with the reported procedures [17][18][19][20]. Seven different concentrations (1.56, 3.125, 6.25, 12.5, 25, 50 and 100 µg/mL) of Z. floridana total methanol extract dissolved in DMSO, were tested against the investigated cancer cell lines as well as one normal cell line (WISH) to test the safety of the plant extract on the normal cells. Then, the most affected cell lines were incubated with different concentrations of petroleum ether, CHCl 3 , EtOAc and n-BuOH fractions using doxorubicin as reference drug. IC 50 was calculated and the cytotoxic potency was assessed according to the classification of Hossan and Abu Melha, 2014 [21].

In Silico Molecular Docking Studies
Molecular docking studies (by MOE 2020.9010 version) were carried out to show the binding mode and interactions of the isolated molecules (1-6) (by Discovery Studio (DS) visualizer program). The Protein Data Bank was used to obtain the crystal structure of TS inhibitor in association with T. gondii TS-DHFR, which has a resolution of 2.79 Å (PDB ID: 4KY4) [22] and CDK2 in association with inhibitor, which has a resolution of 2.20 Å (PDB ID: 1FVT) [23]. We chose a single chain (A) pre-docked with its unique ligand "2-amino-5-(phenylsulfanyl)-3,9-dihydro-4H-pyrimido [4,5-b]indol-4-one" (1UE) and "4-[(2Z)-2-(5bromo-2-oxo-1,2-dihydro-3H-indol-3-ylidene) hydrazinyl]benzene-1-sulfonamide" (106) for TS-DHFR and CDK2, respectively. Both hydrophobic and hydrophilic amino acids were found in the ligand-binding site of the relevant enzymes. At the active sites, (1UE and 106) exhibited both hydrophilic and hydrophobic interactions. The redocking method for the ligands (1UE and 106) was performed with the goal of validating the docking protocol by creating numerous docked poses, one docked pose for each ligand had an RMSD value less than 1 (i.e., 0.8118 and 0.9501 Å for 1UE and 106, respectively), thus confirming the docking procedure. The molecular docking investigation demonstrated that all of the compounds tested fit well into the enzymes' active pockets. Furthermore, based on the results of the binding free energy calculation, the most promising docked conformations of each isolate were analyzed further for binding mode analysis.

Statistical Analysis
All experiments were carried out at least three times, the data are expressed as the mean ± standard error of the mean (SEM).

Toxoplasmocidal Activity
Z. floridana leaves' total methanol extract and its different fractions were screened for toxoplasmocidal activity against T. gondii RH strain tachyzoites. The relative mortality of the parasite incubated with different concentrations of the tested extracts was assessed using trypan blue dye. The results revealed that Z. foridana showed a potent toxoplasmocidal activity with an EC 50 of 8.19 µg/mL compared to that of cotrimoxazole standard drug (EC 50 of 4.18 ± 0.3 µg/mL). Moreover, the n-BuOH fraction showed the highest toxoplasmocidal activity followed by the EtOAc fraction then the CHCl 3 fraction and finally the pet-ether fraction with EC 50 of 7.16 ± 0.4, 9.74 ± 0.5, 16.71 ± 0.8 and 31.95 ± 1.3 µg/mL, respectively. ( Figure 1, Supplementary Material Table S1) inhibitor in association with T. gondii TS-DHFR, which has a resolution of 2.79 Å (PDB ID: 4KY4) [22] and CDK2 in association with inhibitor, which has a resolution of 2.20 Å (PDB ID: 1FVT) [23]. We chose a single chain (A) pre-docked with its unique ligand "2-amino-5-(phenylsulfanyl)-3,9-dihydro-4H-pyrimido [4,5-b]indol-4-one" (1UE) and "4-[(2Z)-2-(5bromo-2-oxo-1,2-dihydro-3H-indol-3-ylidene) hydrazinyl]benzene-1-sulfonamide" (106) for TS-DHFR and CDK2, respectively. Both hydrophobic and hydrophilic amino acids were found in the ligand-binding site of the relevant enzymes. At the active sites, (1UE and 106) exhibited both hydrophilic and hydrophobic interactions. The redocking method for the ligands (1UE and 106) was performed with the goal of validating the docking protocol by creating numerous docked poses, one docked pose for each ligand had an RMSD value less than 1 (i.e., 0.8118 and 0.9501 Å for 1UE and 106, respectively), thus confirming the docking procedure. The molecular docking investigation demonstrated that all of the compounds tested fit well into the enzymes' active pockets. Furthermore, based on the results of the binding free energy calculation, the most promising docked conformations of each isolate were analyzed further for binding mode analysis.

