Metabolomics-Based Profiling of Clerodendrum speciosum (Lamiaceae) Leaves Using LC/ESI/MS-MS and In Vivo Evaluation of Its Antioxidant Activity Using Caenorhabditis elegans Model

We investigated the antioxidant activity of the total methanol extract of C. speciosum leaves (CST), the ethyl acetate (CSE), and the remaining aqueous (CSR) fractions in vitro, in vivo using Caenorhabditis elegans model, and in silico. LC-ESI-MS/MS analysis was employed for metabolic profiling of CST. ADME/TOPAKT prediction was performed to determine the potential pharmacokinetic, pharmacodynamic, and toxicity properties of the major identified phytoconstituents. All examined samples showed considerable antioxidant activity where CST, CSE, and CSR displayed EC50 values of 27.1, 16.2, and 21.3 µg/mL, respectively, in 2,2-diphenyl-1-picrylhydrazyl (DPPH•) assay, whereas they showed 11.44, 16.27, and 12.16 Fe2+ equivalents/mg of sample, respectively, in ferric reducing antioxidant power (FRAP) assay. CST, CSE, and CSR displayed total phenolic content of 262, 326, and 289 mg GAE/g sample, respectively. In vivo antioxidant study revealed that CST at 150 μg/mL increased the survival rate of C. elegans by 71.88% compared to untreated group. Regarding intracellular reactive oxygen species (ROS), worms treated with 150 μg/mL of CSE exhibited 60.42% reduction of ROS compared to the untreated group. Quantitation of hsp-16.2/GFP expression in Caenorhabditis elegans showed that worms treated with 150 μg/mL of CSR exerted 40.43% reduction in fluorescence with respect to the untreated group. LC-ESI-MS/MS of CST revealed the presence of sixteen secondary metabolites belonging mainly to polyphenolics with phenyl propanoids constituting the major detected class. The in silico study showed that rosmarinic acid displayed the best fitting within the active sites of Daf-2 protein with considerable safety profile and limited pharmacokinetic and pharmacodynamic that could be slightly enhanced by certain treatment.


Introduction
Oxidative stress occurs due to a marked imbalance between the amount of free radicals and natural antioxidants inside the human body that ultimately leads to cell and tissue damage. During metabolic processes, the body normally produces free radicals that are

Preparation of the Extract and Different Fractions of C. speciosum Leaves
A total of 2 kg of C. speciosum leaves were air-dried and crushed into coarse powder to give 200 g. They were macerated in distilled methanol (3 L × 3) followed by their filtration. Consequently, the filtrate was completely evaporated under reduced pressure at 45 • C using Rotary evaporator (Heidolph, Germany) until complete dryness and is followed by lyophilization to give 19.43 g of dried total methanol extract (CST). The lyophilized powder was re-dissolved in 70% methanol for fractionation using 3 L of n-hexane, 1 L of dichloromethane, and 1 L of ethyl acetate successively. Drying of the obtained fractions was performed under reduced pressure at 45 • C to give 4.2 g of n-hexane, 1.5 g of dichloromethane, and 4.75 g of ethyl acetate (CSE) dried fractions meanwhile the remaining hydro methanol fraction (CSR) weighed about 7.48 g.

Determination of Total Phenolic Content of the Extract and Different Fractions of C. speciosum Leaves
Total phenolic content was determined for CST, CSE and CSR using Folin-Ciocalteu method that was previously reported by Zhang et al. [13]. Briefly, 20 µL of each of the tested samples (5 mg/mL) were used in this assay and are used to fill a 96-well microplate followed by the addition of 100 µL of Folin-Ciocalteu reagent. Then, the tested samples were maintained at room temperature for 5 min, subsequently followed by the addition of 80 µL of a 7.5% sodium carbonate solution and mixed well. Finally, the plate was maintained for 2 h at room temperature in the dark followed by the determination of absorbance using Biochrom Asys UVM 340 Microplate Reader at 750 nm. Meanwhile, gallic acid was serially diluted in concentration range (0-200 µg/mL) to be used as a standard for the construction of the calibration curve. The total phenolic content of the examined samples was expressed in terms of gallic acid equivalent (GAE)/g sample.  1-picrylhydrazyl) radical scavenging activity of the tested samples was evaluated employing the standard method previously reported by Blois [14] with certain modifications using a 96-well microplate. Briefly, 100 µL of DPPH solution (200 µM) were added to 100 µL within the concentration range (1.25-50 µg/mL). Incubation of the samples was performed at room temperature for 30 min and consequently the absorbance was measured at 517 nm by Tecan Safire II TM spectrophotometer. The % of DPPH scavenging capacity was calculated using the following equation: DPPH scavenging effect (%) = [(A0 − A1)/A0] × 100 A0 and A1 represent the absorbance of the negative control and the tested sample, respectively. The measurements were performed in triplicates where EC 50 value was determined by sigmoid non-linear regression employing the GraphPad Prism version 5 software (GraphPad Software, Inc., La Jolla, CA, USA).

