Biological Evaluation, DFT Calculations and Molecular Docking Studies on the Antidepressant and Cytotoxicity Activities of Cycas pectinata Buch.-Ham. Compounds

Cycas pectinata Buch.-Ham. is commonly used in folk medicine against various disorders. The present study investigated the antidepressant and cytotoxicity activity of methanol extract of C. pectinata (MECP) along with quantitative phytochemical analysis by GC-MS method. Here, the GC-MS study of MECP presented 41 compounds, among which most were fatty acids, esters, terpenoids and oximes. The antidepressant activity was assessed by the forced swimming test (FST) and tail suspension test (TST) models. In contrast, MECP (200 and 400 mg/kg) exhibited a significant and dose-dependent manner reduction in immobility comparable with fluoxetine (10 mg/kg) and phenelzine (20 mg/kg). MECP showed a weak toxicity level in the brine shrimp lethality bioassay (ED50: 358.65 µg/mL) comparable to the standard drug vincristine sulfate (ED50: 2.39 µg/mL). Three compounds from the GC-MS study were subjected to density functional theory (DFT) calculations, where only cyclopentadecanone oxime showed positive and negative active binding sites. Cyclopentadecanone oxime also showed a good binding interaction in suppressing depression disorders by blocking monoamine oxidase and serotonin receptors with better pharmacokinetic and toxicological properties. Overall, the MECP exhibited a significant antidepressant activity with moderate toxicity, which required further advance studies to identify the mechanism.


Introduction
Depression is a condition characterized by a lowering of the mood and dislike for movement that may distress an individual's thoughts, conduct, emotions, and comfort [1]. Depressive behavior is additionally connected with suicide, which ranges from 10 and 20 million each year [2,3]. According to the World Health Organization (WHO) report, around 450 million people have a mental disorder, which may rise to 15% by 2020 [4]. In addition, the physical changes additionally happen in extreme, vital, or melancholia or melancholic depression. These comprise sleep deprivation or hypersomnia, modified eating disorders, anorexia and weight reduction and several endocrine dysfunctions with alterations in body temperature. Depressive behavior is the feature of some psychiatric disorders, which may also be caused by somewhat normal life situations; for example, deprivation of sleep, sicknesses, or an adverse effect of drugs and clinical treatments. Patients with major depressive behavior have several symptoms that may reflect in the brain, monoamine synapses or neurotransmitters, explicitly norepinephrine, serotonin, and dopamine [5][6][7].
There are several antidepressant drugs available to treat depression, but the rate of success of first-line therapy for depression [e.g., selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs)] is low due to several limitations (adverse effects, lower response and the onset of action, etc.), which have been mentioned in several reviews [8]. Thus, it is imperative that new antidepressant drugs demonstrate improvement of these drawbacks. Several phytochemicals (alkaloids, flavonoids, sterol, terpenes) were reported to have an antidepressant effect [9]. Oximes (R 1 R 2 C = NOH) are chemicals containing nitrogen produced by organisms in all kingdoms of life [10]. In recent years, oxime derivatives were reported to have several pharmacological activities: cytotoxicity, antibiotic effect, anticonvulsant, antimicrobial, cardiac dysrhythmia, antinociceptive activities [11][12][13][14].
Presently monoamine oxidase A (MAO-A) is useful in the treatment of depression disorders, because MAO metabolizes serotonin or 5-hydroxytryptamine (5-HT) in the central nervous system (CNS) [15]. SSRIs are effective in depression, but due to their limitations, the eValuation of new bioactive substances is a major target for the researchers [16]. Oxime derivatives are reported to have an antidepressant effect [17][18][19], whereas chalcone oxime ethers are reported to have potent inhibitory activity against MAO-B [20]. Our present study design aimed at the eValuation of the biological activity along with a computational study (DFT, molecular docking, ADME/T), where the MAO-A and serotonin receptor are used as a molecular targets for oxime derivatives in depression disorders.
Cycas pectinata Buch.-Ham. (Family: Cycadaceae), commonly known as moniraj or nagmoni, belongs to the genus Cycas [21]. This plant has traditionally been useful for hair growth, curing stomach aches, and curing ulcers [22,23]. Various ethnopharmacological uses in different treatment aspects are documented for Cycas species. Cycas revoluta Thunb. was used for inflammation, vomiting and tonic conditions [24], while Cycas circinalis L. is used for healing wounds and swollen glands. Cycas rumphii Miq. male pollen and cones are reported to have strong narcotic effects [25]. Like in a previous C. pectinata study, a number of fatty acid methyl esters along with other compounds have been reported for C. revolute [26], whereas 16 different bioactive compounds have been reported for C. circinalis [27]. In our previous study, several secondary metabolites from the methanol extract of C. pectinata exhibited the following pharmacological activities, including antioxidant, anti-inflammatory, thrombolytic, anxiolytic, sedative, antinociceptive and antidiarrheal properties [22]. In the present study we report the antidepressant activity along with the cytotoxicity activity of C. pectinate to find a potential lead compound from C. pectinata in alleviating depression disorders by blocking monoamine oxidase (MAOs) and serotonin receptors. To explain this possible mechanism of action of compounds isolated from C. pectinata, we also performed a quantum chemical analysis (DFT calculations) with molecular docking, and ADME/T studies to reveal the potential target(s) for inhibition of the human MAO and serotonin receptors.

