Anti-SARS-CoV-2 in vitro potential of Castor Oil Plant (Ricinus communis) Leaf Extract: In-Silico Virtual Evidence

doi:10.21203/rs.3.rs-1979637/v1

Background Ricinus communis L. is a medicinal plant displays valuable pharmacological properties. Diverse phytochemical constituents display valuable pharmacological properties, including antioxidant, antimicrobial, analgesic, antipyretic, antibacterial, antiviral, and anti-inflammatory property. This study targeted to isolate and identify some constituents of R. communis leaves using ultra-performance liquid chromatography coupled with mass spectroscopy (UPLC-MS/MS) and different chromatographic techniques, then characterize the potential cytotoxicity, anti-MERS-CoV and anti-SARS-CoV-2 activity in vitro. Isolated phytoconstituents and remdesivir are assessed for in-silico anti-COVID-19 activity by inhibiting the main protease and spike protein using molecular docking tools.

Methods: The CH₂Cl₂ fraction was subjected to repeated chromatographic separation to isolate the phytochemicals, and their structures were elucidated using nuclear magnetic resonance spectroscopy. UPLC-Triple TOF-MS/MS was performed to determine the different phytochemicals in the CH₂Cl₂ fraction. The in vitro anti-MERS and anti-SARS-CoV2 activity for different fractions and for two pure isolated compounds, lupeol (RS) and ricinine (RS1) were evaluated using Plaque reduction assay and IC₅₀ based on their cytotoxic concentration (CC₅₀) from an MTT assay using Vero E6 cell line. Molecular docking studies were carried out for both SARS-CoV-2 spike (S) and main protease (M^pro) receptors then examined the possible mechanisms of action.

Results: The methylene chloride extract exhibited pronounced virucidal effect with more than a 90% viral inhibitory effect, it showed activity against SARS-CoV- 2 (IC₅₀ = 1.76µg/ml) with high safety index, SI = 291.5. It was also shown that ricinine had superior potential activity against SARS-CoV-2, (IC₅₀ = 2.5 µg/ ml). This constituent was less effective for MERS, IC₅₀ = 87.2 µg/ ml. Lupeol displayed the most potency against MERS, (IC₅₀ = 5.28 µg/ ml), SI = 67.27, but was less effective for SARS, IC₅₀ = 19.5 µg/ ml. Ricinine showed significant binding to (3CL^pro) and modest affinity for (S) spike protein, along with a possible interaction with SARS-CoV-2 major protease. Ricinine appeared to be the most biologically active.

Conclusion: The study showed that Ricinus communis and its isolated compounds have potential natural virucidal activity against SARS-COV-2, however, additional exploration is necessary for further chemical modification of these structures, guided by the molecular docking tools and study for their in vivo activity.

Ricinus communis

SARS-CoV-2

MERS-CoV

Virucidal activity

Docking study

UPLC-QTOF-ESI-MS/MS

SARS-CoV-2, the third challenging coronavirus to infect humans in the previous 20 years, is the cause of coronavirus disease 2019 (COVID-19) [1]. The virus is a member of the family of zoonotic viruses known as Coronaviridae, which includes other viruses that caused pandemic outbreaks in 2003 and 2012 due to the SARS-associated coronavirus (SARS-CoV-2) and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), respectively [2, 3]. Both are highly contagious and can lead to high mortality [4, 5]. The greatest global catastrophe since the Second World War, however, has been the spread of COVID-19. Due to this virus's quick spread and high level of contagiousness, a worldwide health disaster resulted. SARS-CoV-2 continues to be an issue for human health in terms of morbidity and mortality as well as financially [6]. The heart [7], kidney [8], liver [9], and brain [10] are just a few of the extrapulmonary organs and tissues that SARS-CoV-2 damages.. The virus also has an effects on the ophthalmic system [11], the gastrointestinal system [12], the musculoskeletal system [13], the skin [14], and the cardiovascular system [15, 16]. Because of the virus's quick spread, scientists and public health organizations are looking into investigate the possibility of repurposing existing drugs for the potential treatment of COVID-19 [17]. Effective treatment for COVID-19 and reduction in mortality rate are foremost priorities for clinical treatment. To date, few specific and efficient pharmacological therapies for COVID-19 have been identified. In order to slow the course of the illness in hospitalized patients, intravenous remdesivir was officially authorized by the FDA in October 2020 [18]. Recently, the FDA approved the oral antivirals Paxlovid (nirmatrelvir tablets and ritonavir(, and molnupiravir for the treatment of COVID-19 in patients who are not hospitalised [19].

Nature provides an immense library of novel chemicals to explore and develop drugs to treat various ailments, including viral diseases [20]. Almost top one-third of the drugs on the market are natural compounds (NPs) or their derivatives [21]. Often, more efficient drugs can be designed based on the structure of natural compounds that exhibit the desired property. NPs isolated from plants and microorganisms are recognized for their antiviral activity via numerous mechanisms to prevent infection and strengthen host immunity [22]. NPs or secondary metabolites are a significant source of compounds required to develop new antiviral agents; NPs include phenolic acids, terpenes, flavonoids, coumarins, lignans, alkaloids and proteins that play an essential role in inhibiting viruses and acting as complementary therapies against viral infection. The low toxicity of herbal medicine further supports screening for anti-COVID-19 agents [23, 24]. Many natural products have been tested in terms of their activity against coronaviruses and showed much potential for the treatment of coronavirus infection. Bioactive compounds use a variety of mechanisms to inhibit coronaviruses; inhibition of ACE2, 3CL^pro, and PL^pro are the three most attractive targets for drug development [25].

Ricinus communis L., commonly known as castor oil plant, is a flowering plant in the Euphorbiaceae. This soft-wooded shrub is found across the tropical world [26]. The plant has a global history in traditional medicine spanning thousands of years. Castor oil plant has long been used to treat liver infections, stomach aches [27], flatulence, constipation [28, 29], inflammation, warts, colic, enteritis, fever, headaches, and as an anti-irritant. Hepatoprotective [30], anti-nociceptive [31], antioxidant [32], anticancer [33], anti-inflammatory [34], antidiabetic [35], antifertility [36], antimicrobial [37], and wound healing activity [38] are among many therapeutic properties attributed to castor oil phytochemicals. From a phytochemical perspective, it has been well investigated; alkaloids, flavonoids, tannins, sterols, and terpenes, including kaempferol, quercetin, gallic acid, ellagic acid, rutin, and ricinin, are hallmark constituents [39, 33, 28]. Numerous phytochemicals from R. communis are well-known antiviral agents [40–42] against HSV and Sindbis virus, confirming their traditional use in treating colds and fever [43]. Moreover, HAV, COXB4, and HSV are all susceptible to the antiviral effects of four fractional extracts of castor oil leaves, which include methanol, methylene chloride, ethyl acetate, and butanol [44].

