In Vitro : Natural Compounds (Thymol, Carvacrol, Hesperidine, and Thymoquinone) against Sars-Cov2 Strain Isolated from Egypt

Background: The current pandemic of the coronavirus disease-2019 (COVID-19) has badly affected our life during the year 2020. SARS-CoV-2 is the primary causative agent of the newly emerged pandemic. Natural flavonoids, Terpenoid and Thymoquinone are tested against different viral and host-cell protein targets. These natural compounds have a good history in treating Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV). Methods: Molecular docking combined with cytotoxicity and plaque reduction assay is used to test the natural compounds against different viral (Spike, RdRp, and Mpro) and host-cell (TMPRSS II, keap 1, and ACE2) targets. Results: The results demonstrate the binding possibility of the natural compounds (Thymol, Carvacrol, Hesperidine, and Thymoquinone) to the viral main protease (Mpro). Some of these natural compounds were approved to start clinical trail from Egypt Center for Research and Regenerative Medicine ECRRM IRB (Certificate No.IRB00012517) Conclusion: Development of an effective anti-viral for SARS-CoV-2 could help to limit the viral load. Benchmarking testing of those natural compounds against other potential antivirals for SARS-CoV-2 with alternative mechanisms of action would thus be important as soon as practicable. Mostfa F et In Vitro : Natural Compounds (Thymol, Carvacrol, Hesperidine, and Thymoquinone) against Sars-Cov2 Strain Isolated from Egypt.


Introduction
By the end of 2019, an outbreak of a novel coronavirus (SARS-CoV-2) in Wuhan city in China was detected and spread all over the world [1]. On 10 th October 2020, the number of confirmed cases of coronavirus disease (COVID-19) reached more than 37 M worldwide with +1M total death, as reported in the World Health Organization (WHO). The associated pneumonia with the novel viral infection, COVID-19, is divided into three phases that correspond to different clinical stages of the disease [2]. Stage 1 is the asymptomatic stage, where the inhaled virus binds to nasal epithelial cells in the nasal cavity and starts replicating. Stage 2 is the upper airway stage, where the virus propagates, migrates down the respiratory tract along the conducting airways, and a more robust innate immune response is triggered. About 20% of the infected patients will progress to stage 3 disease and develop pulmonary infiltrates. Some of these patients will develop a very severe disease as the virus reaches alveoli in the lung and infects alveolar type II cells in peripheral and sub-pleural areas of the lung [3]. SARS-CoV-2 propagates within type II cells, large numbers of viral particles are released, and the cells undergo apoptosis and die. Therefore, the spectrum of symptomatic COVID-19 ranges from mild respiratory tract infection to severe pneumonia that may progress to fatal respiratory syndrome and multi-organ malfunctions [2].
Thymol, known as 2-isopropyl-5-methylphenol, is a natural mono-terpenoid phenol derivative of Cymene. It is found in thyme oil and extracted from Thymus vulgaris [4]. Thymol is a white crystalline substance that has a pleasant aromatic odor. Thymol also provides the distinctive and robust flavor of the culinary herb thyme. Carvacrol is known as monoterpenoid phenol and extracted from Oregano. It has a characteristic pungent and warm odor [5].
Hesperidine is a common flavone glycoside found in citrus fruit such as lemons and sweet oranges [6,7]. It has several pharmacological activities such as antihyperlipidemic, anti-atherogenic, venotonic, antidiabetic, cardioprotective, anti-antihypertensive, and inflammatory actions [6,7]. The anti-inflammatory activity of hesperidin was mainly attributed to its antioxidant defense mechanism and suppression of pro-inflammatory cytokine production [6]. Hesperidin exhibited antiviral activity against the influenza virus through a significant reduction of viral replication.
Nigella sativa (NS) contains many active molecules, such as Thymoquinone (TQ), two forms of alkaloids: isoquinoline alkaloid that includes nigellicimine, nigellicimine n-oxide and pyrazol alkaloid that includes nigellidine and nigellicine [8,9]. TQ is the most abundant constituent in the volatile oil of Nigella sativa seeds, and most of the herb's properties are attributed to it [10,11]. It has been reported that NS oil can decrease the viral count of HCV in patients received capsules of NS oil (450 mg) three times a day over a 3-month period [12]. Moreover, two clinical studies documented to sustained sero-reversion of the HIV virus over treatment period of 6 to 12 months [13][14][15]. Molecular docking represents a promising in silico method used to predict the binding affinities of small molecules to proteins as a first step in structure-based drug design [16][17][18][19][20][21]. This study investigated many active ingredients that showed antiviral activities against SARS-CoV-2, such as Thymol, Carvacrol, Hesperidine, and Thymoquinone. Molecular docking is used to test the binding affinities of these natural product derived compounds against different viral and host cell proteins.
Additionally cytotoxicity assay and plaque reduction assay are used to verify their antiviral activity against SARS-CoV-2 collected from Egyptian patients.

In silico Testing
Before performing the docking studies, the tested compounds are retrieved from the PubChem database and then prepared using PyMOL software [22,23]. The structures of the proteins are downloaded from the protein data bank [24]. Autodock Tools are used to prepare the docking input files after adding charges [25]. Kelch-like ECH-associated protein 1 (KEAP 1) and the Angiotensin Converting Enzyme 2 (ACE2) are targeted due to its fundamental in viral recognition and maintain infectivity for SARS-CoV-2 [32][33][34][35]. For each compound, ten interactions were generated and one with best binding affinity was selected. PyMOL software was used to represent and analyze the docking complexes.

Experimental Section
All the chemical compounds are purchased from different sources as follow; Thymol purchased from upnature as THYME  The plot of % cytotoxicity versus sample concentration was used to calculate the concentration which exhibited 50% cytotoxicity (IC 50 ).

Plaque Reduction Assay
Assay was carried out according to the method of [37] in a six well plate where Vero E6 cells (10 5

Statistical Analysis
Analysis was performed using Graphpad Prism 8.0.2. Data are represented as mean ± SD and statistical significance was evaluated using one-way ANOVA followed by tukey multiple comparison tests.

Results
Many reports showed that different natural product derived compounds have promising results against inflammation and  [30,48]. Table 1 shows the binding affinity calculated using AutoDock Vina software for the docking of the natural compounds (Thymol, Carvacrol, Hesperidine, and Thymoquinone) against the SARS-CoV-2 M pro as a protein target. The standard compound Chloroquine is used to assess the binding affinity of the natural compounds against the M pro . As reflected from the values, Carvacrol, Hesperidine, and Thymoquinone show comparable binding affinities (-7.0, -6.9, and -6.9 kcal/mol, respectively) to SARS-CoV-2 M pro compared to that of the standard compound (-7.2 kcal/mol). Thymol show slightly higher (worse) binding affinity value (-5.8 kcal/mol) compared to Chloroquine but still able to bind the SARS-CoV-2 Mpro tightly.

Compound
Binding Affinity(kcal/mol) Hesperidine -6.9 Thymoquinone -6.9 Chloroquine(standard) -7.2    Benchmarking testing of those natural compounds against other potential antivirals for SARS-CoV-2 with alternative mechanisms of action would thus be important as soon as practicable.

Ethics Approval and Consent to Participate
Not applicable.

Consent to Publication
Not applicable.

Availability of Data and Material
The docking structures are available upon request from the corresponding author.

Competing Interests
All the authors declare that there is no competing interest in this work.

Funding
Not applicable.

A. Mohamed Gomaa Seadawy
In Vitro work, Writing, Editing and Molecular Docking

E. Mostfa Fetooh Mohamed
In vitro study F.