Qualitative and Quantitative Phytochemical Analysis with Acute Toxicity Study
Phytochemical analysis is useful to eValuate the therapeutic and physiological activities of a plant extract. A qualitative phytochemical screen is performed to determine the presence or absence of secondary plant metabolites. The investigation showed positive results for carbohydrates, alkaloids, phenol, proteins, flavonoids, and saponins (data not shown), which was similar to our previous study that reported similar results [22]. The phytochemical analysis of C. pectinata leaves showed the presence of several phytochemicals. Glycosides are a group of compounds with drug-likeness and numerous studies have suggested that they are a fruitful source of potential drugs. Flavonoids are reported to have anti-inflammatory and anti-cancer activity, whereas tannins possess anti-inflammatory and anti-microbial activity [28]. Phenolic compounds are also present, which possess various physiological functions like anti-aging, anti-inflammation, anti-apoptosis, anti-carcinogenic, inhibition of angiogenesis and enhancement of endothelial function [29].
A total of 66 compounds were identified in the GC-MS analysis, whereas 25 compounds were reported by Tareq et al. [22]. In addition, 41 other compounds are presented in Table 1 and Figure S1, most of which were esters, organic compounds and alcohols. The most abundant compounds along with their retention times were (E)-2-decen-1-ol (20.360), chloroacetic acid 4-pentadecyl ester (20.360), glycerol 1-palmitate (20.009), octadecanoic acid 2-hydroxy-1,3-propanediyl ester (20.009), hexadecanoic acid 2-hydroxy-1-(hydroxymethyl)ethyl ester (20.009), docosanoic acid docosyl ester (19.440), cyclopentadecanone oxime (19.440), and 1-O-(16-hydroxyhexadecyl)-d-mannitol (19.440). These compounds isolated from MECP could help develop a new drug for depression and cancer diseases. The antidepressant activity was eValuated in Swiss albino mice, which required a prior toxicity study. Before starting the experiments and the acute toxicity study of MECP at 400-2000 mg/kg dose was conducted in Swiss albino mice. The methanol extract of C. pectinata leaves was determined to be safe. There was no change of behavioral rush or mortality, morbidity in 8 h observation period of 400, 600, 800, 1000, 2000 mg/kg of MECP doses which were similar to the previous study [22].

Antidepressant Activity
Anxiety and depression are mental conditions that may recur and are generally undiagnosed and untreated. Physical problems might join these mental conditions, and patients frequently present in medical care centers with physical problems instead of mental situations or problems [30]. Though several antidepressant drugs available, but the rate of success is falling day by day (e.g., SSRIs and SNRIs) [8,31]. Thus, the phytochemical study is a topic of interest for the researcher to eValuate a lead compound to treat depression. Several phytochemicals (alkaloids, flavonoids, sterol, terpenes) are reported to have antidepressant effects [9]. Additionally, a few medicinal plants such as M. angolensis [32], N. sativa [33] R. rosea [34] are reported to have bilateral anxiolytic and antidepressant effects. In our previous study, MECP showed decreased locomotor activity with a significant anxiolytic activity and also a strong binding affinity against the human serotonin receptor (PDV: 5I6X) suggested by the interacted compounds [22]. Here, the antidepressant activity of MECP was eValuated by a tail suspension test (TST) and forced swimming test (FST), which are the most promising models to assess antidepressant activity.  Moreover, TST is proposed to have a higher pharmacological sensitivity as compared to FST. In these models, the immobility time or the period of time when animals stopped struggling was calculated, where the antidepressant agents reduce the immobility time in rodents [35]. Fluoxetine is a SSRI very widely used as an antidepressant agent, which basically works by inhibiting access of serotonin transporter protein into presynaptic serotonin neurons by inhibiting the transporter protein and also has mild activity at the 5-hydroxytryptamine 2A (5HT2A) and 5-hydroxytryptamine 2C (5HT2C) receptors [36]. Also, phenelzine was used in this study is a monoamine oxidase inhibitor (MAOI) that acts by inhibiting MAO activity and afterward raises the neural concentration of neurotransmitters, thus increasing monoamine secretion in the synaptic cleft and alleviating depression [37].
In our study, both doses of MECP showed significant antidepressant activity, leading to a possibility that MECP may act in the presynaptic serotonin neurons by inhibiting serotonin transporter protein and by inhibiting the activity of MAO. In TST, 200 and 400 mg/kg dose exhibited 46.26% and 51.52% inhibition in immobility, whereas the standard drugs fluoxetine and phenelzine exhibited 55.06% and 39.40%, respectively. Additionally, the FST showed significant (p < 0.001) immobility, whereas the 200 and 400 mg/kg exhibited 28.51% and 32.55% inhibition of immobility. The results are presented in Figure 1. The presence of alkaloids and saponins in MECP may be a possible reason for this antidepressant activity as well as the presence of oxime derivatives [17][18][19]38]. Moreover, TST is proposed to have a higher pharmacological sensitivity as compared to FST. In these models, the immobility time or the period of time when animals stopped struggling was calculated, where the antidepressant agents reduce the immobility time in rodents [35]. Fluoxetine is a SSRI very widely used as an antidepressant agent, which basically works by inhibiting access of serotonin transporter protein into presynaptic serotonin neurons by inhibiting the transporter protein and also has mild activity at the 5-hydroxytryptamine 2A (5HT2A) and 5-hydroxytryptamine 2C (5HT2C) receptors [36]. Also, phenelzine was used in this study is a monoamine oxidase inhibitor (MAOI) that acts by inhibiting MAO activity and afterward raises the neural concentration of neurotransmitters, thus increasing monoamine secretion in the synaptic cleft and alleviating depression [37].
In our study, both doses of MECP showed significant antidepressant activity, leading to a possibility that MECP may act in the presynaptic serotonin neurons by inhibiting serotonin transporter protein and by inhibiting the activity of MAO. In TST, 200 and 400 mg/kg dose exhibited 46.26% and 51.52% inhibition in immobility, whereas the standard drugs fluoxetine and phenelzine exhibited 55.06% and 39.40%, respectively. Additionally, the FST showed significant (p <0.001) immobility, whereas the 200 and 400 mg/kg exhibited 28.51% and 32.55% inhibition of immobility. The results are presented in Figure 1. The presence of alkaloids and saponins in MECP may be a possible reason for this antidepressant activity as well as the presence of oxime derivatives [17][18][19]38].