An investigation of medications that target the viral protease revealed that viral protease was recognized as an appealing target to inhibit COVID-19 replication [24]. 3-chymotrypsin-like protease (3CL^pro), papain-like protease (PL^Pro), and spike (S) proteins are the main targets for anti-SARS-CoV-2 agents [45]. 3CL^pro is regarded as an attractive target among coronaviruses since it is essential in the processing of viral polyproteins translated from viral RNA. PL^pro is an RNA-dependent RNA polymerase. Human angiotensin-converting enzyme (ACE-2) and S-proteins intimately interact, enabling viral entry into cells. For anti-SARS-CoV-2 potential, certain medication candidates have previously undergone in-silico study. Molecular docking has been used to find phytoconstituents that potentially target the primary protease for the treatment of nCoV-2019 [46]. In-silico identification of targets for natural products (NPs) is an efficient, low-cost, time-effective, and valuable virtual screening method. The two in silico approaches that are most extensively used are quantitative structure-activity relationship (QSAR) and molecular docking. The latter recognizes target biomacromolecules (DNA, RNA and proteins) for synthetic compounds and NPs [47, 48]. The technique also aids in addressing the processes by which NPs interact with their target proteins and could offer a plan for creating novel medications generated from NPs. Ligand-protein docking is used to develop several NP based drugs [49]. Understanding COVID-19 replication and infection processes using docking may advance the core idea of structure-based drug design. The latter may lead to the discovery of promising COVID-19 antiviral drugs [20].

Thus, the focus of this work is isolation and identification of constituents of R. communis leaves using ultra-performance liquid chromatography coupled with mass spectroscopy (UPLC-MS/MS) and different chromatographic techniques, then evaluate the potential cytotoxicity, anti-MERS-CoV and anti-SARS-CoV-2 activity. Moreover, we demonstrate in-silico anti-COVID-19 potential for inhibiting the main protease and spike protein using molecular docking tools for phytoconstituents and remdesivir.

Plant material

Ricinus communis L. leaves (castor oil plant) were collected in June 2019 from Bilbeis, Sharqiyah Governorate, Egypt (31.1166⁰ N and 30.63333⁰ E). The plant material was collected in compliance with institutional, national, and international (WHO) guidelines on good agriculture and collection practice (GACP) for medicinal plants. The planters approved the permit for the collection of leaves specimens. and the samples were kindly authenticated by Mrs. Therisa Labib, consultant in plant taxonomy at the Orman Botanical Garden, Giza, Egypt. A voucher specimen (no. R. (03)2017) was deposited at the herbarium of the Pharmacognosy Department, Faculty of Pharmacy (for Girls), Al-Azhar University, Cairo, Egypt. The plant name was verified with The Plant List (http://www.theplantlist.org/) on 15/07/2020.

Preparation of crude extracts

Powdered air-dried leaves of R. communis (1.5 kg) were exhaustively extracted three times at room temperature using 70% methanol (3 L) for three days. The methanolic extract was combined and concentrated under vacuum at 45°C to afford a brown crude extract (560 g). The concentrated extract was suspended in distilled H₂O (700 ml) and filtered through filter paper. The filtrate and washings were combined and defatted with petroleum ether. The process was repeated (3 times × 500 ml), and the obtained defatted extract (350 g) was concentrated using a rotary vacuum evaporator. The aqueous concentrated defatted crude extract was successively partitioned into methylene chloride, and ethyl acetate fractions by extracting four times with 1.5 L of each solvent sequentially. The remaining aqueous layer was dried under a vacuum then extracted with one liter methanol to provide the aqueous methanol fraction. Each fraction was pooled and concentrated under vacuum at a temperature not exceeding 40°C to produce 70 g, 55 g, and 35 g of the methylene chloride, ethyl acetate, and methanol fractions, respectively. Active extract constituents were identified using ultra-performance liquid chromatography coupled with hybrid triple time-of-flight mass spectrometry (UPLC-Triple TOF-MS/MS) analysis and column chromatography [44].

Isolation and purification using column chromatography

The methylene chloride fraction (30 g) was loaded onto a silica gel column, eluted with 100% petroleum ether_, followed by a gradual increase in polarity using dichloromethane (CH₂Cl₂). Similar fractions were combined based on thin-layer chromatography (TLC) patterns into four main fractions (A-D). Fraction B (12 g at 50% Pet. Ether) was further subjected to a series of column chromatographic techniques including silica gel column and eluted with Pet. Ether: CH₂Cl₂ (95:5–10:90). Final purification used gel filtration (Sephadex LH-20) and elution with 100% MeOH to produce compound RS (35 mg). Fraction D (10 g at 100% CH₂Cl₂) was loaded onto a silica gel column and eluted with 100% CH₂Cl₂_, followed by a gradual increase in polarity by adding MeOH until reaching 100% MeOH. Similar fractions were combined based on TLC pattern into six main fractions (a-f). 100% CH₂CL₂ fraction (a, 10 g) was further subjected to a series of column chromatographic techniques, including silica gel column chromatography elution with CH₂Cl₂: MeOH (95:5–10:90) and final purification by gel filtration (Sephadex LH-20) eluted with 100% MeOH to produce compound RS1 (20 mg) [44].

High-resolution UPLC-Triple TOF-MS/MS analysis

The methylene chloride fraction was completely dried and then analyzed using a Triple TOF 5600+ System (AB SCIEX, Canada) [50]. LC columns were used for the separation process; IN-Line filter disks column (0.5 µm × 3 mm; Phenomenex) as a pre-column, and X select HSS T3 ( 2.5 µm, 2.1 × 150 mm; Waters, USA) as a main column. The LC column temperature was set to 40 °C. In 1 ml of mobile phase A working solution (5 mM ammonium formate buffer pH 3 containing 1% methanol) was added to the plant sample (50 mg) to prepare 20 µl of stock solution (50/1000 µl), vortexed for 2 min followed by ultrasonication for 10 min, centrifugation for 5 min at 10000 rpm, and then the stock sample was diluted with 1000 µl of reconstitution solvent. The final injected concentration was 10 µg/µl. The eluents were (A) 5 mM; ammonium formate buffer pH 3 containing 1% methanol for the positive mode, (B) 5 mM ammonium formate buffer pH 8 containing 1% methanol for the negative mode, and (C) 100% acetonitrile for the positive/negative mode. They were applied with multi-step linear gradient as follow; mobile phase A or B with gradient increase from (10%–90%) of mobile phase C over 28 min at a flow rate of 0.3 ml/min to allow comprehensive elution of the different analytes. Analyst software TF 1.7.1 (SCIEX) for LC-QTOF control. Metabolite identification was performed by comparing regions of different classes using the retention time and mass spectrometry data (accurate mass, formula, and fragmentation pattern) in both ion modes with those previously reported in the literature [32, 51-53].