Ferric Reducing Antioxidant Power (FRAP) Assay
Ferric reducing antioxidant assay was done as previously reported by Benzie et al. [15] with certain modification to a 96-well microplate. The assay relied on the tendency of the tested samples to exert reduction of the ferric complex (2,4,6-tripyridyl-s-triazine-Fe 3+ -TPTZ) to ferrous form (Fe 2+ -TPTZ) when experiencing low pH. Briefly, the FRAP working solution is prepared by mixing of 300 mM acetate buffer (pH 3.6), 20 mM FeCl 3 .6 H 2 O, and 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mM HCl with a ratio of 10:1:1 before analysis. Warming of the fresh FRAP working solution was performed for 30 min at 37 • C before the assay. Meanwhile, FeSO 4 .7H 2 O was used as a standard. Then, an addition of 175 µL of freshly prepared FRAP working solution to 25 µL of the prepared samples was performed at 37 • C and maintained for 7 min. Absorbance was measured at 595 nm by Tecan Safire II TM spectrophotometer where reduction is evidenced by the appearance of intense blue color. All the measurements were performed in triplicates and results were expressed as Fe 2+ equivalents/mg of sample.
2.5. In Vivo Antioxidant Determination of the Extract and Different Fractions of C. speciosum Leaves Using C. elegans 2.5.1. C. elegans Strains and Culture Conditions Two types of nematodes, namely, the transgenic strain TJ375 (hsp-16.2/GFP) and the wild type N2 strains, were obtained from the Caenorhabditis Genetic Center (CGC) and were kept under standard conditions. They were maintained at 20 • C on nematode growth medium (NMG) inoculated with living Escherichia coli (OP50). All utilized worms were age-synchronized and this was achieved via the treatment of gravid adults with sodium hypochlorite where the eggs were permitted to hatch in M9 buffer and subsequently the obtained larvae was transmitted to S-medium supplemented with living Escherichia coli (OP50) [16].

Survival Assay of C. elegans Using Juglone Induced Oxidative Stress
Juglone at a concentration (80 µM) was used to induce oxidative stress in synchronized worms which are N2 strain as well as L1 larvae stage that were brought up in S-media supplemented with living E. coli OP50 as a food source at 20 • C. These worms were subsequently treated with 50, 100, and 150 µg/mL of CGT, CSE, and CSR for 48 h except the control group that received no treatment. Meanwhile, epigallocatechin gallate (EGCG) at a concentration of 100 µg/mL was used as a positive control. Consequently, addition of 80 µM of juglone was performed to elicit oxidative stress followed by counting the survivors after 24 h. The survival rate was calculated as percentage of the live worms using one-way ANOVA and subsequently followed by Bonferroni (post-hoc) correction [17].

Assessment of Reactive Oxygen Species (ROS) in C. elegans
Synchronized worms, N2 strain, and L1 larvae stage, that were brought up in S-media supplemented with living E. coli OP50 as a food source at 20 • C, were subsequently treated with 50, 100 and 150 µg/mL of CST, CSE, and CSR for 48 h except for the control group that received no treatment. Careful washing of the worms was achieved by M9 buffer followed by their transfer and incubation in 1 mL of CM-H2DCF-DA (20 µM) at 20 • C for 30 min. Removal of excess dye was achieved via washing the worms again by M9 buffer followed by their analysis with fluorescence microscopy (λ Excitation 480/20 nm; λ Emission 510/38 nm). Paralysis of the worms was accomplished by the addition of 10 mM of sodium azide which were then placed on a glass slide. Images were captured at fixed exposure time from at least 30 worms. The whole body relative fluorescence was estimated using the Image J software densitometrically and the results were expressed as the mean pixel intensity (mean ± SEM) and compared using one-way ANOVA and subsequently followed by Bonferroni post-hoc correction [18].