Statistical Analysis
All experiments were carried out at least three times, the data are expressed as the mean ± standard error of the mean (SEM).

Toxoplasmocidal Activity
Z. floridana leaves' total methanol extract and its different fractions were screened for toxoplasmocidal activity against T. gondii RH strain tachyzoites. The relative mortality of the parasite incubated with different concentrations of the tested extracts was assessed using trypan blue dye. The results revealed that Z. foridana showed a potent toxoplasmocidal activity with an EC50 of 8.19 µg/mL compared to that of cotrimoxazole standard drug (EC50 of 4.18 ± 0.3 µg/mL). Moreover, the n-BuOH fraction showed the highest toxoplasmocidal activity followed by the EtOAc fraction then the CHCl3 fraction and finally the pet-ether fraction with EC50 of 7.16 ± 0.4, 9.74 ± 0.5, 16.71 ± 0.8 and 31.95 ± 1.3 µg/mL, respectively. (Figure 1, Supplementary Material Table S1)  Table S4). Amongst tested fractions, the EtOAc and the n-BuOH fractions showed highest cytotoxic potential against MCF-7 and HCT-116 cell lines with IC 50 of 22.89 ± 1.8 and 9.04 ± 0.8 µg/mL, respectively, for the EtOAc fraction and IC 50 of 12.33 ± 1.1 and 17.88 ± 1.4 µg/mL, respectively, for the n-BuOH fraction. (Figure 2, Supplementary Material Table S5).
viability under the effect of the different tested concentrations is shown in the Supplementary Material Tables S2 and S3. The results showed that total methanol extract of Z. floridana has a cytotoxic potential against MCF-7 and HCT-116 cell lines with IC50 of 20.57 ± 1.7 and 27.33 ± 2.3 µg/mL, respectively, compared to that of doxorubicin as a positive control drug (IC50 of 4.17 ± 0.2 and 5.23 ± 0.3 µg/mL). Interestingly, Z. floridana methanol extract showed a low cytotoxicity effect against normal cell line (WISH) with an IC50 of 40.29 ± 3.2 µg/mL (Supplementary Material Table S4). Amongst tested fractions, the EtOAc and the n-BuOH fractions showed highest cytotoxic potential against MCF-7 and HCT-116 cell lines with IC50 of 22.89 ± 1.8 and 9.04 ± 0.8 µg/mL, respectively, for the EtOAc fraction and IC50 of 12.33 ± 1.1 and 17.88 ± 1.4 µg/mL, respectively, for the n-BuOH fraction. (Figure 2, Supplementary Material Table S5).