UPLC-MS/MS is a sensitive and powerful universal analytical technique for qualitative and quantitative analysis and identification of different classes of secondary plant metabolites. Hence, both negative and positive ionization modes were used to identify R. communis leaf metabolites.

Preparation of stock solutions of tested plant samples

Stock solutions of the tested plant samples were prepared in 10% DMSO in ddH₂O and stored in stock concentrations of 100 mg/ml and 5 mg/ml for crude extracts and pure compounds, respectively. Further dilutions for stock solutions were made with DMEM for use in in vitro experiments.

In vitro studies

Cell line and viruses

African green monkey kidney–derived Vero-E6 cells (ATCC No.CRL-1586) were purchased from the American Type Culture Collection, USA, and sub-cultured in DMEM supplemented with fetal calf serum (FCS; 10% v/v - Gibco), L- glutamine (2 mM) and 2% antibiotics, antimycotic, penicillin (100U/ ml), and streptomycin (100 µg/ml) after subculture in growth media the cells were used for the antiviral assays with infection media 4% bovine serum albumin, and 2% antibiotic Antimycotic. Propagation of cells for up to 4 weeks; then, tissue culture flasks were incubated in a 5% CO₂ humidified atmosphere at 37°C as a cell line. Middle East Respiratory Syndrome (MERS-CoV) related coronavirus isolate NRCE-HKU270 (Accession Number: KJ477103.2), and Severe Acute Respiratory Syndrome SARS-CoV-2 hCoV-19/Egypt/NRC-03/2020 (Accession Number on GSAID: EPI_ISL_430820) were propagated on Vero E6 cells as previously reported [54] and harvested after the appearance of cytopathic effects (CPE). The viral stock was titered using a plaque assay [55] and stored at −80°C. All experimental work involving the SARS-COV-2 virus was performed in a biosafety level 3 (BSL3) containment laboratory.

MTT Cytotoxicity assay

This assay assesses concentrations of compounds that cause toxicity to 50% of cells (CC₅₀). Stock solutions of tested compounds were prepared in 10% DMSO in ddH₂O and diluted with Dulbecco’s Modified Eagles Medium (DMEM). Cytotoxic activity of the extracts was tested in VERO-E6 cells using the 3-(4, 5-dimethylthiazol -2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method with minor modification [56]. Briefly, Vero-E6 cells were cultivated in 96 well-plates (100 µl/well at a density of 3×10⁵ cells/ml) and incubated for 24 h at 37°C in 5% CO₂. After 24 h, cells were treated with various concentrations of the tested compounds diluted with DMEM in triplicates. 24 h later, supernatants were discarded, and cell monolayers were washed with sterile 1x phosphate buffer saline (PBS) 3 times. MTT solution (20 µl of 5 mg/ml stock solution) was added to each well and incubated at 37°C for four h followed by media aspiration. In each well, the formed formazan crystals were dissolved with 200 µl of acidified isopropanol (0.04 M HCl in absolute isopropanol = 0.073 ml HCL in 50 ml isopropanol). A multi-well plate reader measured the absorbance of formazan solutions at λ max 540 nm with 620 nm as a reference wavelength. Percent cytotoxicity compared to untreated cells was determined as:

A plot of percent cytotoxicity versus sample concentration was used to calculate the concentration, which produced 50% cytotoxicity (CC₅₀).

Screening for Antiviral activity by Plaque reduction assay

Assays were performed as previously described [55] and modified [57]. In a six well plate where Vero E6 cells (10⁵ cells/ml) were cultivated for 24 h. at 37°C. MERS-CoV and SARA-CoV-2 were diluted to give 10³ PFU/ well, and mixed with safe concentrations of tested compounds and incubated for 1 hour at 37°C before being added to the cells. Growth medium was removed from the cell culture plates and cells were inoculated with (100 µl / well) viral suspension with the tested compounds, After one hour for virus adsorption, 3 ml of DMEM supplemented with 2% agarose, and the tested compounds were added onto the cell monolayer, agarose was allowed to solidify and plates were incubated at 37°C until the formation of viral plaques (3 to 4 days). Formalin (10%) was added, and incubation continued for two hours plates were stained with 0.1% crystal violet in distilled water. Control wells were included where untreated virus was incubated with Vero E6 cells as a negative control, and Remdesivir was used as a positive control for antiviral assays. All experiments were performed in triplicate. Finally, plaques were counted and percentage reduction in plaque formation in comparison to control wells was recorded as

% inhibition = viral count (untreated) - viral count (treated)/viral count (untreated) x 100

Inhibitory concentration 50 (IC₅₀) determination

In 96-well tissue culture plates, 2.4×10⁴ Vero-E6 cells were distributed in each well and incubated overnight at 37°C in a humidified incubator with 5%CO₂ condition. Cell monolayers were then washed once with 1x PBS and subjected to virus adsorption for one h at room temperature (RT). Cell monolayers were further overlaid with 100μl of DMEM containing varying concentrations of the test compounds and incubated at 37°C in a 5% CO₂ incubator for 72 h. Cells were fixed with 100 μl of 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet in distilled water for 15 min at RT. The crystal violet dye was then dissolved using 100 μl absolute methanol per well and optical density measured at 570 nm using an Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The IC₅₀ of the compound is the concentration required to reduce virus-induced cytopathic effects (CPE) by 50% relative to virus controls [57].

Mode of action of virus inhibition

The possible mode of action of virus inhibition by the selected plants’ extracts was examined at three different stages of the virus propagation cycle and based on three main possible modes of action: (i) Inhibition of budding and viral replication. (ii) The ability of each extract to inhibit the attachment of the virus to infected cells-membrane fusion known as blocking the viral entry (viral adsorption); and (iii) The direct effect of each extract to inactivate the virus viability (virucidal activity) [57]. For consistency, each mode was repeated three times independently of each other and the mean value of the three experiment of each mode was presented. Additionally, the above-mentioned mode of action could account for the recorded antiviral activities either independently, or in combinations. In this regard, the interaction between the selected plants’ extracts.