Quantitation of hsp-16.2/GFP Expression in Caenorhabditis elegans
Synchronized worms of the transgenic strain TJ375 that express hsp-16.2/GFP) were brought up in S-media supplemented with living E. coli OP50 as a food source at 20 • C. They were subsequently treated with 50, 100, and 150 µg/mL of CST, CSE, and CSR for 48 h except the control group that received no treatment. Exposure of these worms to oxidative stress was achieved by adding 20 µM of juglone for 24 h and consequently the worms were analyzed using fluorescence microscopy (λ Excitation 480/20 nm; λ Emission 510/38 nm). Hsp-12.6 promoter that linked to gene encoding GFP (green fluorescence protein) exists in the mutant strain, TJ375, and its expression is directly determined quantitatively via determining fluorescence intensity of the GFP reporter that exists in the pharynx of the worm. Then the worms were treated with 10 mM of sodium azide to reduce the motility of the worms which were then placed on a glass slide. Images were captured by 20X objective lens at fixed exposure time from at least 30 worms. The pharynx relative fluorescence was estimated using the Image J software densitometrically and the results were expressed as the mean pixel intensity (mean ± SEM) and compared using one-way ANOVA and subsequently followed by Bonferroni (post-hoc) correction.
2.6. Phytochemical Profiling of C. speciosum Leaves Total Methanol Extract Using LC/ESI/MS-MS An analysis of a total methanol extract from C. speciosum leaves was carried out using HPLC Agilent 1200 series instrument. The used column was Gemini C18 with 3 µm and 110 Å obtained from Phenomenex possessing 100 × 1 mm i.d as dimensions. This is in turn is protected by a guard column formed of RP C18 100 Åpossessing 5 mm × 300 µm i.d., 5 µm as dimensions. Elution was done in a gradient manner at a flow rate of 50 µL/min where the mobile phase is composed of 2% acetic acid (A) and 90% MeOH in 2% acetic acid (B). The elution started with 5% B at 0 min that gradually increased to 50% B in 60 min and then consequently elevated to 90% B within 60 min and maintained for 5 min. Mass spectrometry was performed via the utilization of Fourier transform ion cyclotron resonance mass analyzer supplemented with anelectrospray ionization (ESI) system. Controlling of the system was done using the X-calibur ® software where the data were gathered in the negative ion mode as previously described by Sobeh et al. The mass in the range of 150 to 2000 m/z was selected for the full mass scan with resolution up to 100,000 [19].

In Silico Molecular Docking
To get a deep insight into the molecular antioxidant effect of the identified compounds from C. speciosum leaves total methanol extract in Caenorhabditis elegans, molecular docking was done for all of them against daf-2 protein (PDB ID: 4JDE; 1.90Å) obtained from Caenorhabditis elegans using Discovery Studio 4.5 (Accelrys Inc., San Diego, CA, USA) employing C-Docker protocol. The X-ray crystal structure of the protein used in this study was obtained from protein data bank in pdb format. The default protocol for the preparation of protein implemented in Discovery Studio 4.5 (Accelrys Inc., San Diego, CA, USA) was utilized for the preparation of the studied protein. This was achieved by removal of water molecules, addition of hydrogen atoms followed by cleaning of the protein from any unwanted interactions. CHARMm was selected as the forcefield; however, MMFF94 was chosen for calculation of partial charge and then minimization of the added hydrogen in nearly 2000 steps. The active centers were selected relied upon the previous data approaching the catalytic domain. ChemDraw 13.0 was used to construct the 2D structures of the compounds and then they were converted to 3D using default ligand preparation protocol constructed Discovery Studio 4.5 (Accelrys Inc., San Diego, CA, USA). Then, docking of the prepared compounds in the active center was done employing C-Docker protocol. CHARMm force field was determined and the binding energies were computed using distance dependent dielectric implicit solvation model for the selected docking poses. Meanwhile the following equation was used to determine binding energies (∆G) [3,20].
where ∆G binding : The ligand-protein interaction binding energy; E complex : The potential energy for the complex of protein bound with the ligand; E protein: The potential energy of protein alone; and E ligand : The potential energy for the ligand alone.