Cytotoxicity Activity
The eValuation of the bioactivity of plant products by the brine shrimp lethality bioassay is an effective, safe and economical method. A good correlation is found in the brine shrimp lethality bioassay with solid human tumors for cytotoxic and pesticidal activity, which is useful for the discovery of active antitumor agents and natural pesticides [39]. This method is also used as a pre-screening test for antitumor research. Generally, the higher the ED 50 , the lower the toxicity of the extract is and vice versa [40]. In our study, the ED 50 of the test samples was calculated using a concentration against the viability of the nauplii. Vincristine sulfate demonstrated the viability of nauplii when the concentration gradually decreased from 10 µg/mL (zero viability) to 0.125 µg/mL (90% viability). MECP has an ED 50 of 358.65 µg/mL, which is weakly toxic, whereas the standard drug vincristine sulfate exhibited 2.39 µg/mL (highly toxic). The results are presented in Figure 2. This moderate toxicity level of MECP may be due to the presence of oxime derivatives, which reported to have cytotoxicity and antitumor activity [11,41]. . The values are shown as mean ± standard error of the mean (SEM). * p < 0.001 statistically significant compared with the control by Dunnett's test (n = 5).

Cytotoxicity Activity
The evaluation of the bioactivity of plant products by the brine shrimp lethality bioassay is an effective, safe and economical method. A good correlation is found in the brine shrimp lethality bioassay with solid human tumors for cytotoxic and pesticidal activity, which is useful for the discovery of active antitumor agents and natural pesticides [39]. This method is also used as a pre-screening test for antitumor research. Generally, the higher the ED50, the lower the toxicity of the extract is and vice versa [40]. In our study, the ED50 of the test samples was calculated using a concentration against the viability of the nauplii. Vincristine sulfate demonstrated the viability of nauplii when the concentration gradually decreased from 10 µg/mL (zero viability) to 0.125 µg/mL (90% viability). MECP has an ED50 of 358.65 µg/mL, which is weakly toxic, whereas the standard drug vincristine sulfate exhibited 2.39 µg/mL (highly toxic). The results are presented in Figure 2. This moderate toxicity level of MECP may be due to the presence of oxime derivatives, which reported to have cytotoxicity and antitumor activity [11,41].

Molecular Geometry
The sTable configurations of 5-chloro-1-(trimethylsilyl)-1H-indole-2,3-dione 3-[O-(trimethylsilyl)oxime], cyclopentadecanone oxime; and trans-2-dodecen-1-ol trifluoroacetate obtained from the conformational analysis which has been used for reactivity analysis are shown in Figure 3 with the numbering of atoms. From the structural point of view, these three compounds belongs to the C1 point group symmetry group and hence all the calculated frequencies transform to the same A symmetry species.

Molecular Geometry
The stable configurations of 5-chloro-1-(trimethylsilyl)-1H-indole-2,3-dione 3-[O-(trimethylsilyl)oxime], cyclopentadecanone oxime; and trans-2-dodecen-1-ol trifluoroacetate obtained from the conformational analysis which has been used for reactivity analysis are shown in Figure 3 with the numbering of atoms. From the structural point of view, these three compounds belongs to the C1 point group symmetry group and hence all the calculated frequencies transform to the same A symmetry species. The total energies of the three compounds calculated by the B3LYP method are -1845.68068, 718.77081 and -997.04879 Hartree, respectively (Table 2). Among the three compounds trans-2-dodecen-1-ol trifluoroacetate showed a higher dipole moment value. Dipole moments tell us about the charge separation in a molecule. The larger the difference in electronegativity of bonded atoms, the larger the dipole moment [42]. Among the three isolated compound, has higher polarizability value. Generally, polarizability increases as the volume occupied by electrons increases. In atoms, The total energies of the three compounds calculated by the B3LYP method are −1845.68068, 718.77081 and −997.04879 Hartree, respectively (Table 2). Among the three compounds trans-2dodecen-1-ol trifluoroacetate showed a higher dipole moment value. Dipole moments tell us about the charge separation in a molecule. The larger the difference in electronegativity of bonded atoms, the larger the dipole moment [42]. Among the three isolated compound, Pharmaceuticals 2020, 13, 232 9 of 25 5-chloro-1-(trimethylsilyl)-1H-indole-2,3-dione 3-[O-(trimethylsilyl)oxime] has higher polarizability value. Generally, polarizability increases as the volume occupied by electrons increases. In atoms, this occurs because larger atoms have more loosely held electrons than smaller atoms with tightly bound electrons [43]. Table 2. Optimized energies of 5-chloro-1-(trimethylsilyl)-1H-indole-2,3-dione 3-[O-(trimethylsilyl)-oxime]; cyclopentadecanone oxime and trans-2-dodecen-1-ol trifluoroacetate with dipole moment and polarizability.