Post infection treatment (Viral replication)

In a 6-well plate where VERO-E6 cells were cultivated (105 cells/mL) for 24 h at 37 ^oC. The virus was diluted to 103 PFU/well and applied directly to the cells and incubated for 1 h at 37 ^oC for complete adsorption. Unabsorbed viral particles were removed by washing cells 3 successive times with supplements free-medium. The extract was applied at different concentrations, after 1 h contact time, add 3 mL of 2X DMEM medium supplemented with 2% agarose to the cell monolayer. Plates were left to solidify and incubated at 37 °C till the appearance of viral plaques. Cell monolayers were fixed in 10% formalin solution for 2 h, and stained with crystal violet. Control wells were included where Vero-E6 cells were incubated with the virus and didn’t treat with the extract. Finally, plaques were counted and percentage reduction in plaques formation in comparison to control wells was recorded [57].

Cell pretreatment (protective activity) Viral adsorption

This protocol was used to test for virus entry into the host cells by blocking attachment to the cell surface. Vero-E6 cells were cultivated in a 6 wells plate (105 cells/mL) for 24 h at 37 ^oC for the viral adsorption assay. The plant extract was applied at different concentrations in a 200 µL medium without supplements and co-incubated with the cells (pre-treatment) for 2 h at 4 ^oC. The unabsorbed extract was removed by washing cells 3 successive times with supplements free-medium then virus diluted was co-incubated with the pretreated cells for 1 h followed by adding 3 mL DMEM supplemented with 2% agarose. Plates were left to solidify and then incubated at 37 ^o C to allow the formation of viral plaques, fixed and stained as above mentioned to calculate percentage reduction in plaques formation in comparison to control wells where untreated Vero-E6 cells were directly infected with the virus [57].

Virus pretreatment (Virucidal)

The virucidal assay was carried out in a 6 wells plate where Vero-E6 cells were cultivated (105 cells/mL) for 24 h at 37 ^o C. A volume of 200 µL serum-free DMEM containing virus was added to the concentration of the tested extract. After 1 h incubation, the mixture was diluted using serum-free medium 3 times each 10-fold which still allows the existence of viral particles to grow on Vero-E6 cells but leaves nearly no extract and 100 µL of each dilution was added to the Vero-E6 cell monolayer. After 1 h contact time, DMEM over layer was added to cell monolayer. Plates were left to solidify and then incubated at 37 ^oC to allow the formation of viral plaques, fixed and stained as above mentioned to calculate percentage reduction in plaques formation in comparison to control wells where cells were infected with the virus that was not pretreated with the tested extract [57].

Molecular Docking

Molecular docking used MOE-Dock 2014 software [58]. Chemical structures of compounds RS, RS1, and remedisvir were drawn with MOE builder, after that minimization was done by using the force field MMFF94x. Water molecules were removed, and hydrogen atoms were added. Molecular docking was then accomplished using London dG and GBVI/WSA dG. Meanwhile, refinement was done by force field. 2D visualization of protein–ligand interactions, used “Ligand Interactions”. And the best pose was selected based on binding energy and binding interactions at the active site.

Identification of isolated compounds

Two compounds were isolated and identified from R. communis: lupeol and ricinine (Fig. 1, table 1). The isolated compounds were identified using spectroscopic techniques, including ultraviolet-electrospray ionization mass spectrometry, ¹H-nuclear magnetic resonance (NMR), and ¹³C NMR-DEPT 135 (distortionless enhancement by polarization transfer 135).

Ricinine: (4-methoxy-1-methyl-2-oxopyridine-3-carbonitrile) (RS1) was isolated as white powder. Positive ESI/MS showed m/z 165.068 [M + H]⁺for molecular formulaC₈H₈N₂O₂ and m/z 138 [M -C₂H₂]. The ¹HNMR (DMSO-d6, 500MHz) spectrum (Table1) showed δ ppm: 6.43 (1H, d, J= 8 HZ, H-5), 8.1 (1H, d, J= 8 HZ, H-6), 3.42 (3H, s, CH₃), 3.97 (3H, s, OCH₃). ¹³C-NMR (125 MHz, DMSO-d6) spectrum (Table1) showed δ ppm: 161.36 (s, C-2), 86.14 (s, C-3), 173.09 (s, C-4), 94.18 (d, C-5), 146.58 (d, C-6), 37.19 (q, C-7), 115.14 (s, C-8), 58.07 (q, C-9). The results are consistent with those reported in the literature [59]

Lupeol: (RS) it is isolated as white powder. Positive ESI/MS showed m/z 427.18[M+H]⁺for C₃₀H₅₀O molecular formula and m/z 137 [M+H-C₂₀H₃₃O]. The ¹HNMR (DMSO-d6, 500MHz) spectrum (Table1) showed δ ppm 4.28 (1H, d, J= 4.25 HZ, H-3), 0.773 (3H, s, CH₃), 0.876(3H, s, CH₃), 0.766 (3H,s, CH₃), 0.658 (3H,s, CH₃). ¹³C-NMR (125 MHz, DMSO-d6) spectrum (Table1) showed δ ppm 38.72 (C-1), 27.62(C-2), 77.25 (C-3), 38.97 (C-4), 55.31 (C-5), 18.42 (C-6), 34.30 (C-7), 40.81 (C-8), 50.29 (C-9), 37.14 (C-10), 20.87 (C-11), 25.14 (C-12), 38.03 (C-13), 42.83 (C-14), 27.45 (C-15), 35.53 (C-16), 43.02 (C-17), 48.24 (C-18), 47.87 (C-19), 150.68 (C-20), 29.65 (C-21), 39.94 (C-22), 28.57 (C-23), 16.16 (C-24), 16.40 (C-25), 16.28 (C-26), 14.80 (C-27), 18.24 (C-28), 110.16 (C-29), 19.42 (C-30). ). The results are consistent with those reported in the literature [60]

Table 1[1]

¹H and ¹³C- NMR Spectral Data for Identification of Lupeol and Ricinine (500 / 125 MHz, DMSO-d6) [59, 60]

Remark	Ricinine		Lupeol			No.
Remark	¹³C-NMR / DPT135	¹H-NMR	Remark	¹³C-NMR / DPT135	¹H-NMR
		^--	CH₂(t)	38.72	^-	1
Q(s)	161.36	-	CH₂(t)	27.62	-	2
Q (s)	86.14	-	CH(d)	77.25	4.28 (d, J = 4.25)	3
Q(s)	173.09	-	Q(s)	38.97	-	4
CH(d)	94.18	6.43(J = 8 HZ)	CH(d)	55.31	-	5
CH(d)	146.58	8.1(J = 8 HZ)	CH₂(t)	18.42	-	6
CH₃(q)	37.19	3.42	CH2(t)	34.30	-	7
Q(s)	115.14	-	Q(s)	40.81	-	8
CH₃(q)	58.07	3.97	CH(d)	50.29	-	9
			Q(s)	37.14	-	10
			CH₂(t)	20.87	-	11
			CH₂(t)	25.14	-	12
			CH(d)	38.03	-	13
			Q(s)	42.83	-	14
			CH₂(t)	27.45	-	15
			CH₂(t)	35.53	-	16
			Q(s)	43.02	-	17
			CH(d)	48.24	-	18
			CH(d)	47.87	-	19
			Q(s)	150.68	-	20
			CH₂(t)	29.65	-	21
			CH₂(t)	39.94	-	22
			CH₃(q)	28.57	α 0.773(s)	23
			CH₃(q)	16.16	ß 0.876(s)	24
			CH₃(q)	16.40	ß 0.766(s)	25
			CH₃(q)	16.28	ß 0.658(s)	26
			CH₃(q)	14.80	α 1. 26(s)	27
			CH₃(q)	18.24	-	28
			CH₂(t)	110.16	α 0.84(s)	29
			(CH₃)(q)	19.42	ß 1.646(s)	30