ADME/TOPAKT Prediction
The identified compounds from C. speciosum leaves total methanol extract were subjected to AMET prediction (absorption, distribution, metabolism, excretion, and toxicity) in addition to toxicity determination (TOPKAT) using Discovery Studio 4.5 (Accelrys Inc., San Diego, CA, USA). This was performed in an effort to evaluate their pharmacokinetic, pharmacodynamic and toxicity behavior. Blood-brain barrier penetration (BBB), human intestinal absorption (HIA), aqueous solubility, plasma protein binding prediction (PPB), hepatotoxicity level, and cytochrome P450 2D6 were selected as ADMET descriptors. Meanwhile, the carcinogenic effect on male and female rat NPT (National Toxicology Program), Ames mutagenicity, eye and dermal irritation, and Chronic LOAEL were chosen as TOPKAT parameters [21,22].

Determination of the Total Phenolic Content of C. speciosum Leaves Extract and Different Fractions
The total phenolic content (TPC) of C. speciosum leaves extract and different fractions, namely, CST, CSE, and CSR was determined using Folin-Ciocalteu method. All of the tested samples showed considerable values of total phenolic content with the ethyl acetate fraction (CSE) showed the highest value followed by the remaining hydro methanol fraction (CSR) and CST where they showed TPC values of 326 ± 9.7, 289 ± 6.8, and 262 ± 3.8 mg GAE/g sample, respectively. Phenolic compounds possess redox features that enable them to act as antioxidants. The presence of free hydroxyl groups in the phenolic compounds enhanced their free radical scavenging potential and thus the total phenolic content determination could serve as a primary marker that overview the antioxidant capacity of the tested sample [23][24][25].