Phytochemical Investigation
The n-BuOH fraction was subjected to several chromatographic columns to separate six compounds (1-6) (

Phytochemical Investigation
The n-BuOH fraction was subjected to several chromatographic columns to separate six compounds (1-6) ( Compound (1) was isolated as an amorphous yellow powder. It gave a yellow color with 5% AlCl3 and a UV λmax at 222, 271 and 328 nm, which suggested that compound (1) is a flavonoid. The IR spectrum showed a strong band at 3415 cm −1 for phenolic hydroxyl

Identification of the Compounds (1-6)
Compound (1) was isolated as an amorphous yellow powder. It gave a yellow color with 5% AlCl 3 and a UV λ max at 222, 271 and 328 nm, which suggested that compound (1) is a flavonoid. The IR spectrum showed a strong band at 3415 cm −1 for phenolic hydroxyl (OH) groups stretching and at 1644 cm −1 for a carbonyl (C=O) group. The 1 H-NMR spectrum of compound (1) proposed a biflavonoid structure consisting of two units (I and II). The 1 H-NMR spectrum of compound (1) showed an AA'BB' coupling system of the para substituted ring B of unit II at δ H 6.92 (2H, brs, H-3 , H-5 ) and 7.56 (2H, brs, H-2 , H-6 ). In addition, the 1 H-NMR spectrum showed an ABX coupling system at δ H 7.33 (1H, brs, H-5 ), 7.98 (1H, brs, H-2 ) and 8.09 (1H, brs, H-6 ) of ring B of unit I indicating that C-3 was the position of linkage of the two flavonoid units. Signals for the two meta-coupled protons at δ H 6.22 (1H, brs, H-6) and 6.45 (1H, brs, H-8) were ascribed to ring A of unit I. The DEPT-Q NMR of compound (1) showed a downfield shift for C-3 , C-8 signals at δ DEPT-Q 121.6 and 104.2, respectively compared to the apigenin 13 C-NMR spectral data [24]. The 1 H-NMR signal at δ H 6.40 (1H, s, H-6 ) indicated that there is no meta coupling between H-6 , H-8 , all of these signals support the interflavonoid linkage between C-3 and C-8 . Therefore, compound (1)  Compound (2) was isolated as an amorphous yellow powder. It gave a yellow color with 5% AlCl 3 and a UV λ max at 234, 270 and 330 nm, which suggested that compound (2), is a flavonoid compound. The IR spectrum showed a strong band at 3417 cm −1 for phenolic hydroxyl (OH) groups stretching and at 1643 cm −1 for a carbonyl (C=O) group. The 1 H NMR spectrum of compound (2) showed the pattern of a biflovonoid pattern as in compound (1) (2) showed a downfield shift for C-3 , C-8 signals at δ APT 122.0 and 103.8, respectively, compared to the apigenin 13 C-NMR spectral data [24]. The 1 H NMR signal at δ H 6.37 (1H, s, H-6 ) indicated that there is no meta coupling between H-6 , H-8 , these signals support the interflavonoid linkage between C-3 and C-8 . Therefore, a 3 , 8 biapigenin structure was proposed for compound (2). Signals at δ H 3.82 (3H) and at δ APT 55.0 were also observed indicating a methoxy group. The location of the methoxy group was confirmed at C-4 due to the upfield shifts of ∆δ 4.6 ppm at C-5 (δ APT 111.1) and the downfield shift of ∆δ 2 ppm at C-1 (δ APT 122.9) compared to the apigenin 13 C NMR spectral data [24]. The ESIMS of (2) showed a pseudo molecular ion at m/z 575.4 for [M + Na] + and 551.1 for [M − H] − suggesting a molecular formula for (2) as C 31 H 20 O 10 which is consistent with an amentoflavone methoxy derivative. The IR spectrum of compound (2) was found identical to an authentic sample of bilobetin ( Figure S8B). By comparing our data to those reported in the literature [2,24,27,28], compound (2) was identified as amentoflavone 4 -O-methyl ether (bilobetin).