UPLC-Triple TOF-MS/MS analysis

Retention times, molecular weight, and fragment ions for each metabolite and their identities are presented in (Tables 2 a, b).

Mass spectral data for tentatively identified compounds in R. communis

UPLC-MS/MS was performed in positive and negative mode on methylene chloride fraction of R. communis leaves (Table 2 a, b). UPLC-MS/MS chromatogram shows the relationship between retention time of metabolites and intensity (Fig. 2). Several classes of compounds were identified, including flavonoids, flavonoid glycosides, isoflavonoids, coumarins, alkaloids, phenolic acids, triterpenoids, amino acids and fatty acids. Five classes of flavonoids were identified in UPLC-MS/MS negative mode flavanones, isoflavone, flavone, flavanol, and dihydrochalcone. Flavanone, narigenin was eluted at 1.39 min. Isoflavone, diadzein-8-C-glucoside was eluted at 1.26 min. Flavone, luteolin, apigenin, acacetin (4'-methylated apigenin), and acacetin-7-O-rutinoside were eluted at 5.23, 12.01, 15.73, and 15.37 min, respectively. Flavonol, myricetin, quercitrin, kaempferol-3-O-α-L-rhamnoside, kaempferol-3-O-α-L- arabinoside, and 3'-methoxy-4',5,7- trihydroxyflavonol, were eluted at 1.46, 5.16, 5.41, 7.18, and 13.37 min, respectively. Dihydrochalcone, phlorizin (glucoside of dihydrochalcone) and neohesperidin dihydrochalcone were eluted at 15.22 and 15.18 min, respectively. Most of these compounds were previously reported in R. communis [61, 39, 33, 28, 44], Phlorizin (glucoside of dihydrochalcone), acacetin (4'-methylated apigenin) and acacetin-7-O-rutinoside were identified for the first time in R. communis.

In UPLC-MS/MS positive mode, six classes of flavonoids were tentatively identified; flavanone, flavanonol, isoflavone, flavone, flavone without a hydroxyl group in the B ring, and flavonol. Flavanone, 3' 4' 5 7- tetrahydroxyflavanone, and Isosakuranetin-7-O-neohesperidoside were eluted at 5.91, 7.24 min, respectively. The Flavanonol; Taxifolin was eluted at 1.39 min., Isoflavone, 4'-hydroxyisoflavone-7-O-glucoside was eluted at 1.33 min., Flavone, 3-5 7-trihydroxy-4'-methoxyflavone, Acacetin (4'-methylated apigenin), Acacetin-7-O-rutinoside, Some phenolic acids, such as Caffeic acid, para-aminobenzoic acid, 4- Methoxy cinnamic acid, 3,4-dimethoxycinnamic acid, and chlorogenic acid were eluted at 1.4, 4.3, 17.89, 8.25, and 1.4 min, respectively. These latter compounds were among the most active in the methylene chloride fraction from R. communis leaves via UPLC-MS/MS. Two alkaloids identified in UPLC-MS/MS positive mode; trigonelline and ricinine which were eluted at 1.38 and 4.25 min, respectively . Ricinine was the major metabolite identified in methylene chloride fraction. Also, coumarins, scopoletin, and daphnetin, were identified in R. communis. Furthermore, lupeol, (a triterpene), eluted at 5.63 min. The compounds were structurally identified by comparing retention times, high-resolution mass spectrometry data, and fragment ions with reference substances and literature data.

Antiviral assay

Determination of cytotoxicity (CC₅₀) and antiviral activity for Three fractions [MeOH, Methylene chloride, and Ethyl acetate] against MERS-COV2 by plaque reduction assay

CC₅₀ values for MeOH, CH₂Cl₂, and ethyl acetate fractions were 17.1, 0.4, and 8.2 mg/ml, respectively. Cytotoxicity tests used Vero-E6 cells and analysis using nonlinear regression analysis of plots of log concentration against percent cell viability with GraphPad Prism software (version 5.01).

Plaque reduction assay data were used to calculate percent inhibition against MERS-CoV for each extract (Table 3). Methylene chloride crude extract exhibited 92.5% antiviral activity. So the same. This extract was also tested for antiviral activity against SARS-CoV-2 to determine IC₅₀.

Table 3 Plaque reduction assay for extracts against MERS-CoV (NRCE-HKU270)

Extract	Concentration µg/ml	Viral Count (PFU/ml)	Viral Count after treatment (PFU/ml)	Inhibition %
MeOH	50	27x10³	22x10³	18.5
	25		25x10³	7.4
	12.5		30x10³	0
Methylene chloride	50	27x10³	2x10³	92.5
	25		10x10³	62.9
	12.5		22x10³	18.5
Ethyl acetate	50	27x10³	25 x10³	7.4
	25		28 x10³	0
	12.5		29 x10³	0

Antiviral activity of methylene chloride extract against SARS-CoV-2 (NRC-03-nhCoV)

The methylene chloride extract showed promising antiviral activity against SARS-CoV-2, IC₅₀ = 1.76 µg/ml with high safety index, SI = 291.5.

Antiviral activity of pure compounds [lupeol and ricinine)] for MERS-CoV and SARS-CoV-2.

Methylene chloride extract exhibited promising activity against MERS-CoV virus. Evaluation of pure compounds was performed against MERS and SARS-CoV-2 and calculated cytotoxicity concentration (CC₅₀), Inhibitory concentration (IC₅₀) and Safety index (SI) showed high antiviral activity of Ricinine (RS1) against SARS-CoV2, and a significant safety index, IC₅₀ = 2.5 µg/ ml, and SI = 18098; activity against MERS was lower, IC₅₀ = 87.2 µg/ ml (Table 4). In contrast, Lupeol (RS) had greater antiviral activity against MERS, IC₅₀ = 5.28 µg/ ml, than SARS-CoV2, IC₅₀ = 19.5 µg/ ml. (Table 4 & Fig. 4).

