Chemical and spectroscopic characterization of (Artemisinin/Querctin/ Zinc) novel mixed ligand complex with assessment of its potent high antiviral activity against SARS-CoV-2 and antioxidant capacity against toxicity induced by acrylamide in male rats

Ethical approval

The inhibitory effect of Art/Q/Zn novel synthesized complex against the SARS-CoV-2 was tested at the Centre of Scientific Excellence for Influenza Viruses, National Research Centre, Dokki, Egypt using the SARS-CoV-2 isolate hCoV-19/Egypt/NRC-03/2020 (EMBL-EBI, accession number SAMN14814607). The biological activity of the novel complex Art/Q/Zn was tested in male rats. The experiment was approved by the ethical approval committee of Zagazig University (approval number ZU-IACUC/2/F/61/2022). This study was also approved for evaluation of Art on lung functions by the Taif University ethical committee, accredited by the national committee for Bioethics (approval No. HAO-02-T-105). The SARS-CoV-2 strain sample used in the research has ethical approval and the accession number: (hCoV-19/Egypt/NRC-03/2020 (Accession Number on GSAID: EPI_ISL_430820). The bio-sample’s record history can be accessed through the link: https://www.ebi.ac.uk/biosamples/samples/SAMN14814607. The available phylogenetic analysis is accessible at: http://purl.obolibrary.org/obo/NCBITaxon_2697049.

Chemicals and instrumental analyses

Quercetin (Q, CAS Number: 117-39-5) (Fig. 2A), artemisinin (Art, CAS Number: 63968-64-9, Fig. 2B), zinc chloride (ZnCl₂, CAS Number: 7646-85-7), and acrylamide (Acy, CAS Number: 79-06-1) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and then used with no more purification. The analytical instruments employed are mentioned in Table 1.

Synthesis of Artemisinin/Quercetin/Zinc novel mixed ligand complex

The Art/Q/Zn complex was formed and produced by mixing an ethanolic solutions of zinc chloride (ZnCl₂) (0.001 mole, 0.137 g) with Q (0.001 mole, 0.302 g) and Art (0.001 mole, 0.283 g). Next, this mixture was completely dissolved in 25 ml of ethanol. The pH was adjusted to 8.0–9.0 using 10% ammonia solution. The solution was refluxed for 5 h using a refluxing system. Next, the solution was filtered using a semi-thin filter (25 µm) and washed with ethanol (96%) and purified distilled water. Finally, the resulting solid mixed ligand complex was dried in an oven at 80 °C and then kept in a desiccator (Fig. 3).

Cytotoxicity concentration assay

To assess the maximal (CC₅₀) half cytotoxic concentration of the newly synthesized complex of Art/Q/Zn, a stock solution of Art/Q/Zn was produced by using a 10% mixture of Dimethyl sulfoxide (DMSO), diluted with DMEM and di-distilled water. The cytotoxic activity of Art/Q/Zn was tested on VERO-E6 cells by using the MTT assay as previously described in our previous study (Al-Salmi, El-Megharbel & Hamza, 2023). Briefly, VERO-E6 cells were seeded at 100 µl per each well and were all incubated at 37 °C in a CO₂ environment for approximately 24 h. Following this, varying concentrations of Art/Q/Zn were administered to VERO-E6 cells in triplicates. VERO-E6 cells were all treated with Art/Q/Zn novel complex at different concentrations and tested in 3 times (triplicates). Subsequently, the obtained supernatants were suddenly discarded, and the cellular monolayers underwent three washes using MTT solution (5 mg/mL) of Art/Q/Zn solution in combination with sterile phosphate buffer saline. The cells were then incubated at 37 °C for 4 h. The formazan crystals were dissolved in 200 µl of isopropanol and HCl mixture. The absorbance of the formazan crystals was measured within the wavelength range of 540 to 620 nm using a multi-well plate reader. The calculation of cytotoxicity percentage was performed according to the equation:

% Cytotoxicity = (absorbance of cells without Art/Q/Zn (Control) − absorbance of VERO-E6 cells with Art/Q/Zn) ×100/absorbance of VERO-E6 cells without Art/Q/Zn.