In Vitro Antioxidant Determination of the Extract and Different Fractions of C. speciosum Leaves
Exposure to stress, chemical substances as well as smoking markedly exaggerates oxidative stress causing the generation of a huge amount of free radicals. This in turn triggers the occurrence of many life threatening diseases exemplified by cancer, diabetes, neurological, and cardiovascular disorders in addition to exacerbating ageing [17]. Thus, in vitro antioxidant evaluation of CST, CSE, and CSR was performed using 2,2-diphenyl-1-picrylhydrazyl radicle (DPPH • ) scavenging capacity assay as well as ferric reducing antioxidant power (FRAP) assay. These assays are highly used to assess the antioxidant potential of plant extracts, their pure isolated compounds in addition to food products as their long-lived radicals represented by DPPH • and FeSO 4 are reliable and sensitive [26]. Concerning 2,2-diphenyl-1-picrylhydrazyl radicle (DPPH • ) scavenging capacity assay, it relied upon bleaching the color of DPPH methanol solution (purple) where it converted gradually to yellow color depending on the antioxidant potential of the tested sample. Thus, the results are highly correlated to the antioxidant capacity of the examined sample where antioxidants possess the ability to donate hydrogen atom or electron that consequently reduces the radical solutions [27]. Besides, FRAP is an easy, rapid, and reproducible method that accurately assesses the reduction of ferric ion (Fe 3+ ) to ferrous (Fe 2+ ) that is perfectly linked to the antioxidant power of phytoconstituents and is determined spectrophotometrically [22]. Results displayed in Table 1 revealed that all of the tested samples showed marked antioxidant ability in both assays with the ethyl acetate fraction (CSE) exhibited the highest activity. CST, CSE, and CSR displayed EC 50 values of 27.1, 16.2, and 21.3 µg/mL, respectively, in DPPH • assay, whereas they showed antioxidant capacity estimated by 11.44, 16.27, and 12.16 Fe2 + equivalents/mg of sample, respectively. Juglone, 5-hydroxy-1,4-naphthoquinone, is isolated from Juglans regia and is considered as a natural toxin that elicits oxidative stress acting as pro-oxidant. Thus, exposure to a high concentration of juglone is drastic to Caenorhabditis elegans that promptly causes its death meanwhile the presence of antioxidant agents that capture free radicals possibly hinder this effect [5,28]. Results displayed in Figure 1 showed that C. elegans pretreated with different concentrations (50, 100, and 150 µg/mL) of extracts and fractions of C. speciosum leaves markedly improved the survival rate of the nematode. C. speciosum total methanol extract (CST) at 150 µg/mL exerted the highest in vivo antioxidant potential as evidenced by the high survival rate estimated by 71.88% compared to untreated control group that received juglone alone (24.82% survival rate) and approaching that pretreated with 100 µg/mL of EGCG (epicogallocatechin gallate) that exerted 80.26% survival rate. From Figure 1 it is clearly obvious that CST, CSE and CSR exerted 64.44%, 56.59%, and 63.43% survival rate at 50 µg/mL, respectively; meanwhile, they showed 65.03%, 56.78%, and 57.72% survival rate at 100 µg/mL, respectively, whereas they exhibited 71.88%, 63.53%, and 55.85% survival rate at 150 µg/mL, respectively. It is worthy to highlight that the decreased in the survival rate in the group pretreated with CSR by increasing the concentration of the administered drug reflects its increased toxicity at high concentrations, besides all the tested samples revealed toxicity at 200 µg/mL. untreated control receives juglone treatment; EGCG = Epigallocatechin gallate (100 µg/mL) after juglone treatment; CST, CSE, and CSR groups receive (50, 100, and 150 µg/mL) of CST, CSE, and CSR after juglone treatment; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Assessment of Intracellular Reactive Oxygen Species (ROS) in Caenorhabditis elegans
In order to evaluate the amount of reactive oxygen species (ROS) within the cells that in turn help in the determination of the in vivo antioxidant potential of the administered samples, 2 ,7 -dichlorofluorescindiacetate (CMH 2 DCF-DA), which acts as a membrane permeable reagent, was utilized. The presence of intracellular esterases causes the deacetylation of the reagent to a non-fluorescent compound that consequently oxidized in the presence of ROS, in particular H 2 O 2 producing 2 ,7 -dichlorofluorescein (DCF), a compound with high fluorescence that can be analyzed using fluorescence microscopy. Herein treatment of the worms with different concentrations (50, 100, and 150 µg/mL) of extract and fractions of C. speciosum leaves for 48 h resulted in a notable reduction in the concentration of intracellular ROS as evidenced by fluorescence microscopy analyses in a dose dependent manner in most of the samples that in turn reflects their ability to scavenge ROS in vivo (Figure 2). Images revealed that worms treated with 150 µg/mL of CSE experienced the lower fluorescence intensity when compared to untreated control group showing 60.42% reduction in fluorescence compared to untreated control group and approaching that pretreated with 100 µg/mL of EGCG (epigallocatechin gallate) that revealed 70.44% decrease in fluorescence. CST, CSE, and CSR exerted 41.21%, 36.59%, and 34.34% reduction in fluorescence compared to untreated group 50 µg/mL, respectively; meanwhile, they showed 41.49%, 38.79%, and 48.16% reduction in fluorescence compared to untreated group 100 µg/mL, respectively, whereas they exhibited 51.67%, 60.42%, and 40.38% reduction in fluorescence compared to untreated group 150 µg/mL, respectively. From the displayed results it is obvious that CSR at 50 µg/mL displayed the lowest reduction in fluorescence with respect to all of the examined samples meanwhile it showed higher reduction in fluorescence at 100 µg/mL than 150 µg/mL that may indicate its toxicity at a high concentration.