Compound (3) was isolated as an amorphous white powder. It gave a blue color with FeCl 3 spray reagent and a UV λ max at 234 and 260 nm suggesting that compound (3) has a phenolic acid nucleus. The 1 H NMR spectrum of compound (3) showed a typical signal for two symmetric aromatic protons at δ H 7.54 (2H, s, H-2, H-6) which suggested that this compound has 1,3,4,5-tetra-substituted aromatic ring. Another signal at δ H 3.72 integrating for 6 carbons (6H, s, 3, 5-OCH 3 ) indicated the presence of two methoxy groups in this compound. The APT-NMR spectrum showed the presence of two equivalent olefinic methine carbons and two equivalent methoxy carbons at δ APT (108.8, 55.6), respectively. Additionally, the APT-NMR spectrum showed five quaternary carbon signals including three oxygenated olefinic carbons two of them are equivalent at δ APT (147.3, 140.0) and were assigned to C-3, 5 and C-4, respectively. Another signal at δ APT (168.5) indicated the presence of carboxyl carbon (C-7) and a signal at δ APT (120.0) for the aromatic carbon C-1. The ESIMS of compound (3) showed a pseudo molecular ion at m/z 199.1 for [M + H] + with a molecular formula C 9 H 11 O 5 , 197.1 for [M − H] − , which is consistent with syringic acid. All of these spectral data were identical to those previously reported of syringic acid [29][30][31]. This is the first report of syringic acid from Z. floridana.
Compound (4) was obtained as an amorphous yellow powder. It gave a yellow color with 5% AlCl 3 and UV λ max at 232, 274 and 329 nm suggesting that compound (4) is a flavonoid structure. The IR spectrum showed a strong band at 3417 cm −1 for phenolic hydroxyl (OH) groups stretching and at 1651 cm −1 for a carbonyl (C=O) group. The APT NMR analysis showed signals for 30 carbons, including two carbonyl group signals at δ APT 182.3 and 182.7 of (C-4, C-4 , respectively). These signals suggest that compound (4) is a biflavonoid. The 1 H NMR data showed typical signals for AA'BB' coupling pattern at δ H 7.39 (2H, d, J = 8 Hz, H-2 , 6 ) and 6.59 (2H, d, J = 8 Hz, H-3 , 5 ), which suggested the presence of 1, 4-disubstituted benzene ring B of unit II and typical signals for an ABX coupling system at δ H 6.97 (1H, d, J = 8 Hz, H-5 ), 7.73 (1H, brd, J = 8 Hz, H-6 ) and 7.85 (1H, brs, H-2 ) of ring B of unit II suggesting that C-3 was the position of linkage of the two flavonoid units. Signals at δ H 6.06 (1H, brs, H-6) and 6.29 (1H, brs, H-8) indicated a meta coupling of H-6, H-8 of ring A of unit I. Additionally, only one aromatic proton singlet at δ H 6.24 was assigned to H-6 with the absence of the proton signal for C-8 suggested that C-8 is involved in the interflavonoid linkage. These proton signals in addition to the downfield shift of C-3 and C-8 which appeared at δ APT 120.3 and 104.0, respectively, compared to the apigenin 13 C-NMR spectral data [24], suggested that C-3 and C-8 were involved in the linkage between the two flavonoids moieties of the biflavonoid structure which is consistent with amentoflavone in the literature [24,32]. The ESIMS of (4) showed a pseudo molecular ion at m/z 561.4 for [M + Na] + with a molecular formula C 30 H 18 O 10 Na, and at m/z 537.1 for [M − H] − with a molecular formula C 30 H 17 O 10 suggesting a molecular formula for compound (4) as C 30 H 18 O 10 , which matches amentoflavone. The IR spectrum of compound (4) was found identical to an authentic sample of amentoflavone ( Figure S15B). The spectral data of compound (4) was identical to those reported in the literature for amentoflavone [2,24,32].