Table 4 Antiviral activity of ricinine and lupeol against MERS- (NRCE-HKU270) and SARS- (NRC-03-nhCoV)

Compound

CC₅₀ (Vero-E6)

µg/ml

IC₅₀ SARS- (NRC-03-nhCoV)

µg/ml

IC₅₀ MERS NRCE-HKU270

µg/ml

SI- SARS

SI MERS

Ricinine

45753

2.528

87.2

18098

524.69

Lupeol

355.2

19.59

5.28

18.13

67.27

Mode of action

Percent inhibition for various mode of actions are shown in (Fig. 5). Interestingly, the results showed that the best mechanism of action for antiviral activity was virucidal effect for ricinine and crude. Ricinine had a combination of viral inhibitory effect on SARS-CoV-2 at different viral stages. Ricinine has >99% virucidal effect indicating that it possibly acts directly on the virion causing inactivation. Additionally, it showed 76% inhibitory effect during viral adsorption stage. Furthermore, the crude methylene chloride extract exhibited the virucidal effect with more than a 90% viral inhibitory effect. Lupeol showed almost nearly an approximately 65% inhibitory effect on virucidal as well as virus adsorption. This result confirmed the results of crystal violet assay which gave IC₅₀equal 2.5, 1.7, and 19.5 for ricinine, crude, and lupeol respectively.

Molecular Docking

Docking into COVID-19 3CL^pr°

The main protease (M^PRO) is a critical enzyme encoded in SARS-CoV-2. The protein is also referred to as 3C-like protease (3CL^pro), an essential enzyme in viral RNA translation and maturation. Hence, the protease is crucial for viral infection and replication processes and is thus an attractive therapeutic target for developing anti-coronavirus drugs [62]. In this study we examined the antiviral activity of isolated pure compounds for SARS-CoV-2 via targeting 3CL^pro using molecular docking to evaluate molecular binding affinity of both compounds with 3CL^pro (PDB ID code: 6LU7). Lupeol and ricinine are anchored closely in the protease enzyme's active site, with binding energies of −5.342 and −7.875 Kcal/mol, respectively (Table 5). By comparison, remedisvir displayed a binding energy pf −8.602 Kcal/mol. Both tested compounds were able to form hydrogen bonds with a key amino acid, Cys145. Further, additional amino acid residues, Met49, Gly143, His163, and Ser144, were involved in hydrogen bonding (Fig. 6-8). Lupeol docking indicated one hydrogen bond interaction of the OH group with the Cys145 residue (Fig. 6). Ricinine was bound in the active site with four hydrogen bond interactions (Fig.7). Interestingly, the CN group participated in two hydrogen bonds with Gly143 and Cys145 residues, and C=O formed one hydrogen bond with the Ser144 residue. An additional hydrogen bond was formed by the interaction of the methyl group of OCH₃ with the Cys145 residue.

Table 5: The molecular docking results for lupeol, ricinine, and remedisvir in the active site of SARS-CoV-2 (3CL^pr°) (PDB ID: 6LU7)

Compound	Docking score (kcal/mol)	Interacting residues Type of Interaction
1- Lupeol	−5.342	Cys145 (1H bond)
2- Ricinine	−7.875	Gly143, Ser144, Cys145 (4H bonds)
Remedisvir	−8.602	Met49,Gly143, Cys145, His163 (4H bonds)

Docking into COVID-19 (S) Glycoprotein

SARS-CoV-2 attacks the host cell when the surface spike proteins (S-proteins) recognize and bind to angiotensin-converting enzyme 2 (ACE2) receptors, leading to fusion between the viral envelope and host cell membrane, resulting in successful infection. Active S-protein inhibitors may reduce the severity of such virulent virus. Molecular docking can estimate the binding affinity of isolated compounds with the S-protein receptor-binding domain [63]. Molecular binding modes in the active site of S glycoprotein (PDB ID: 6VXX) identified docking energy scores and hydrogen bond interactions with essential amino acids residues (Fig. 9–11) (Table 6). Remedisvir exhibited the highest binding affinity with an energy of −7.201 Kcal/mol. This drug forms three hydrogen bonds with Lys304, Glu309, and Gln957, all essential amino acids residues. In contrast, lupeol showed the lowest binding affinity, −4.103 Kcal/mol, via a single hydrogen bond with the Glu309 residue. Ricinine affinity, −6.622 Kcal/mol, reflected two hydrogen bonds. The C=O group formed a hydrogen bond with the Lys964 residue, and the nitrogen atom of the CN group formed a covalent hydrogen bond with the Thr961 residue. Additionally, an arene-H interaction was observed with the Gln957 residue.

Table 6: Molecular docking results of lupeol, ricinin and remedisivir in the active binding site of SARS-CoV-2 (S glycoprotein) (PDB ID: 6VXX)

Compound	Docking score (kcal/mol)	Interacting residues Type of Interaction
1- Lupeol	−4.103	Glu309 (1H bond)
2- Ricinine	−6.622	Thr961, Lys964 (2H bonds) Gln957 (arene–H)
Remedisvir	−7.201	Lys304, Glu309, Gln957 (3H bonds)

[1] Q= quaternary carbon, J= J-coupling value, S=singlet, D= doublet, T= tertiary.

The frequent failure of conventional drugs against viral infections combined with specific viral resistance has increased interest in plants for providing alternative antiviral agents. Successful constituents are natural chemicals with excellent bioavailability, efficacy, and low cytotoxicity [64]. The efficacy of natural products against COVID-19 is known from previous in vitro studies of natural compounds, including phenolic acids, terpenes, flavonoids, coumarins, lignans, and alkaloids. Such compounds may be important in complementary therapy against viral infections and might demonstrate bioactivity against coronaviruses [17, 65].

The current study clarified the phytochemical profile of R. communis leaf constituents and evaluated cytotoxicity, anti-MERS-CoV, and anti-SARS-CoV-2 activity of three fractional extracts (methanol, methylene chloride, and ethyl acetate) and two pure isolated compounds (lupeol and ricinine). The antiviral activity of the methylene chloride extract and two pure constituents was determined by examining the anti-replicative, protective and anti-infective effects of the extract in infected cells in culture. Each experiment was performed three times for consistency. Cytotoxic concentrations (CC₅₀) for each assay were calculated and compared with remdesivir as a positive control.