Compounds
Energy (a.u) Dipole Moment (Debye) Polarizability (a.u) The atomic charges (Mulliken and NBO) play an important role in molecular polarizability, dipole moment, electronic structure, molecular reactivity and a lot of related properties of molecular systems. The charge distributions over the atoms suggest the formation of donor and acceptor pairs involving the charge transferring the molecule. The charges on the atoms of the present 5-chloro-1-(trimethylsilyl)-1H-indole-2,3-dione 3-[O-(trimethylsilyl)oxime]; cyclopentadecanone oxime and trans-2-dodecen-1-ol trifluoroacetate; were calculated by Mulliken population analysis [44] and NBO charges [45] using B3LYP method with 6-31G+ (d,p) basis set, the tabular representation of the results are presented in Tables S1-S3. For it can be easily seen that the highest positive Mulliken charge value of 1.710 a.u was accommodated on the Si 16 atom that is attached to the pyrrole ring, while in NBO charges the highest positive value was 1.905 a.u on the Si 30 atom which connect with O atom. Also, the Mulliken charge with the highest negative value of (−0.862~−0.877) a.u was on the methyl group C atom wherein NBO charges provide the highest negative value of (−1.219~−1.225) a.u on the methyl group C atom. Due to the electron-withdrawing nature of the methyl group, its C atom is pulling electrons towards it.
As for cyclopentadecanone oxime, it showed the highest positive Mulliken charge value of 0.428 a.u accommodated on the H 16 atom, which is bonded to the O atom. The highest negative Mulliken charge value of −0.638 a.u belongs to the O 15 atom which is attached to the N atom. The natural atomic charges value is in excellent agreement with the highest positive and negative Mulliken charge values for the same atom of the molecule. From the Table it can be easily seen regarding the Mulliken charge values for trans-2-dodecen-1-ol trifluoroacetate, the highest positive value of 0.769 a.u was accommodated on the C 1 atom which is bond with CF 3 . This natural atomic charge value also agreed with the obtained result for the same carbon. It shows the highest positive value was 0.983 a.u. In Mulliken charges, the highest negative value of −0.480 a.u is accommodated on the O 3 atom. This natural atomic charge value does not also agree with the obtained result for the same atom, whereas it shows the highest negative value of −0.524 a.u is accommodated on O 42 atom at molecule. From the charges calculation the highest positive and negative value of Mulliken and NBO charge of atoms sometimes did not agreed with each other due to the two methods used.
The molecular electrostatic potential (MESP) surface [46] from Figure 4 illustrates the molecules' charge distributions three-dimensionally. This map allows us to visualize variably charged regions of a molecule. The knowledge of the charge distributions can be used to determine how molecules may interact with one another and it is also used to determine the nature of their chemical bonds [47]. The MESP map was checked out by theoretical calculations using the B3LYP/6-31G+ (d,p) level. Molecular electrostatic potential shows the electronic density and is useful in recognizing sites for electrophilic attack, nucleophilic reactions, and hydrogen bonding interactions. Different colors represent the different values of the electrostatic potential at the surface. The negative areas (red, orange and yellow color) of MESP were related to electrophilic reactivity, the positive areas (blue color) ones to nucleophilic reactivity and green color are neutral regions. This Figure also provides a visual representation of the chemically active sites and the comparative reactivity of atoms. The computed 3D plot of MESP for the title compounds is depicted in the figure, based on the electron density at different points on the molecule. However, potential values of the three isolated compounds ranges from −6.383 × 10 −2 a.u (deepest red) to +6.383 × 10 −2 a.u (deepest blue), −5.902 × 10 −2 a.u (deepest orange) to +5.902 × 10 −2 a.u (deepest blue), −4.638 × 10 −2 a.u (deepest red) to +4.638 × 10 −2 a.u (deepest blue) respectively.

FMOs and Global Descriptors
The frontier molecular orbitals, HOMO and LUMO, are the most important orbitals in a molecule. They play an important role in the optical and electric properties, as well as in quantum chemistry and the UV-Vis spectra [48]. The highest occupied molecular orbital (HOMO), represents the ionization potential of the molecule and lowest occupied molecular orbital (LUMO), corresponding electron affinity value is called the frontier molecular orbitals (FMOs) showed in Figure 5 were calculated at the B3LYP/6-31G+ (d,p) level for the three isolated compounds.