Quantitation of hsp-16.2/GFP Expression in Caenorhabditis elegans
Heat shock proteins (HSPs) are present in all living organisms where elevated levels of HSP is highly correlated with many stress conditions comprising high temperature as well as of presence of oxidants that result in protein damage induction that in turn influences aging and longevity as well [29]. Suppression of hsp-16.2/GFP expression in C. elegans pretreated with the extract and different fractions of C. speciosum leaves was estimated using transgenic strain TJ375 that expresses hsp-16.2/GFP upon exposure to juglone treatment. Treatment of the transgenic strain TJ375 with different concentrations (50, 100, and 150 µg/mL) of extract and fractions of C. speciosum leaves elicited a substantial reduction in the expression of hsp-16.2/GFP as evidenced by fluorescence microscopy analyses in a dose dependent manner that is in turn correlated with the ability of the tested samples to enhance the worm survival rate and to reduce the intracellular ROS (Figure 3). Images showed that the worms treated with 150 µg/mL of CSR exerted the lower fluorescence intensity with respect to the untreated control group displaying 40.43% reduction in fluorescence with respect to the untreated group meanwhile that pretreated with 100 µg/mL of EGCG (epigallocatechin gallate) revealed 82.79% decrease in fluorescence. In contrast, CSE at 50 µg/mL displayed the lowest reduction in fluorescence with respect to all of the examined samples estimated by 7.14%. CST, CSE, and CSR exerted 15.06%, 7.14%, and 15.23% reduction in fluorescence compared to untreated group 50 µg/mL, respectively; meanwhile, they showed 16.73%, 15.35%, and 20.48% reduction in fluorescence compared to untreated group 100 µg/mL, respectively, whereas they exhibited 17.68%, 22.12%, and 40.43% reduction in fluorescence compared to untreated group 150 µg/mL, respectively.
Phenylpropanoid glycosides were identified constituting the highest abundance in C. speciosum leaves total methanol extract represented by compounds (3-5), (10-11), and (13) where they showed molecular ions [M − H] − at m/z of 451, 609, 623, 667, 721, and 649, respectively, assigned to phenylpropanoid glycosides. More than one type of phenylpropanoid glycosides exist in the total methanol extract in which one type represented by verbascoside consists of caffeoyl and dihydroxy phenylethanol moieties linked together with two sugars, hexose and deoxy hexose. The deoxy hexose moiety of verbascoside is rhamnose. Another type represented by calceolarioside C consists of caffeoyl and dihydroxy phenylethanol moieties linked together with two sugars, hexose and pentose. In MS/MS fragmentation, both types are characterized by elimination of 162 atomic mass units (amu) corresponding to caffeoyl ion and the appearance of a fragment at m/z 161. Meanwhile another type of phenylpropanoid glycosides scroside B is characterized by the presence of feruloyl and hydroxy methoxy phenylethanol moities linked together with two hexose sugar moieties where its MS/MS analysis by elimination of 176 amu corresponding to feruloyl moiety and the appearance of a fragment at m/z 175. Furthermore an additional type of phenylpropanoid glycosides, 2 ,4 -diacetyl leucosceptoside (11), is characterized by the presence of caffeoyl and dihydroxy phenylethanol moieties linked together with two acetylated sugars, hexose and deoxy hexose, and it is characterized by elimination of 162 atomic mass units (amu) corresponding to caffeoyl ion and the appearance of a fragment at m/z 161. Meanwhile tubuloside E is characterized by the presence of coumaroyl and dihydroxy phenylethanol moieties linked together with two sugars, an acetylated hexose and deoxy hexose moieties and showed fragmentation pattern at m/z 163 which is characteristic to coumaric acid. However, four flavonoid glycosides were detected in total methanol extract of C. speciosum leaves represented by tiliroside, rhamnazin-3O-rutinoside, quercetin methyl galloyl-hexoside, and acacetin trioside. Rhamnazin-3-O-rutinoside was detected as a flavonol linked to a disaccharide, rutinose, at position 3 displaying molecular ion peak [M − H] − at m/z of 637.   The results of LC/MS analysis of C. speciosum comes in accordance with other previously investigated Clerodendrum species where phenylpropanoids constitute the major observed class of secondary metabolites. Phenylpropanoid glycosides were isolated in considerable quantities from several Lamiaceae species as shown by Erdtman and are considered as crucial taxonomic markers in members of Lamiaceae [44]. LC/MS analysis previously conducted on C. inerme and C. splendens revealed that phenylpropanoids, flavonoids, phenolic acids, iridoid glycosides, as well as diterpenoids and fatty acid derivatives represents the major classes of secondary metabolites. Verbascoside, markhamioside B, magnoloside A or D, as well as markhamioside are the predominate phenylpropanoid in C. inerme and C. splendens. However, rhamnazin-3-O-rutinoside was identified in the leaves of C. splendens in addition rosmarinic acid was detected only in the methanol extract of the leaves of C. splendens that further confirms the relation between our studied C. speciosum and C. splendens. Besides, flavonoids such as scutellarein, naringenin, 4 -methyl scutellarein-7-O-hexuronide, and acacetin-7-O-hexuronide were identified in C. inerme leaves methanol extract [35]. Furthermore, phenylpropanoids represented by isoacteoside, acteoside mar-tynoside, leucosceptoside A, incanoside C, jionoside C, and jionoside D were αpreviously identified from C. infortunatum in addition to the presence of acetylated phenylpropanoids such as 2 -O-acetyl-martyonside and 3 -O-acetyl-martyonside which further confirms our results. Regarding flavonoids, apigenin 7-O-glucuronide and acacetin 7-O-glucuronide were also present in C. infortunatum [45].
3.5. Computer Aided Drug Design Studies 3.5.1. In Silico Molecular Docking Molecular docking was done for all of the identified compounds from C. speciosum leaves total methanol extract within the active sites of DAF-2 protein where survival and combating oxidative stress is highly crucial for many organisms as this proceeds via the stimulation of various signaling pathways. DAF-2 protein is an insulin receptorlike protein that was linked to the antioxidant potentiality of Caenorhabditis elegans in several reports [46,47]. Caenorhabditis elegans depends on daf-2 protein to regulate longevity and resist oxidative stress [48]. Consequently, daf-2 protein (PDB ID: 4JDE) has been utilized as a target in our docking study using Naringenin as a reference ligand due to its reported activities [49,50]. In this study, we depended on the binding free energy (∆G) between the docked molecules and the active site, in addition to the correct binding mode. The binding free energies were summarized in Table 3. Results of molecular docking illustrated in Table 3 revealed that rosmarinic acid (9) followed by fuhsioside (3) showed the best fitting within the active sites of DAF-2 protein employing both pH-based and rule-based ionization modes. Table 3. Binding energies (kcal/mole) of major identified phytoconstituents present in the total methanol extract of C. speciosum leaves within the active sites of daf-2 protein employing both pH-based and rule-based ionization modes. Positive values indicate unfavorable interaction; FD: fail to dock.
They showed binding energies (∆G) of −41.99 and −17.70 kcal/mole, respectively, in pH-based ionization mode meanwhile they exerted ∆G estimated by −41.24 and −28.73 kcal/mol, respectively, in rule-based ionization mode. Employing pH-based ionization mode that actually mimic the physiological pH, the tight fitting of rosmarinic acid (9) to the active site, is attributed to the formation of multiple bonds manifested by one conventional H-bond with Asn37; 1 π-cation interaction with Arg129 in addition to two ionic salt bridge formation and two attractive charges between the carboxylic acid group and His136 and Arg129 amino acid residue ( Figure 5A). Meanwhile fuhsioside (3) forms four conventional H-bonds with His136, Leu39, Glu143; one C-H bond with Ala38; one π-cation interaction with Arg129 in addition to the formation of an amide −π stacked bond ( Figure 5B). The binding mode of naringenin as a refrence ligand showed a binding free energy of −19.45 kcal/mol. It forms one π-δ bond with Ala38; one π-cation bond with Arg129; two C-H bonds with Leu39 and Arg129; two π-alkyl bonds with Ala41 and Leu39 in addition to π-π-T-shaped bond with His136 ( Figure 5C). , and naringenin (C) within the active sites of daf2 protein employing pH-based ionization mode; H-bonds (heavy green dotted bonds); C-H bonds (light green dotted bonds); π-ionic bonds and attractive charges (orange dotted bonds); π-δ bond (violet dotted bonds); π-π bonds (heavy purple dotted bonds) and π-alkyl bonds (light purple dotted bonds).
Besides, the presence of rosmarinic acid within the active pocket of Daf-2 protein illustrating hydrogen bond type, with receptor donors in green and receptor acceptors in cyan.; hydrophobicity of the receptor residues, from blue for hydrophilic to brown for hydrophobic; solvent accessibility of the receptor residues from blue for exposed to green for buried and the ionizability of the receptor residues, from blue for basic to red for acidic is displayed in Figure 7. Rosmarinic acid was previously proved to act as a potent natural antioxidant evidenced by many assays as DPPH and ABTS assays showing 72.3 ± 3.3 µM as IC 50 in the former and 3.7 ± 0.7 mM Trolox as TEAC in the latter. It acts as free radicle scavenger besides, it showed a significant activity versus lipid peroxidation [51,52]. Figure 7. The presence of rosmarinic acid within the active pocket of Daf-2 protein illustrating (A) hydrogen bond type, with receptor donors in green and receptor acceptors in cyan.; (B) hydrophobicity of the receptor residues, from blue for hydrophilic to brown for hydrophobic; (C) solvent accessibility of the receptor residues from blue for exposed to green for buried; and (D) the ionizability of the receptor residues, from blue for basic to red for acidic.