Compound (5) was isolated as an amorphous white powder. It gave a blue color with FeCl 3 spray reagent and a UV λ max at 227 and 266 nm suggesting that compound (5) has a phenolic acid nucleus. The IR spectrum indicated the presence of a carboxylic group through a strong band at 3494 cm −1 , hydroxyl phenolic groups at 3414 and 3285 cm −1 , a carbonyl group at 1645 cm −1 . The 1 H NMR spectrum of compound (5) showed a singlet integrating for two protons of two similar methine carbons in the aromatic range at δ H 6.93 which suggested that this compound has 1,3,4,5-tetra-substituted aromatic ring similar to compound (3). The APT-NMR spectrum showed five signals for seven carbons including a carbonyl carbon at δ APT 167.6 (C=O), 3 oxygenated quaternary carbons at δ APT 145.1 (C-3, 5) and 138.4 (C-4), a signal for two equivalent methine carbons at δ APT 108.6 (C-2, 6) and another quaternary carbon signal at δ APT 120.0 (C-1). This pattern proposed 3, 4, 5-trihydroxy benzoic acid which is known as gallic acid. The ESIMS of compound (5) showed a pseudo molecular ion at m/z 171.1 for [M + H] + with a molecular formula C 7 H 7 O 5 , and at m/z 169.1 for [M − H] − with a molecular formula C 7 H 5 O 5 suggesting a molecular formula for compound (5) as C 7 H 6 O 5 , which is consistent with gallic acid. All of these spectral data were identical to the previous literature of gallic acid [33][34][35]. This is the first report of gallic acid from Z. floridana.
Compound (6) was isolated as an amorphous yellow powder. It gave a yellowish green color with 5% AlCl 3 spray reagent and brown color with 10% H 2 SO 4 spray reagent and a UV λ max at 241, 265 and 322 nm suggesting that compound (6) is a flavonoid glycoside. The IR spectrum showed a strong band at 3416 cm −1 for phenolic hydroxyl (OH) groups stretching and at 1652 cm −1 for a carbonyl (C=O) group. The 1 H NMR data showed typical signals for an AA'BB' coupling pattern at δ H 7.99 (2H, d, J = 8 Hz, H-2 , 6 ) and 6.95 (2H, d, J = 8 Hz, H-3 , 5 ), which suggested the presence of 1, 4-disubstituted benzene ring B. In addition, a singlet at δ H 6.64 (1H, s, H-3) was observed in the 1 H-NMR spectrum, which indicated that compound (6) is a flavone. The appearance of two anomeric doublets signals at δ H 5.01 (d, J = 9.6 Hz, Glu H-1 , 1H) and 5.05 (d, J = 9.6 Hz, Glu H-1 , 1H) also, the presence of other sugar signals in the range of δ H 3.44-4.12 suggested the presence of two sugar moieties. In addition, the absence of the proton signals for H-6 and H-8 suggested that the two sugar moieties are linked to C-6 and C-8. The APT-NMR spectrum showed signals for 27 carbons including 12 signals for the two sugars moieties. The two anomeric carbons were assigned at δ APT 74.8 and 73.7 through their correlation to signals at δ H 5.01 and 5.05 in the HSQC spectrum, which suggested the C-glycosidic linkage of these two sugars molecules. The other sugar resonances were identical to glucose moiety [36]. The aglycon carbon signals were assigned with using the HSQC and HMBC experiments which are identical to that of an apigenin moiety [24]. However, the downfield shift of C-6 and C-8 of compound (6) compared to that of apigenin supported that these two carbons are linked to sugars moieties. The HMBC correlation data confirmed the linkage of the two glucose moieties at C-6 and C-8 through the correlation of an anomeric proton signal at δ H 5.01 and δ C 108.3 (C-6), 159.1 (C-5), 104.6 (C-10), while the other anomeric proton signal at δ H 5.05 was correlated to δ C 104.7 (C-8) and 156.2 (C-9). The ESI-MS of (6) 15 . The configuration of glucose at the glycosidic bonds was determined as β based on the large J value for the anomeric protons (9.6 Hz) and by comparing the resonances of the carbons and protons, the HSQC and the HMBC correlations to the published data of vicenin-2 [37,38]. Based on these data, compound (6) was identified as apigenin 6, 8-di-C-β-D glucoside (vicenin-2). This is the first report of vicenin-2 from Z. floridana.