FMOs and Global Descriptors
The frontier molecular orbitals, HOMO and LUMO, are the most important orbitals in a molecule. They play an important role in the optical and electric properties, as well as in quantum chemistry and the UV-Vis spectra [48]. The highest occupied molecular orbital (HOMO), represents the ionization potential of the molecule and lowest occupied molecular orbital (LUMO), corresponding electron affinity value is called the frontier molecular orbitals (FMOs) showed in Figure 5 were calculated at the B3LYP/6-31G+ (d,p) level for the three isolated compounds. These orbitals determine the way how the molecule interacts with other species and give information about the reactivity/stability of specific regions of the molecule. The energy of HOMO characterizes the electron-donating ability of a molecule, while LUMO energy determines the ability to accept an electron. Therefore, higher values of EHOMO indicate a better tendency towards the donation of an electron. From Figure 5, trans-2-dodecen-1-ol trifluoroacetate is the better molecule which has the ability to accept electrons while the energy value of HOMO (EHOMO = −7.39524 eV) that allows it to be the best electron donor molecule. The energy gap between the HOMO and LUMO is very important in determining a molecule's chemical reactivity. A high value of the energy gap These orbitals determine the way how the molecule interacts with other species and give information about the reactivity/stability of specific regions of the molecule. The energy of HOMO characterizes the electron-donating ability of a molecule, while LUMO energy determines the ability to accept an electron. Therefore, higher values of E HOMO indicate a better tendency towards the donation of an electron. From Figure 5, trans-2-dodecen-1-ol trifluoroacetate is the better molecule which has the ability to accept electrons while the energy value of HOMO (E HOMO = −7.39524 eV) that allows it to be the best electron donor molecule. The energy gap between the HOMO and LUMO is very important in determining a molecule's chemical reactivity. A high value of the energy gap indicates that the molecule shows high chemical stability; indicates a hard molecule, while a small HOMO-LUMO gap means small excitation energies to the manifold of excited states, and action as a soft molecule. Among three isolated compounds, 5-chloro-1-(trimethylsilyl)-1H-indole-2,3-dione 3-[O-(trimethylsilyl)oxime] shows the lowest energy gap indicating it is more reactive than the two other compounds.
Using Koopmans' theorem [49,50] (I) and (A) values can be correlated with the frontier orbitals by the relation: I = −E HOMO and A = −E LUMO . Ionization potential (I) is defined as the amount of energy needed to remove an electron from a molecule. High ionization energy indicates high stability, chemical inertness and small ionization energy indicating high reactivity of the atoms and molecules. Trans-2-Dodecen-1-ol, trifluoroacetate has the lowest ionization potential value (I = 7.39524 eV), which indicates that it is the best electron donor. The electronic affinity (A) is defined as the energy released when an electron is added to a neutral molecule. A molecule with high (A) values tends to accept electrons easily. From Table 3 it is clear that 5-chloro-1-(trimethylsilyl)-1H-Indole-2,3-dione 3-[O-(trimethylsilyl)oxime] is the most reactive. The global chemical reactivity descriptors such as chemical potential (µ), electronegativity (χ), hardness (η), softness (S), and electrophilicity index (ω) which were calculated from the HOMO and LUMO energies were obtained at the level of theory B3LYP/6-31G+ (d,p) and are incorporated in Table 3. Table 3. Global reactivity descriptors values in the gas phase.

trans-2-Dodecen-1-ol Trifluoroacetate
Ionisation potential (I) eV 6 According to these parameters, the chemical reactivity varies with the structural configuration of the molecules. Global reactivity descriptors such as chemical potential denote as (µ = -χ), the absolute electronegativity (χ) is given by the relation (χ = (IP + EA)/2), global hardness and global softness (S) are defined as (η = (E LUMO − E HOMO )/2) and (S = 1/2η), the electrophilicity (ω) can be calculated using the electronic chemical potential and the chemical hardness (ω = µ 2 /2η) [51][52][53][54][55]. Hardness (η) and softness (S) are useful concepts for understanding the behavior of chemical systems. A hard molecule has a large energy gap and a soft molecule has a small energy gap [56]. Therefore, soft molecules will be more polarizable than hard molecules. From the established theoretical calculations cyclopentadecanone oxime has the highest hardness value (η = 3.16210 eV), which indicates that it is the hardest molecule. 5-Chloro-1-(trimethylsilyl)-1H-indole-2,3-dione 3-[O-(trimethylsilyl)oxime] has the highest softness (S = 0.53083eV), so it is the softest molecule. The chemical potential µ (eV) measures the escaping tendency of an electron and it can be associated with the molecular electronegativity [57] then, as µ becomes more negative, it is more difficult to lose an electron but easier to gain one. As shown in Table 3, trans-2-dodecen-1-ol trifluoroacetate is the least sTable and the most reactive among all the compounds. Electronegativity (χ), represents the ability of molecules to attract electrons. The (χ) values displayed in Table 3 show that cyclopentadecanone oxime; has the higher electronegativity (4.47546 eV) value compared to all the other molecules. Electrophilicity (ω), that gives an idea of the stabilization energy when the system gets saturated by electrons, which come from the external environment. This reactivity information shows if a molecule is capable of donating charges. A good, more reactive nucleophile is characterized by a lower value of (ω), while higher values indicate the presence of a good electrophile. Our results indicate that cyclopentadecanone oxime has lower values of (ω), so that compound is a good nucleophile, whereas trans-2-dodecen-1-ol, trifluoroacetate is a good electrophile.

Vibrational Spectral Analysis
The vibrational band assignments had been performed based on the normal coordinate analysis. Internal coordinates of three isolated compounds had constructed according to Pulay's recommendations [58]. The calculated wavenumbers were selectively scaled according to the scaled quantum mechanical (SQM) method recommended by Rauhut and Pulay [59] using a scale factor with the root mean square (RMS) wavenumber error, which is in the reasonable limit for proper assignment. The observed FT-IR and simulated theoretical spectra calculated at the B3LYP/6-31G+ (d, p) basis set are shown in Figure S2. The calculated wavenumbers and their assignments are also presented in Table 4. The detailed analyses of vibrational wavenumbers for various functional groups are discussed below.