ADME/TOPAKT Prediction
The identified compounds from C. speciosum leaves total methanol extract were subjected to ADME/TOPAKT estimation in an effort to evaluate their pharmacokinetic, pharmacodynamic, and toxicity behavior as revealed by theoretical estimates performed by Discovery Studio 4.5 (Accelrys Inc., San Diego, CA, USA) built in protocol. Results displayed in Table 4 showed that methyl ligstroside aglycone (16) showed good intestinal absorption, whereas rosmarinic acid (9) revealed low human intestinal absorption and thus lies within the 99% absorption ellipse as shown in ADMET plot (Figure 8). In contrast all other identified compounds showed very low absorption characteristics and consequently lie outside in the 99% absorption ellipse in ADMET plot. Besides, all the identified compounds showed undefined BBB behavior lying outside BBB 99% confidence eclipse, whereas methyl ligstroside aglycones showed low penetration via BBB and thus present within the 95% confidence eclipse of BBB. Concerning the solubility level, serratoside A (2) showed optimal solubility; meanwhile, fuhsioside (3), rosmarinic acid (9), and methyl ligstroside aglycone (16) exhibited good solubility, whereas other compounds showed low solubility pattern. Regarding plasma protein binding, all of the examined compounds revealed less than 90% binding. Besides, they showed no inhibition to CPY2D6 and did not exhibit hepatotoxicity (Table 4). Table 4. ADMET (Absorption, distribution, metabolism, excretion, and toxicity) properties of the major compounds identified in the total methanol extract of C. speciosum leaves. 0, 1, 2, and 3 indicates good, moderate, low and very low absorption, respectively; 0, 1, 2, 3, 4, and 5 indicates extremely low, very low but possible, low, good, optimal, and too soluble, respectively; 0, 1, 2, 3, and 4 denote very high, high, medium, low, and undefined, penetration via BBB respectively. PBB, plasma protein binding; false = less than 90%, true = more than 90%; NI: non-inhibitor; NT: non-toxic. Figure 8. ADMET Plot for major compounds identified in the total methanol extract of C. speciosum showing 95% and 99% confidence limit ellipses with respect to the human intestinal absorption and the blood-brain barrier (BBB) models; rosmarinic acid (9) (triangle) and methyl ligstroside aglycone (16) (large filled square).