Investigation of the Toxoplasmocidal Effect of Compounds (1-6) via In Silico Studies
In silico molecular docking studies were carried out for these pure compounds to study their possible toxoplasmocidal and cytotoxic mechanisms. T. gondii has a number of viable targets that can be inhibited by several drugs [39,40]. Thymidylate synthasedihydrofolate reductase (TS-DHFR) was selected as a target for drugs that can eradicate this parasite [41][42][43] and 1UE ligand (the co-crystalized ligand inside active site) was chosen as a positive control compound. According to what was previously published, we found that there is a direct relationship between the inhibition of cyclin dependent kinase 2 (CDK2) and flavonoids in cancer therapy [44][45][46][47]. Thus, we selected (CDK2) as a target protein for a molecular docking cytotoxic evaluation in comparison to the ligand (106) (the co-crystalized ligand inside active site) as a positive control compound. Docking results, binding modes, and the interactions of pure compounds isolated from Z. floridana n-BuOH fraction with the critical amino acids in the active site of TS-DHFR (PDB ID: 4KY4) and CDK2 (PDB ID: 1FVT) are recorded in Tables 1 and 2  Phenolic ring (cent.) Ile402 Phenolic ring (term.)

Discussion
This study reports an in vitro assessment for the potential toxoplasmocidal and cytotoxic activities of Z. floridana leaves for the first time. The results revealed that the methanol extract of Z. floridana leaves showed a significant toxoplasmocidal effect against T. gondii tachyzoites, however is less potent compared to a cotrimoxazole drug. Therefore, its different fractions were also tested, and the results showed that n-BuOH fraction was the most potent fraction against T. gondii but less potent than cotrimoxazole. In addition, Z. floridana methanol extract showed moderate cytotoxic activity against MCF-7 and HCT-116 according to the classification of Hossan and Abu Melha, 2014 [21], compared to a doxorubicin drug as a positive control. Interestingly, the total methanol extract showed more selectivity to cancer cells rather than normal cells than doxorubicin. The different fractions of Z. floridana extract were tested against the most affected cell lines (MCF-7 and HCT-116). The results showed that the EtOAc and n-BuOH fractions were the most potent fractions against the tested cell lines. The EtOAc fraction of Z. floridana has very strong cytotoxic activity against HCT-116 and moderate cytotoxic activity against MCF-7, while n-BuOH fraction showed strong cytotoxic activity against the two tested cell lines. Based on this biological evaluation, n-BuOH fraction is the most potent fraction amongst the tested plant extracts but less potent than doxorubicin. Thus, this motivated us to investigate this fraction for its phytochemicals that may be responsible for these biological activities. The phytochemical investigation of n-BuOH fraction of Z. floridana led to the isolation of six compounds identified as isoginkgetin, bilobetin, syringic acid, amentoflavone, gallic acid, and vicenin-2. Four compounds of them were isolated for the first time from Z. floridana leaves. These compounds were tested previously in other studies against breast and colon cell lines in vitro and they showed cytotoxic effects [2,[48][49][50][51][52][53][54][55][56][57], which could account for the cytotoxic effect of the n-butanol fraction of Z. floridana. The potent toxoplasmocidal effect of n-BuOH fraction encouraged us to investigate and predict the possible mechanisms of these compounds to inhibit T. gondii using in silico molecular docking study. The results indicated that compound (2) "bilobetin" followed by compound (6) "vicenin-2", then compound (1) "isoginkgetin", and finally compound (4) "amentoflavone", had the highest binding affinity to the target protein compared to (1UE). According to the docking scores of bilobetin, isoginkgetin and amentoflavone (−8.95, −8.54 and −7.63 kcal/mol), respectively, the presence of one methoxy group in bilobetin at the central phenyl ring at position 4 and two methoxy groups in isoginkgetin increase binding affinity over the presence of a hydroxyl group amentoflavone at that position. Where the bilobetin methoxy group makes hydrophobic interaction (with Trp403) and isoginkgetin methoxy groups