Hydroxyl (O-H) Group Vibrations
Bands due to O-H stretching are of medium to strong intensity in the infrared spectrum, although it may be broad. For solids, liquids and concentrated solutions a broad band of less intensity is normally observed [60]. The very weak FT-IR band at 3696 cm −1 is assigned to the O-H stretching vibrations. Normally free O-H stretching vibrations appeared around 3600 cm −1 for phenols [61]. The observed broad intense IR band for cyclopentadecanone oxime at corresponds to O-H stretching mode, which is calculated at 3884 cm −1 .

C-N Vibrations
The identification of C = N vibrations is a difficult task since mixing of vibrations is possible in this region. Silverstein et al. [67] assigned the C = N stretching absorption in the 1690-1640 cm −1 range for aromatic amines. The present work shows that the theoretically computed value of C = N stretching vibrations band observed at 1593 cm −1 and 1759 cm −1 in the FT-IR spectrum for 5-chloro-1-(trimethylsilyl)-1H-indole-2,3-dione 3-[O-(trimethylsilyl)oxime] and cyclopentadecanone oxime, respectively.

Carbonyl (C=O) Group Vibration
The C=O stretching vibrations give rise to the characteristic bands in IR spectra, and the intensity of these bands can increase owing to the conjugation or formation of hydrogen bonds. The C=O stretching of ketones is expected in the region 1760-1730 cm −1 [69]. C=O stretching mode is not an independent vibrational mode because of it coupled with the vibrations of adjacent groups. The FT-IR band with strong intensity at 1717 cm −1 alone was assigned to the carbonyl stretching mode of 5-chloro-1-(trimethylsilyl)-1H-indole-2,3-dione 3-[O-(trimethylsilyl)oxime].

NMR Analysis
After the optimization of molecular geometry of the three isolated compounds the 1 H and 13 C nuclear magnetic resonance (NMR) chemical shift values calculated at the B3LYP/6-31G+ (d,p) level in chloroform solvents by comparing their observed values in CDCl 3 solvent with respect to TMS as an internal reference [70]. The theoretically calculated 1 H-and 13 C-NMR chemical shift values are presented in Tables 5 and 6. The theoretically determined 1 H-and 13 C-NMR spectra are shown in Figures S3 and S4, respectively.
The 1 H atoms chemical shift values of 1H-Indole-2,3-dione, 5-chloro-1-(trimethylsilyl)-, 3-[O-(trimethylsilyl)oxime] are divided into two ranges; the first range is approximately 0~6.5 ppm, the second range is around 0~−0.956 ppm. The first group is due to the H atoms in the benzene ring and methyl group. The second group is due to the H atoms in the methyl group attached to Si atoms. Also, 1 H atoms chemical shift values of cyclopentadecanone, oxime divide into two ranges; the first one is around 0~4.5 ppm, the second one is greater than 4.5 ppm. The first group is due to the H atoms in the cyclic alkyl chain and that atoms have slightly positive charges. The highest chemical shift was found for H 16 atom which associated with the O atom. Lastly, the chemical shift values of 1 H atoms for trans-2-Dodecen-1-ol, trifluoroacetate are divided into two ranges; the first one is around 0~5 ppm, the second one is greater than 5 ppm. Frist range chemical shift values determined those H atoms in the alkyl chain and showed a slightly positive charge. The highest chemical shifts were found for H 35 , H 36 atoms which associated with the C atom nearly O atom. Due to different chemical atmospheres at various regions around the H atoms for 1H-Indole-2,3-dione, 5-chloro-1-(trimethylsilyl)-, 3-[O-(trimethylsilyl)oxime]; cyclopentadecanone, oxime; and trans-2-Dodecen-1-ol, trifluoroacetate the chemical shift inequality had originated.

Proton
No.

Proton
No.

Proton
No. The calculated 13 C chemical shift values of for 5-chloro-1-(trimethylsilyl)-1H-indole-2,3-dione 3-[O-(trimethylsilyl)oxime] are in the −15~143 ppm range. This range is divided into two parts; the first range is greater than 100 ppm for C 7 , C 8 , C 1 , C 4 , C 5 , C 2 , C 3 atoms which are located in the benzene ring. The second range is less than 100 ppm for C 6 , C 17 , C 33 , C 19 , C 18 , C 32 , C 31 atoms that alkyl chain carbons attached to silicon atoms. Also, the 13 C chemical shift values of cyclopentadecanone oxime are in the 11~147 ppm range. This range divided into two part; firstly 146.55 ppm was found for O bonded C 12 atom and below 100 ppm corresponds to the C 11 , C 10 , C 8 , C 36 , C 1 , C 13 , C 3, C 4 , C 31 , C 5 , C 6 , C 7 , C 2 , C 9 atoms in the cyclic alkyl chain. Finally, the trans-2-dodecen-1-ol trifluoroacetate chemical shift values are found in the 3~152 ppm range. The highest chemical shift values were found at 151.74, Pharmaceuticals 2020, 13, 232 16 of 25 129.32, 121.59 and 105.65 ppm for the C 2 , C 6 , C 1 , and C 5 atoms that are bonded with a highly negative charge O atom. Besides, less than 100 ppm values are found for the C 4 , C 7 , C 13 , C 12 , C 11 , C 10 , C 9, C 8 , C 14 and C 15 atoms that are located in the straight alkyl chain. Table 6. Calculated 13 C-NMR isotropic chemical shift (TMS and chloroform solution) by the DFT/B3LYP/6-31G+ (d,p) method (atom positions numbered in the table).