Determination of (IC₅₀) inhibitory concentration 50%

In tissue cultures’ healthy plates, the Vero-E6 cells were all distributed and incubated (Temp. 37 °C) under CO₂ conditions overnight. Three times with phosphate buffer and then exposed to the virus adsorption (hCoV-19/Egypt/NRC-03/2020 (EPI-ISL-430820)) for 1 hr at the room temperature (RT). At 37 °C for only 1 hr, the monolayers were overlaid with DMEM media containing concentrations of Art/Q/Zn. After incubation at 37 °C for 72 h, VERO-E6 cells were treated with paraformaldehyde that was freshly prepared as (4% paraformaldehyde/PBS), just warming PBS (Phosphate buffer saline) then, with continuous vigorous stirring, slowly add paraformaldehyde, then gradually add a few drops of 0.1 M NaOH based on (O’Neil, 2006) that confirmed that paraformaldehyde dissolved in fixed alkali hydroxide solution, then filter with filter paper till obtain solution with pH 7.9 for 20 min at room temperature (RT) and stained with 0.1% crystal violet stain in distilled water for about 15 min. Then, we added methanol for each well to dissolve the crystal violet dye, and the color optical intensity of Art/Q/Zn was then measured at absorbance of 570 nm by using multi-plate readers. The IC₅₀ of Art/Q/Zn novel complex is needed to decrease the “SARS-CoV-2” cytopathic effect by 50% percentage ratio, which is relative to control SRAS-CoV-2 (El-Megharbel et al., 2021).

Treatment of animals and animal ethics approval

The animals were treated using a procedure approved by the ethical approval committee of both Zagazig and Taif Universities (the ethical approval numbers ZU-IACUC/2/F/61/2022 and HAO-02-T-105, respectively). Male rats weighing 150–180 g were obtained from the animal house of the Faculty of Pharmacy, Zagazig University. Healthy pathogen-free animals aged 6 weeks were then immediately kept at the animal house of the Zoology Department (Physiology division), Faculty of Science. The animals were housed under standard laboratory conditions (27 °C and a normal daylight cycle). Food and water were provided ad libitum.

The experiment was conducted in 40 male rats who were randomly divided into four groups. The rats were administered the treatments via the oral route using an oral gastric tube for 30 days. The treatment administered in the four groups is mentioned as follows:

Group I: control group: animals received normal physiological saline (0.9% NaCl).

Group II: animals were administered Acy (500 mg kg⁻¹) in saline solution (Hamza, Al-Motaani & Malik, 2019).

Group III: animals were administered Art/Q/Zn (30 mg Kg⁻¹) that was dissolved in normal physiological saline (Refat et al., 2021).

Group VI: animals were orally administered Acy and the Art/Q/Zn complex after 30 min, as described previously, (Figs. 4 and 5) for 30 successive days.

Samples collection

Blood samples were centrifuged at 5000 r.p.m for about 5 min to obtain the serum. The serum was used for further analysis, and it was persevered at −20 °C. The experimental male rats were suddenly decapitated after light anesthesia with xylene/ketamine (I.P), and both the liver and lung tissues were removed, 1^st part of liver and lung tissues was immediately used for the histological, ultra- structural and immune testing, Meanwhile the 2^nd part of liver tissues was weighed, and homogenized and used immediately for the evaluation of antioxidant enzyme capacities (Fig. 1). The supernatants of hepatic tissue homogenates were obtained after centrifugation at 3,000 g for 1/4 hr at 4 °C, then the supernatants were immediately collected and preserved at −20 °C for further analysis.

Biochemical investigation

Histological and immunohistochemically assessment of both liver and lung tissues

Fixed samples were processed by using hematoxylin/eosin staining of liver and lung tissue sections. Photomicrographs of the tissues were captured by light microscope. The excessed liver and lung slices (four mm thickness) were blocked with within 0.1% mix of water and methanol for about 1/4 hr to study apoptosis-related proteins. After the blocking process, both tissue sections were treated at 4 °C overnight with polyclonal caspase-3 antibody. The color intensity of caspase-3 in the immunohistochemical sections was used to classify the intensity as follows: (+) means (weak immunoreactivity), (++) means (moderate immunoreactivity), (+++) means (high and strong immunoreactivity), and (++++) means (very strong immunoreactivity).

Measurement of blood pressure

Systolic, diastolic and mean arterial blood pressures (BP) were measured using noninvasive BP measurement system (NIBP 250, Serial No: 21202-108, BIOPAC system, Inc., Goleta, CA, USA) (Abubakar, Ukwuani & Mande, 2015).