Compounds
Regarding TOPKAT analyses, all the examined compounds showed to be non-mutagenic in Ames prediction without carcinogenic effects in both male and female rats NTP except for rhamnazin-3O-rutinoside (7) that revealed certain carcinogenicity towards male rats NTP. In addition, all of the tested compounds revealed mild to none ocular and skin irritation except tiliroside (6) that exerted a moderate ocular irritation. Besides, the compounds displayed rat oral LD50 values in the range of 0.67-10.57 g/kg body wt. where fuhsioside (3) and rosmarinic acid (9) showed LD50 of 5.82 and 3.17 g/kg body wt., respectively. Similarly, the analyzed compounds showed rat chronic LOAEL (The lowest-observed-adverse-effect level) in the range of 0.01-0.16 g/kg body wt. where fuhsioside (3) and rosmarinic acid (9) showed LOAEL of 0.16 and 0.13 g/kg body wt., respectively, that in turn reflects their safety ( Table 5). From ADME/TOPAKT analyses, it was clearly obvious that fuhsioside (3) and rosmarinic acid (9) that showed the highest docking scores reflecting their high antioxidant potential. Besides, they displayed considerable safety profile evidenced by their toxicity criteria with limited pharmacokinetic and pharmacodynamic parameters. Meanwhile, certain treatment is required to slightly enhance their pharmacokinetic and pharmacodynamic parameters and thus can be formulated with C. speciosum leaves in pharmaceutical dosage forms to alleviate oxidative stress. Table 5. TOPKAT prediction of the major compounds identified in the total methanol extract of C. speciosum leaves. and editing, S.S.E. and M.L.A.; supervision, M.W.; funding acquisition, H.A.B., A.E.K. and S.S.E. All authors have read and agreed to the published version of the manuscript.