Carbon
No.

Carbon
No. Computer-aided drug design (CADD) plays a significant role in developing new drugs. There are mainly two types of drug design methods available, namely: structure-based and ligand-based drug design [71]. In our previous study, we used ligand-based interactions to select a lead compound with sedative activity, which exhibited a significant binding affinity towards the human serotonin receptor (PDB: 5I6X) [22]. Here, the MAO receptor is used because MAO-A is generally targeted to treat depression and anxiety, whereas MAO-B useful for Alzheimer's disease (AD) and Parkinson's disease [72]. As oxime derivatives were reported to have antidepressant effects [17][18][19], in our present study, human monoamine oxidase A (PDB: 2Z5X) was used for a molecular docking study of antidepressant activity. The antidepressant activity is presented in Table 7. In the present study, cyclopentadecanone oxime and trans-2-dodecen-1-ol trifluoroacetate showed the highest and lowermost binding affinity against human monoamine oxidase A (PDB: 2Z5X), with docking scores of −4.333 kcal/mol and −3.155 kcal/mol, respectively. 5-Chloro-1-(trimethylsilyl)-1H-indole-2,3-dione 3-[O-(trimethylsilyl)oxime] did not show any interaction, whereas the standard drug phenelzine showed −5.324 kcal/mol binding affinity. Cyclopentadecanone oxime interacted with the monoamine oxidase A (PDB: 2Z5X) by one π-alkyl interaction to Phe 112. The interaction of the compounds is presented in Figure S5.

trans-2-Dodecen-1-ol
Human serotonin receptor (PDB: 5I6X) used also for the molecular docking study, where cyclopentadecanone oxime and trans-2-dodecen-1-ol trifluoroacetate exhibited the highest and lowermost binding affinity, with docking scores of −6.537 kcal/mol and −2.387 kcal/mol, respectively The standard drug fluoxetine shows a −9.07 kcal/mol interaction. Cyclopentadecanone oxime interacted with the human serotonin receptor (PDB: 5I6X) by one H-bond to Asp 98 and one alkyl interaction to Ile 172. The interaction of the compounds is presented in Figure S6. Table 7. Docking scores of the identified compounds from methanol extract of C. pectinata leaves.

Compounds
Docking Score (kcal/mol) The molecular docking study of cytotoxicity activity was performed against the human estrogen receptor (PDB ID: 1ERR) and epidermal growth factor receptor tyrosine kinase (PDB ID: 1M17). Cyclopentadecanone oxime gave a −7.685 kcal/mol and −4.59 kcal/mol binding interaction against the human estrogen receptor (PDB ID: 1ERR) and epidermal growth factor receptor tyrosine kinase (PDB ID: 1M17), whereas the standard drug vincristine sulfate exhibited −3.896 kcal/mol and −3.85 kcal/mol interactions, respectively. Cyclopentadecanone oxime interacted with the human estrogen receptor (PDB: 1ERR) through one H-bond to Glu 353, one π-alkyl interaction to Phe 404 and an alkyl-interaction to Leu 346. In addition cyclopentadecanone oxime interacted with the epidermal growth factor receptor tyrosine kinase (PDB ID: 1M17) through one H-bond to Met 769, and two alkyl-interactions to Val 702 and Leu 820. The interactions of the compounds are presented in Figures S7 and S8. 2.4.13. ADME/T and Toxicological Properties Analysis ADME properties and drug toxicity are important in preventing the early introduction of drugs into the commercial market. From a business point of view, it is necessary to remove the poor pharmacokinetic profile compounds, which reduces the cost of the drug development stage. As a result, over the previous decade, ADME/T screening has been applied in the early drug discovery phase [73]. The selected isolated compounds from the methanol extract of C. pectinata were subjected to the ADME/T profiling by following Lipinski's (Rule of Five) [74] and Veber's rules [75]. The three compounds 5-chloro-1-(trimethylsilyl)-1H-indole-2,3-dione 3-[O-(trimethylsilyl) oxime]; cyclopentadecanone oxime; and trans-2-dodecen-1-ol trifluoroacetate satisfy Lipinski's Rule of Five, whereas trans-2-dodecen-1-ol trifluoroacetate violated Veber's rules (Table 8). Table 8. ADME/T properties of the selected compounds in MECP by SwissADME.

Plant Materials and Preparation of Crude Extract
The details of the C. pectinata leaves (MECP) plant material were described in our earlier study [22]. The freshly collected leaves were ground into a coarse powder using a grinder (NOWAKE, Hokuto, Japan). The maceration of powder and methanol solvent was followed in a 1:4 ratio, with filtration by Whatman filter paper (#1) after seven days. The filtration was followed by eVaporation in a water bath (40 • C) to obtain a crude extract. The crude extract was kept under refrigeration at 4 • C until further use.