Molecular docking, physicochemical, and pharmacokinetics studies

In the current study, we analyzed the pharmacophoric features of SARS-CoV ACE2 receptor co-crystallized inhibitor novel complex Art/Q/Zn to synthesize novel compounds using the approach of ligand-based design (Daina, Michielin & Zoete, 2019). We synthesized a novel Art/Q/Zn formula that can bind with ACE2 and M^pro receptors that bind SARS-CoV-2.

Generalized molecular docking simulations of complex. We used Swiss docking tool and based on the previous study of Abo Elmaaty et al. (2022).

Statistical and data analysis

The data were expressed as mean ± SE., a one-way analysis of variance was employed for comparing several groups, using post-hoc test. Statistical significance at P ≤ 0.05 (Dean, Sullivan & Soe, 2013). We assumed that CAT in hepatic tissue homogenates in Acy group versus Acy+ Art/Q/Zn group are 2.49 ± 0.86 versus 4.21 ± 0.95 (U/g). At power 80% and confidence level 95%, sample size is 40 (10 in every group). This sample was calculated by OPEN EPI software package (Dean, Sullivan & Soe, 2013).

Physical measurements data

Zinc chloride salt reacted with the mixed ligands of Artemisinin and quercetin according to this equation: ZnCl₂ + Artemisinin (Art) + quercetin (Q) = [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O.

The proposed structure for zinc mixed ligand complex [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O (molecular formula: (C₃₀H₄₁O₁₉ClZn)) was in (Fig. 6) and elemental analysis values: (%C = 44.72, %H = 5.09, %Cl = 4.40) which shows that the molar ratio of Zn:Q:Art is 1:1:1. The novel mixed ligand Art/Q/Zn complex was stable in the air and was soluble in DMSO. The value of molar conductivity (Λ_m) was 23 ohm⁻¹⋅cm²⋅mol⁻¹, supporting the non-electrolytic nature of the mixed ligand complex (El-Megharbel & Hamza, 2022).

Infrared spectra

Infrared for quercetin (Q), artemisinin (Art), and their zinc complexity are shown in Fig. 7, the assignments for prominent vibrational bands:

For free ligand (quercetin), broad, strong bands appear at 3,563 and 3,388 cm⁻¹, referring to hydroxyl (OH) stretching vibration for polyphenolic groups. For Q with Zn(II), a robust and broadband appeared at 3,372 cm⁻¹ referring to molecules of water, which is convenient with the suggested structure (Fig. 6) of mixed ligand complex [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O. Also, for free (Q), the ν(C4=O) stretching vibration of the carbonyl group appeared at 1,679 cm⁻¹, while for [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O complexity, shifting occurred for this band in the range 1,613–1,660 cm⁻¹. It confirmed that Zn (II) can be chelated through C(4)=O and C(3)−OH which are the carbonyl oxygen atom of of the (Q) ligand (Nakamoto, 1986). The stretching vibration band for ether group ν(COC) of in the ring II for (Q) appeared at 1,269 cm⁻¹, where no shift occurred for this band after chelation. Also, ether group ν(COC) in the ring of Art appeared at 1,260 cm⁻¹. No change occurred for this band after chelation, which confirmed that this band is not involved in coordination. The ν(C−OH) for free (Q) appears at 1,409 cm⁻¹, where shifting occurred from 1,361–1,383 cm⁻¹ for [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O complexity, and this confirms the involvement of C(3)−OH phenolic oxygen group (ring II) in chelation process.

For the Art free ligand, many bands (strong and broad) appeared at 3,752 and 3,692 cm⁻¹, referring to ν(O−H) of the formed O—H hydrogen bond between the ring’s hydrogen atom and oxygen atom. Also, for free Art, the carbonyl group’s ν(C=O) stretching vibration appeared at 1,736, 1,689 cm⁻¹. In contrast, for [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O complexity, shifting for this band appeared in the range 1,652–1,722 cm⁻¹ and confirming that Zn (II) can be bonded via carbonyl (O) atom C=O of the Art ligand. Stretching vibration bands for ν(C–H), aliphatic appeared at the range 2,851–2,967 cm⁻¹, for free Art ligand and [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O complex.