R. communis leaves are rich in phytochemicals; extracts showed substantial content of phenolic compounds, flavonoids, terpenes, and alkaloids. Isolation of kaempferol, quercetin, their O-3-glycosides, naringenin, tannins, and the alkaloid, ricinine, from R. communis, is reported [27]. Many R. communis phytochemicals are known antiviral agents [66, 40 - 42, 44]. In this study, UPLC- QTOF-ESI-MS/MS negative and positive modes were used to analyze the methylene chloride fraction of R. communis leaves; different classes of flavonoid and phenolic acid compounds were tentatively identified. Most of these compounds were previously reported in R. communis [61, 39, 33, 28, 44], Phlorizin (glucoside of dihydrochalcone), acacetin (4'-methylated apigenin), and acacetin-7-O-rutinoside were identified for the first time in R. communis.

Interestingly, the results showed that the best mechanism of action for antiviral activity was virucidal effect for ricinine and crude. Ricinine had a combination of viral inhibitory effect on SARS-CoV-2 at different viral stages. Ricinine has >99% virucidal effect indicating that it possibly acts directly on the virion causing inactivation. Additionally, it showed 76% inhibitory effect during viral adsorption stage. Furthermore, the crude methylene chloride extract exhibited the virucidal effect with more than a 90% viral inhibitory effect. Lupeol showed almost nearly an approximately 65% inhibitory effect on virucidal as well as virus adsorption.

Among the three different extracts, the methylene chloride extract of R. communis leaves showed potent in vitro activity against MERS-CoV. Viral replication was reduced by 92.5% (Table 3). Further, this extract exhibited more than 90% virucidal anti-SARS-CoV-2 activity (CC₅₀= 513.1 µg/ml and IC₅₀ = 1.76 µM) (Figures 3, 5). This activity might be attributed to substantial flavonoid, phenolic acid, terpene, and alkaloid content identified in this fraction. This interpretation is consistent with our results with molecular docking studies that suggest good affinity of two constituents of R. communis extract SARS-CoV-2. Substantial evidence supports significant antiviral properties of pure flavonoids and flavonoid-containing plant extracts against MERS-CoV, and SARS-CoV-2. The polyphenols from Curcuma (curcumin and its derivatives) and citrus sp. (hesperetin, hesperidin) exhibit a greater affinity for the S-protein than the reference compound, nafamostate, according to molecular docking studies [67]. Compared to Remdesivir, Naringenin demonstrated greater binding to viral spike proteins [68]. Flavonoids, such as kaempferol and iso-liquiritigenin, synergistically inhibited the SARS-CoV-2 Mpro and PLpro in vitro [69]. In a computational analysis of a library of 80 flavonoids from natural phenolic compounds, certain flavonoids shown potential for binding to the active site of the SARS-CoV-2 major protease. The greatest binding affinity was demonstrated by hesperidin [25]. Also, rutin in silico showed notable inhibitory activity against SARS-CoV-2 main protease. The flavonoids, myricitrin, baicalin, and scutellarin, showed strong binding with SARS-CoV-2 M^pro. Interestingly, these compounds also showed strong binding with other potential SARS-CoV-2 targets, such as viral receptor ACE-2 and block entry of the virus [70, 71].

Additionally, epigallocatechin gallate, gallocatechin gallate, and quercetin were discovered to be potent inhibitors of the SARS-CoV 3CL^pro by in vitro and in silico analysis [72]. Several phenolic acids, including caffeic acid, para-aminobenzoic acid, 4-methoxy cinnamic acid, 3,4-dimethoxycinnamic acid, and chlorogenic acid, were among the most effective compounds identified in the methylene chloride fraction and tested for their antiviral activity. Moreover, the antiviral effects of gallic acid, chlorogenic acid, quinic acid, and caffeic acid were studied, and they demonstrated a considerable efficacy against HSV1, HSV2, and influenza virus [40]. Further analysis of the caffeic acid in elderberry stem extracts suggested that binding to S-protein may hinder HCoV adhesion to host cells [73].

UPLC-MS/MS analysis in the present study identified several alkaloids and terpenes in the methylene chloride fraction of Ricinus leaves, including isolation and identification of two pure compounds, the triterpene, lupeol, and the alkaloid, ricinine, eluted at 5.63 min and 4.25, respectively. Ricinine, a natural pyridine alkaloid, showed superior virucidal and adsorption activities against SARS-CoV-2 (IC₅₀= 2.528 µg/ml, SI = 18098) compared to MERS-CoV (IC₅₀= 87.2 µg/ml). Lupeol, a lupane-type triterpene, was more potent against MERS-CoV (IC₅₀= 5.28 µg/ml, SI = 67.27) than SARS-CoV-2 (IC₅₀= 19.59 µg/ml) (Figure 4). These results fully agree with many previous studies of triterpene and alkaloid antiviral activity against SARS-CoV-2. The activity of pure terpenes has also been extensively studied [74, 17, 75, 20]. Glycyrrhizin, the primary triterpenoid derived from licorice (Glycyrrhiza glabra L.) roots, is a potent inhibitor of SARS-CoV replication in Vero cells, with a selectivity index of 67. Despite having a poor selectivity index, it dramatically reduced the ability of all tested viruses to replicate while exhibiting little harmful effects [76]. Moreover, terpenoids from Tripterygium regelii, including celastrol, pristimerin, tingenone, and iguesterin inhibited SARS-CoV 3CL^pro [77]. Tanshinone IIA, tanshinone IIB, methyl tanshinonate, cryptotanshinone, tanshinone I, dihydrotanshinone I, and rosmariquinone are examples of abietane-type diterpenoids isolated from ethanol extracts of Salvia miltiorrhiza that inhibited SARS-CoV 3CL^pro as well as PL^pro. Interestingly, this inhibition was selective because no inhibition of other proteases was observed [78]. To our knowledge, no any prior reports of lupeol's antiviral activity against CoVs. However, EGYVIR extract (a curcumin-piperine infusion) demonstrated immunomodulatory activity in vitro during SARS-CoV-2 infection. Lupeol comprised 8.86% of infusion constituents and is probably directly or indirectly responsible for some of its overall antiviral activity [79].

Ricinine is assumed to be the hallmark of the methylene chloride extract. This conclusion is consistent with a study that showed R. communis seeds, which are rich in ricinine, are active against HSV and Sindbis virus. This finding supports their traditional use in treating colds and fever [43]. Many alkaloids have been studied for their anti-coronavirus activity. Lycorine and emetine alkaloids exhibited in vitro inhibitory activity against coronavirus replication, and emetine can block MERS-CoV entry to host cells [80]. Many alkaloids are specifically active against SARS-CoV [17, 75, 20]. Indigo is an alkaloid isolated from Isatis indigotica and can block the cleavage of 3CL^pro[81]. Most previously mentioned phytochemicals from different classes (flavonoids, phenolic acids, terpenes, and alkaloids) are identified in Ricinus leaves; this literature reinforces our current finding of significant antiviral activity of the methylene chloride fraction and two leaf compounds. Phenolics, terpene, and alkaloid compounds are likely responsible for these results.