make hydrophobic interaction (with Leu486 and Met608), but the amentoflavone hydroxyl group did not undergo. Additionally, one of the reasons for the high value of the docking score for bilobetin is that it makes twice the number of hydrogen bonds (6 H-bonds with Lys371, Asn406, Gln509, Asp513, Ala609, and Val610) that isoginkgetin and amentoflavone do (3 H-bonds for each), as shown in Figure 4. Additionally, "vicenin-2" made three hydrogen bonds through hydroxyl groups and oxygen atom in the two glucopyranose rings, which proves the importance of the glucopyranose rings [58]. Further, the phenolic and flavone rings of vicenin-2 had seven hydrophobic interactions with Lys371, Phe374, Ile402, Leu516, Phe520, Arg603, and Met608, as shown in Figure 4. Additionally, the previously published data of cytotoxic activities of the isolated six compounds from n-BuOH fraction of Z. floridana leaves motivated us to study and predict the possible anticancer mechanism of these compounds. Thus, we performed a molecular docking study for these isolates. The docking results revealed that compound (6) "vicenin-2" followed by compound (1) "isoginkgetin", then compound (4) "amentoflavone", and finally compound (2) "bilobetin", had the highest binding affinity to the target protein compared to the ligand (106). The ability of vicenin-2 (docking score, −8.38 kcal/mol) to form six hydrogen bonds with four residues (His84, Asp86, Lys89, and Asp145), three of them interacting with the hydroxyl groups in one glucopyranose ring and one hydrogen bond with the phenolic ring, may be the reason for its excellent inhibitory activity [58]. Additionally, Ile10, Val18, Ala31, Phe80, Leu134, and Ala144 exhibited hydrophobic interactions with the phenolic and flavone rings ( Figure 5). The almost similar docking scores of isoginkgetin, amentoflavone, and bilobetin (−7.62, −7.60 and −7.58 kcal/mol, respectively), is due to their ability to make from two to four hydrogen bonds and hydrophobic interactions with at least four amino acids ( Figure 5). Moreover, these computed binding energy values also confirm and support the in vitro results of the n-BuOH effectivity against T. gondii and the different cancer cell lines tested.

Conclusions
In conclusion, the biological screening for Z. floridana methanol extracts and its different fractions indicated that although it is less potent than the control drugs, n-BuOH fraction has noticeable toxoplasmocidal and cytotoxic activities against two different cell lines. Therefore, the phytochemical investigation of the n-BuOH fraction of Z. floridana leaves was carried out and resulted in the isolation of six compounds, four of them were isolated for the first time from Z. floridana leaves. Various spectroscopies were used to identify these chemicals, and the results were compared to published data. An in silico molecular docking study was used to study the possible toxoplasmocidal and cytotoxic

Conclusions
In conclusion, the biological screening for Z. floridana methanol extracts and its different fractions indicated that although it is less potent than the control drugs, n-BuOH fraction has noticeable toxoplasmocidal and cytotoxic activities against two different cell lines. Therefore, the phytochemical investigation of the n-BuOH fraction of Z. floridana leaves was carried out and resulted in the isolation of six compounds, four of them were isolated for the first time from Z. floridana leaves. Various spectroscopies were used to identify these chemicals, and the results were compared to published data. An in silico molecular docking study was used to study the possible toxoplasmocidal and cytotoxic mechanisms of these isolated compounds. The results showed that among all compounds, compounds (1, 2, 4, and 6) have the highest docking score. Future research is required to assess these actions in vivo.  Table S4: Cytotoxic effect of Z. floridana methanol extract against different cell lines; Table S5: Cytotoxic effect of Z. floridana different fractions against MCF-7, HCT-116 and WISH cell lines; Figure S1: Extraction and fractionation steps of Z. floridana leaves; Figure S2: Isolation steps of six pure compounds from Z. floridana n-BuOH fraction; Figure S3: UV spectrum of compound (1) in MeOH; Figure S4: IR spectrum of compound (1) in KBr disc; Figure S5