For Art, the vibrational stretching band for ether group ν(COC) has appeared at 1,260 cm⁻¹, where no shift occurred after chelation, confirming that this band is not involved in coordination. The [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O has new bands appearing within wavenumbers 611–645 cm⁻¹, that are referring to stretching vibration for zinc-chloride bond (Nakamoto, 1970; Bellamy, 1975). The bands appeared at the range 497–511 and 473–449 cm⁻¹, referring to ν(Zinc−O), the stretching vibration which confirmed the formation of metal complexity (Bellamy, 1975).

UV–Vis spectra and magnetic measurement

The electronic spectrum of the free ligand quercetin has absorption bands appeared at 290 and 365 nm referring to π → π* and n →π* transitions. In comparison, Art free ligand has absorption bands appeared at 295 and 354 nm posted to π → π* and n →π* transitions, respectively (Lever, 1984). The UV-Vis spectrum for [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O novel complex has two absorption bands at 290 nm and 345 nm due to π → π* and n →π* transitions respectively (Fig. 8), (Table 2) and regarded as diamagnetic due to formation of the novel complex Art/Q/Zn caused by the conjugated system by complexation that made the bathochromic shift (Charisiadis et al., 2014). Other bands have appeared at 415 nm, 440 nm, and 455 nm for zinc-mixed ligands. These bands can be due to the M →L charge transfer transition. The measured magnetic moment value obtained for the novel Zinc mixed ligand complex lies at 3.98 BM, corresponding to the octahedral field.

¹H-NMR spectra

¹H-NMR spectrum for free Q ligand shows the following signals: (1H, C5-OH): δ 12.52, (1H, C7-OH):10.80, (1H, 3-OH): 9.54,(1H, 4′-OH): 9.61, (1H, 3′-OH):9.32, (1H, 2′-H): 7.62, (1H, 6′-H) :7.54,(1H, 5′-H): 6.89,(1H, 8-H):6.37, (1H, 6-H):6.15 (Sanna et al., 2016). For mixed ligand complex [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O (Fig. 9) the prominent signal bands appeared are: (1H,5-OH): δ 12.492, (1H, 7-OH):10.806, (1H, 4′-OH):9.67, (1H, 3′-OH):9.383, (1H, 2′-H):7.664, (1H, 6′-H) 7.661, (1H, 5′-H): 7.534,7.521, (1H, 8-H)6.874, (1H, 6-H):6.856, 6.390, 6.387. According to these data, the band appeared at δ 9.54 for free Q assigned to (1H, 3-OH) disappeared for [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O, and other signal bands not shifted, confirming that hydroxyl group at C-3OH is deprotonated and involved in coordination with Zn(II). Also, there is a new signal band appeared at δ 3.335 ppm for [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O,which is assigned to protons of H₂O coordinated and uncoordinated. Based on the above data, the complexation structure for [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O was confirmed.

X-ray diffraction analysis

X-ray powder diffraction (XRD) patterns were used to determine the crystallinity of the novel mixed ligand Zinc complex at the value of (2θ) 4–80° and, also used to examine the nanostructural form of the zinc mixed ligand complex. For X-ray diffractogram of [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O, a broad peak at 2θ = 23° appeared, confirming that the zinc mixed ligand complex has an amorphous structure (Hamza et al., 2022; Al-Thubaiti et al., 2022), as shown in Fig. 10. All our attempts to prepare single crystals failed.

Scanning electron microscopy SEM and EDX

The technique of SEM determine the microscopic and physical character of Art, (Figs. 11A, 11B); Q, (Figs. 11C, 11D), and [Zn(Q)(Art)(Cl) (H₂O)₂]⋅3H₂O. (Figs. 11E, 11F). SEM can be taken as an indication for the presence of a single component for [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O. The images of [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O clarify nano-feature products with small particle size. The morphological surface for the novel mixed ligand Zinc complex was checked using SEM, showing a small particle with a high more ability to form agglomerates with different shapes. EDX clarified the elemental analysis of the novel complex with percentage (Fig. 12).

Transmission electron microscopy

TEM images for ligands: free Art, (Figs. 13A, 13B); Q (Figs. 13C, 13D), and synthesized [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O complex Fig. 13 (E,C2). The uniform matrix for [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O complex was cleared in the pictograph and this confirms that [Zn(Q)(Art)(Cl)(H₂O)₂]⋅3H₂O complex has a homogeneous phase where spherical black spots like shape is observed for Zn(II) complex with the particle has size ranged of 31.99–48.13 nm.