Experimental Animals
The average weight of 25-35 g of six-seven weeks old Swiss albino mice of both sexes was obtained from the animal house of Department of Pharmacy, International Islamic University of Chittagong (IIUC), Chittagong, Bangladesh. The animals were adapted with the laboratory condition (room temperature 25 ± 2 • C, relative humidity 55-60%) by supplying food pellets and water. For the use of the experiment, all the animals were adapted for 14 days with laboratory conditions. The study was approved by the Institutional Animal Ethical Committee, Department of Pharmacy, International Islamic University Chittagong, Bangladesh, according to governmental guidelines under the reference (Pharm/p&d/138/13-19,22/12/2019) [76].

GC-MS (Gas Chromatography-Mass Spectroscopy) Analysis of MECP
The detailed gas chromatography-mass spectroscopy (GC-MS) analysis of the methanol extract of C. pectinata leaves (MECP) were described in the earlier study of Tareq et al. [22].

Acute Toxicity Study
The acute oral toxicity of methanol extract of C. pectinata was determined by the OECD (2002) guidelines No. 423 method [77]. Mice were divided into six groups, where each group contained five animals. The first group received 1% Tween-80 in normal saline. The other groups were received 400, 600, 800, 1000, 2000 mg/kg of MECP dose. Then all the animals were observed for 8 h to detect early symptoms such as behavioral changes or mortality, morbidity and later for 3 days.

Phytochemical Screening
In the preliminary phytochemical screening of freshly prepared methanol leaves crude extract was qualitatively tested for the determination of carbohydrates, alkaloids, glycosides, tannins, terpenoids, flavonoids, and saponins [78,79].

Experimental Design for Anti-Depressant Activity
The antidepressant activity of the extract eValuated by the tail suspension test and forced swimming test. The mice were divided into four groups (n = 5). Administration of extract/control to the animals was followed after 60 min prior to study [80,81]: The antidepressant activity of MECP was executed by the method described by Steru et al. [80]. The treatment was followed as described in Section 3.7.1. After 60 min of treatment, each mouse was suspended by using adhesive tape at the tip of the tail over the rim of a box. Then the immobility time was recorded from the 6 min suspended period, whereas the first 2 min for initial adjustment and last 4 min for immobility time: where, A = immobile time in the control group; B = immobile time in the test group.

Forced Swimming Test (FST)
The antidepressant activity of MECP was eValuated by the forced swimming test, as described by Porsolt et al., [81]. A glass box (25 × 15 × 25 cm 3 ) filled to 15 cm with water (25 ± 2 • C) was utilized as a test apparatus for swimming. The treatment was followed as described in Section 3.7.1. After 60 min of treatment, each mouse was forced to swim in the apparatus. The immobility time was calculated from the 6 min swimming period. When the mice stopped struggling and remained suspended in water was considered as the immobility time and the period is recorded.
where, A = immobile time in the control group; B = immobile time in the test group.

Brine Shrimp Lethality Bioassay
The brine shrimp lethality bioassay was followed to eValuate the cytotoxicity of methanol extract of C. pectinata leaves by Meyer et al. [39]. In 1000 mL distilled water, 38 g NaCl was dissolved to prepare artificial seawater. NaOH was added to maintain the pH at 8.0. Then serially diluted concentrations of 50, 100, 200, 400, 600 and 800 µg/mL were obtained. Vincristine sulfate used as a positive control as the preceding method in a serial concentration dilution 0.125, 0.25, 0.5, 1, 5 and 10 µg/mL. Then ten matured live shrimp were placed in all test tubes at room temperature (25 ± 1 • C) and after 24 h, each test tube was assessed, and the number of alive nauplii was counted and recorded.

Quantum Chemical Analysis
Quantum chemical analysis was performed with the Gaussian 09 software package [82] via the Gauss view 6.0.10 [83] molecular visualization program on a Pentium IV/3.02Hz personal computer. The selected isolated compounds 5-chloro-1-(trimethylsilyl)-1H-indole-2,3-dione 3-[O-(trimethylsilyl) oxime], cyclopentadecanone oxime and trans-2-dodecen-1-ol trifluoroacetate were fully optimized at the level of density functional theory (DFT) using the B3LYP with the 6-31G+ (d,p) basis set. The minima of the potential energy hypersurfaces were considered to be the stationary points and confirmed from the absence of any imaginary frequency. Electronic properties, such as HOMO-LUMO energies, molecular electrostatic potential (MESP) were calculated using the B3LYP method, based on the optimized structure in the gas phase. Furthermore, Mulliken and natural bond orbital (NBO) charges and global reactivity descriptors of the proposed compounds were analyzed. Calculated vibrational frequencies were multiplied by a suiTable scaling number (0.964) [84] to better match experimental frequencies. Besides, the 1 H and 13 C nuclear magnetic resonance (NMR) chemical shift [85] (with respect to a TMS reference and chloroform solution) of the proposed compounds were also carried out by GIAO method in same method and level of basis set.

Statistical Analysis
The values are shown as mean ± standard error mean (SEM). * p < 0.001 statistical significance was calculated by one-way ANOVA (Dunnett's test) using the GraphPad Prism (version 8.4.) software (San Diego, CA, USA).

Conclusions
This study reports that methanol leaves extract of C. pectinata could be a potential source of compounds with antidepressant and cytotoxicity activity due to the presence of secondary metabolites. In addition, the computational study of the oxime derivatives by DFT and molecular docking study unveiled better binding interaction against the MAO and serotonin receptor with good pharmacokinetic and toxicological properties. Further advanced studies are recommended to identify the mechanism of action of C. pectinata.