Numerous studies have revealed that plant derived natural compounds might suppress SARS-CoV-2 replication by blocking 3CL^pro activity [80, 21]. Our findings show that ricinine binds to 3CL^pro with high affinity, −7.875 Kcal/mol, due to the formation of four hydrogen bonds. Interestingly, the CN group participated in two such bonds with Gly143 and Cys145 residues, and C=O formed a hydrogen bond with the Ser144 residue. The remaining hydrogen bond formed between the methyl group of OCH₃ and the Cys145 residue. Cys145 is a critical amino acid in the catalytic dyad of SARS-CoV-2 3CL^pro. 3CL^pro is, thus, a promising anti-SARS-CoV-2 target. Lupeol showed a lower binding affinity for protease 3CL^pro, −5.342 Kcal/mol. This terpene formed one hydrogen bond between the OH group and the Cys145 residue, suggesting anti-SARS-CoV-2 activity.

Several natural compounds block SARS-CoV entry into host cells [22]. Lupeol in this study showed a binding affinity for −4.103 Kcal/mol via a single hydrogen bond interaction with the Glu309 residue of the S-protein. Ricinine showed a higher relationship, −6.622 Kcal/mol, and two hydrogen bonds. The C=O group formed a bond with the Lys964 residue, and the nitrogen atom of the CN group began another bond interaction with the Thr961 residue. Furthermore, an arene-H interaction was observed with the Gln957 residue.

Notably, both lupeol and ricinine showed interactions with SARS-CoV-2 main protease and moderate affinity for spike protein. These interactions may form a valuable basis for their development as natural candidates to counter coronaviruses. However, additional exploration is necessary to expand in silico results with in vivo activity.

This study is the first, to our knowledge, to show in vitro activity against SARS-CoV-2 and MERS-CoV by constituents in R. communis leaves, including two pure isolated compounds. This beneficial activity might be due to various phytochemicals, particularly in the methylene chloride fraction of leaf extracts. This fraction showed the highest antiviral activity in MERS-CoV plaque assays. The most common compounds isolated and identified from the methylene chloride fraction were ricinine and lupeol. The latter, a natural triterpene, showed the greatest activity against MERS-CoV with IC₅₀ 5.28 µg/ml and SI-MERS 67.27. In addition to, almost nearly moderate an inhibitory effect on virucidal as well as virus adsorption. While ricinine, a natural alkaloid, showed superior virucidal antiviral activity against SARS-CoV-2 with an IC₅₀ 2.528 µg/ml and SI SARS 18098. Ricinine showed significant binding to 3CL^pr°, a modest S-spike protein affinity in molecular docking studies, and a possible interaction with SARS-CoV-2 major protease. This compound exhibited the greatest affinity for two critical targets for anti- SARS-CoV-2 activity in silico and to be the most biologically active. These results might be a promising basis for developing these constituents or their derivatives as antiviral agents for the treatment of coronavirus infections. Additional research will be required to assess the effects of chemical modification of their structures, guided by the molecular docking tools, and evaluate their in vivo activity to determine safe therapeutic levels before clinical trials in humans can be considered.

3CL^PRO: 3-chymotrypsin-like protease; ACE 2: angiotensin-converting enzyme; APT: Attached proton test; BPC: base peak chromatogram; BSL 3: a biosafety level 3; C=O: carbonyl group; CC50: 50 % cytotoxic concentration; CH₂Cl₂: methylene chloride; COVID-19: Coronavirus disease 2019; CPE: cytopathic effects; Cys: cysteine amino acid; D: doublet; DEPT: distortionless enhancement by polarization transfer 135; DMEM: Dulbecco’s Modified Eagles Medium; DMSO: Dimethyl sulfoxide; Gln: glutamine amino acid; Glu: glutamic acid; Gly: glycine amino acid; H bond: hydrogen bond; His: histidine amino acid; IC50: 50% inhibitory concentration; J: J- coupling value; Lys: lysine amino acid; Mass: kg, g, μg; MeOH: methanol; MERS-CoV: Middle East Respiratory Syndrome Coronavirus; Met: methionine amino acid; MOE, 2014: MOE-Dock 2014; M^PRO: main protease; MTT: 3-(4, 5-dimethylthiazol -2-yl)-2, 5-diphenyltetrazolium bromide; NPs: natural products; Nuclear magnetic resonance: ¹H NMR and ¹³C NMR; OH: hydroxyl group; PBS: phosphate buffer saline; PFU: plaque forming units; PL^PRO: papain-like protease; Q: quaternary carbon; RT: Retention time; RT: Room temperature; S: singlet; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; Ser: serine amino acid; SI: safety index; S-proteins: spike proteins; T: tertiary; Thr: Threonine; TLC: thin-layer chromatography; UPLC-Triple TOF-MS/MS: ultra-performance liquid chromatography coupled with hybrid triple time-of-flight mass spectrometry; Volume: L (liter), ml, μl.

Ethics approval and consent to participate:

The plant material was collected in compliance with institutional, national, and international (WHO) guidelines on good agriculture and collection practice (GACP) for medicinal plants. The planters approved the permit for the collection of leaves specimens. NO animals nor human used in this study. (Not applicable)

Consent for publication

Not applicable.

Availability of data and materials

All the necessary data supporting the result and conclusion of the study have been incorporated in the manuscript.

Competing interest

The authors declare that they have no competing interests

Funding

Not applicable

Authors’ contributions

RH and ZS conceived and designed the research proposal. MA and OK contributed to the design of the virology experiments, the latter performing the practical part. RH and SE participated in the practical chromatographic section and contributed to data collection. RH, OK and SE analyzed the data during the experiments and interpreted the results. RH and SE wrote the manuscript with assistance, reviewing, editing and feedback from all. ZS and MA checked the theoretical results and approved the manuscript as the final version. All authors have read and approved the final manuscript.

Acknowledgments

The authors gratefully acknowledge Dr Rehab Sabour, assistant professor at the Department of Pharmaceutical Chemistry, Faculty of Pharmacy for her assistance in docking experiments performance.

Patent

This work has been submitted for patent application to patent office Academy of Scientific Research and Technology, Egypt under number 20220406000106

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Table 2 is available in the Supplementary Files section.

No competing interests reported.

Table2.doc

Anti-SARS-CoV-2 in vitro potential of Castor Oil Plant (Ricinus communis) Leaf Extract: In-Silico Virtual Evidence

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Version 1

Abstract

Figures

Background

Materials And Methods

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Table 2

Additional Declarations

Supplementary Files

Status:

Version 1