Cytotoxicity assay

Art/Q/Zn novel complex showed highly potent inhibitory activity against the SARS-CoV-2 in very low concentration (IC₅₀ = 10.14 µg/ml). This assay assessed the possible cellular cytotoxicity of Art/Q/Zn (CC50 = 208.5 µg/ml) (Fig. 14) and attached an image for crystal violet analysis with silver crystals formed, successive dilution and high anti-viral activity of Art/Q/Zn by appearance of pale purple color as cells that undergo cell death lose their adherence. They are subsequently lost from the population of cells that indicate inhibition of SARS-CoV-2 cell growth.

Biochemical evaluation

AST, ALT, and LDH serum enzyme levels were significantly increased in the Acy group, as shown in Table 3 and Fig. S1. The current findings indicated disruption of the hepatic cellular membranes. Meanwhile, Art/Q/Zn mixed ligand novel complex at a dose of 30 mg/kg was found to be safe for hepatic enzyme activity. Male rats were given Acy and then treated with Art/Q/Zn, which resulted in a decrease in hepatic enzymatic and function activity.

CRP, IL-6, and TNF-α levels were significantly elevated in the Acy treated group as compared with control group (Table 4). The CRP, IL-6, and TNF-α levels were markedly reduced in groups either treated with Art/Q/Zn alone or combined with Acy administration (Table 4).

Table 5 clarified that Acy afforded oxidative injury in the hepatic tissues, the level of the antioxidant enzymes were significantly declined due to treatment with Acy. Acy markedly declined CAT, SOD, and GPx activities. The Acy-group showed higher MDA levels than the control group, which is an indicator of an increment of oxidative injury. Meanwhile, administration of Art/Q/Zn afforded significant increase in the antioxidant enzymes CAT, SOD and GPx activities with reduction of MDA level (Table 5).

Histological and ultrastructural examination of liver and lung tissues (histological, TEM, and immunostaining sections) with live sections clarifying variations in the structure of studied tissues

Following Acy administration, hepatic tissues displayed toxicity in the form of fatty change with considerable and elevated hepatocyte degeneration, as well as a congested portal vein with severe hemorrhage and infiltration of blood sinusoids by mononuclear inflammatory cells (Fig. 15). After the administration of Art/Q/Zn, the liver showed alleviation of hepatotoxicity with restoration of normal hepatic tissues (Fig. 15).

TEM examination of hepatic tissues showed normal structure in the control group, and Art/Q/Zn treated group with normal nucleus with clear nuclear boundaries, normal-sized mitochondria, and endoplasmic reticulum. Meanwhile, the treated group with Acy and Art/Q/Zn showed restoration of the normal hepatic structures with regular nuclear boundaries and significantly alleviated the hepatotoxicity (Fig. 16).

Immunostaining of the hepatic tissue sections showed high immunoreactivity for caspase-3 in the hepatic tissues of Acy treated group. Meanwhile, the immunoreactivity for caspase-3 was very weak in the Acy treated group and followed by Art/Q/Zn, as shown in (Fig. 17).

After administration of Acy and Art/Q/Zn, rat lung tissues showed pulmonary toxicity in the form of congested blood vessels with scattered aggregates of inflammatory cells, and appearance of granular debris of fibrin and congested blood vessels with areas of hemorrhage with non-specific inflammatory cells (Fig. 18). Administration of novel Art/Q/Zn after Acy improved the structure of lung tissues and showed alleviation of pulmonary fibrosis with restoration of normal structure of the pulmonary tissues (Fig. 18).

TEM of lung tissues showed normal structure in the control and the Art/Q/Zn treated groups with appearance of normal nucleus, basement lamina, alveolar sacculus, and normal Bronchioles. Meanwhile, the Acy treated group showed sizeable red blood cells that entirely blocking of the air sacs with appearance of small granules with detaching of most pulmonary tissues with pulmonary fibrosis. Meanwhile, normal pulmonary sacculus and alveolar epithelial cells were restored in Acy and Art/Q/Zn treated group with alleviation of the pulmonary fibrosis and hemorrhage (Fig. 19).

Immunostaining of the pulmonary tissues showed high immunoreactivity for caspase-3 in the group treated with Acy. Meanwhile, the immunoreactivity for caspase-3 was very weak in Acy treated group and followed by Art/Q/Zn, as shown in (Fig. 20).

Figure 21 demonstrated various morphological alterations in both lung and liver tissues of different treated groups. Lung tissues of Acy treated group as shown in Fig. 21A showed large lesions with congested tissues and bleeding. On the other hand, the lung tissues of Art/Q/Zn treated groups as shown in Fig. 21B are of normal appearance with a bright red color and without any lesions or any structural changes. Liver of Acy treated group as shown in Fig. 21 (C1, C2, and C3) showed hepatomegaly, dark oxidative color, and some lesions. Meanwhile, liver tissues of Art/Q/Zn treated groups as in Fig. 21 (D1, D2, and D3) showed bright normal sized hepatic tissues without any appeared lesions.

Effect of Art/Q/Zn on blood pressure levels

The control group showed standard heart rate with normal blood pressure recorded by systolic pressure 134.9 mmHG, diastolic pressure 108.7 mmHG and heart rate 263.2 beats/min (bpm) (Fig. 22A). Acy induced elevation in systolic and diastolic blood pressure by 188.20 mmHG and 162.02 mmHG, respectively with slightly higher heart rate recorded as 297.0 beats/min (bpm) than that of the control group (Fig. 22B). The Art/Q/Zn complex caused a significant reduction in systolic pressure in the control group. The Acy group recorded 102.05 mmHG as a systolic blood pressure and diastolic blood pressure as 82.94 mmHG after successive 30 days of treatment. Still, with a significant increment in heart rate by 379.78 beats/min (bpm) (Fig. 22C). However, treatment of male rats with a combination of Acy followed by the Art/Q/Zn complex induced a more pronounced reduction in systolic and diastolic blood pressures (by 134.95 and 263.54 mmHg) than in the Acy-only treated group with the lowering of heart rate recorded as (263.54 beats/min) (bpm) (Fig. 22D), All measurements for systolic and diastolic blood pressure with recording heart rate for all treated groups were carried out by digital blood pressure measurement system (NIBP250, BIOPAC systems, Inc., Goleta, CA, USA) and values were measured in rats after putting in the streamer (Fig. 22E). Systolic and diastolic values were shown in both (Fig. S2 and Table 6).

Molecular docking assays

In the initial phase of the docking assay, the program was set up by redocking the novel formula (Art/Q/Zn) against two receptors. Angiotensin-converting enzyme-2 (ACE2). This receptor may generate a highly potent protective effects in the COVID-19 patients by reducing the severe respiratory symptoms risk, and M^pro targets the main protease receptor for SARS-CoV-2. It is the the key and vital enzyme of coronaviruses and has a vital role in mediating the highly viral replication and transcription, as shown in Fig. 23A (v1-v6). Also, lipophilicity capacities were shown as most therapeutics essentially used specialized transport systems of the body, and instead of that mechanism, others tend to diffuse via the cellular membrane. To do so, therapeutics must be sufficiently lipophilic. So, if therapeutics showed lipophilicity affinity, this enables easy cellular diffusion, thus getting a much better effect of the therapeutics. Figure 23 showed high lipophilicity capacities of novel synthesized complex (Art/Q/Zn) with both SARS-CoV-2 receptors (ACE2) (Fig. 23B) and primary SARS-CoV-2 inhibitor (M^Pro) (Fig. 23C).

Molecular dynamic simulation

The molecular dynamic (MD) simulation was performed for Art/Q/Zn at (100 ns) by using, a Package of Schrödinger (Desmond) Fig. 23A. Simulations were carried out to predict the ligand binding status in the physiological environment to mimic the expected binding of the novel complex formula (Art/Q/Zn) with the primary receptors of ACE2 and M^Pro (The main protease of SARS-CoV-2) (Figs. 23B and 23C).

Histogram and heat map analyses

Histograms were described in Fig. 24 (A1 and B1) and heat maps are shown in Fig. 24 (A2 and B2) for the SARS-CoV-2 ligand-protein of Art/Q/Zn novel complex with ACE2 and M^Pro, respectively, during the simulation time (100 ns).

Ligand properties

Regarding the novel complex formula Art/Q/Zn with ACE2 Fig. 24 (A3), GLN-72 contributed mainly about 99%, besides GLN-59 contributed mainly about 49%, followed by LYS-51, LEU-54 and PHE-55 contributed mainly as follows, respectively (41,66, 41,38 and 31%) of the interactions as H-bonding; However, ILE-61, VAL-93, ILE-19 and MET-62 formed essentially the hydrophobic interactions. LYS-51, LEU-54, PHE-55, GLN-59, and GLN-72 were the most members contributing to the water-bridges, with no record of ionic bonds. GLN-72 was the most essential participating amino acid in the interactions through hydrogen bonds.

By analyzing the docking results for our synthesized complex against ACE2 and M^pro receptors of SARS-CoV-2, we can conclude that the protein-ligand contact is as follows: the reduced cocrystallised formula formed hydrogen bonds in case of ACE2 with GLN-18, ILE-19, GLN-24, LYS-51, LEU-54, PHE-55, LEU-57, GLY-58, GLN-59, ILE-61, MET-62, TYR-67, GLN-72, VAL-75, VAL-93, LYS-94, HIS-96 and TYR-100 incase of ACE2 receptor (Fig. 24 (A3)) and PHE-8, LYS-102, VAL-104, ARG-105, ILE-106, GLN-107, GLY-109, GLN-110, THR-111, ASN-151, ILE-152, ASP-153, TYR-154, CYS-156, SER-158, CYS-160, ASN-203, ASP-248, PHE-294, THR-292, ASP-295, ARG-298 and GLN-306 incase of M^Pro with a very high rate of activity and the novel complex Art/Q/Zn showed a binding interaction energy as shown in Fig. 24 (B3).

Additionally, the SARS-CoV-2 ligand-protein of the novel complex Art/Q/Zn with the receptor M^pro during the simulation time (100 ns) as described in Fig. 24 (B3). Regarding the novel complex Art/Q/Zn with M^Pro, GLN-10 contributed about 99%, besides PHE-8 contributed only about 60%, followed by ASP-298 and THR-111, which contributed as follows, respectively (58 and 57%) of the interactions as H-bonding. However, PHE 294 and LYS-102 formed mostly the hydrophobic interactions. ASP-248, THR-111, CYS-156 and GLN-110 were the main members contributing to the water-bridges, no record of ionic bonds in both ILE-152 and ARG-298. Obviously, GLN-10 was the most participating amino acid in the interactions through hydrogen bonds.

Root mean square deviation analysis

The root mean square deviation (RMSD) values of Cα atoms were evaluated the novel complex Art/Q/Zn to monitor the effect of this novel complex on the conformational the high stability of ACE2 and M^pro receptors during the current simulations—the results as seen in Fig. 24 (A4 and B4). The fluctuation of the proteins was within acceptable variation with RMSD values that were less than 2.00° A in the case of ACE2 and less than 3.00° A in the case of M^Pro, indicating the stability of the protein conformation.

The features of the ligand, including the RMSD, radius of gyration (rGyr), solvent accessible surface area (SASA), the intramolecular hydrogen bond (intraHB), molecular surface area (MolSA), and polar surface area (PSA), all these features are shown in Fig. 24 (A4 and B4). Other ligand characteristics are reported in the root mean square deviation (RMSD).

The RMSD and rGyr for Art/Q/Zn novel complex with ACE2 were determined to be within the range of (0.7–1.9) Å. Also, no intraHB bands were observed during the 100 ns of simulation, and the MolSA range was within (65–512 Å2). Additionally, the SASA was within the (35–400 Å2). Additionally, its PSA range was within the range of 10 and 130 Å2 (Fig. 24) (A4).

The RMSD and rGyr for Art/Q/Zn novel complex with M^Pro were observed within the (0.3–2.6)Å range, respectively. Also, intraHB with bands were observed range to∼2 during the 100 ns of the current simulation, and the MolSA range was within (70–285 Å2). Furthermore, the SASA was within the range of (80–320 Å2). Moreover, its PSA range was between 25 and 260 Å2 (Fig. 24) (B4).

The ligand properties showed fluctuation and torsion at the beginning of the simulation Fig. 24 (A5 and B5) before reaching the equilibrium state, indicating the high stability of the Art/Q/Zn complex to the main active site of the SARS-CoV-2 main protease active sites.