Natural Compounds (Thymol, Carvacrol, Hesperidine, and Thymoquinone) Against SARS- CoV-2 Strain Isolated From Egyptian Patients


 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). 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. 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)


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 con rmed 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 in ltrates. 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 .
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 avor 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 avone 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 in ammatory actions 6,7 . The anti-in ammatory activity of hesperidin was mainly attributed to its antioxidant defense mechanism and suppression of pro-in ammatory cytokine production 6 . Hesperidin exhibited antiviral activity against the in uenza virus through a signi cant 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 a nities of small molecules to proteins as a rst 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 a nities 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.

Materials And Methods
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  and the Angiotensin Converting Enzyme 2 (ACE2) are targeted due to its fundamental in viral recognition and maintain infectivity for SARS-CoV-2 32-35 . For each compound, ten interactions were generated and one with best binding a nity 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 100% pure and natural 118 ml, Carvacrol purchased from Zane Hellas as oil of Oregano 30 ml, Hesperidin purchased from science-based nutrition as hesperidin methyl chalcone 500 mg -60 veggie caps, and Thymoquinone purchased from prime natural black seed USDA organic Absorbance of formazan solutions were measured at λ max 540 nm with 620 nm as a reference wavelength using a multi-well plate reader. The percentage of cytotoxicity compared to the untreated cells was determined with the following equation. The plot of % cytotoxicity versus sample concentration was used to calculate the concentration which exhibited 50% cytotoxicity (IC50).

B) Plaque reduction assay
Assay was carried out according to the method of 37  -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.   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 M pro tightly. Figures 3A and 3B show the 3D poses for the docking complexes. The four compounds (Thymol, Carvacrol, Hesperidine, and Thymoquinone) are able to bind to the active site of the M pro (His41 and Cys145). Table 1 The binding a nity (in kcal/mol) of the natural compounds against the main protease of SARS-CoV-2 calculated using AutoDock Vina software.

Compound
Binding a nity(kcal/mol) Hesperidine -6.9 Thymoquinone -6.9 Chloroquine(standard) -7.2 The half maximal inhibitory concentration (IC50) of Thymol, Carvacrol, Hesperidin, and Thymoquinone The effect of different concentrations of the compounds on the cellular proliferation of Vero E6 cell line following 24 h of treatment was determined using MTT assay.
In vitro study for accessing the antiviral effect of natural products against SARS-CoV-2: Our results revealed that Hesperidine was the most affective natural product against SARS-CoV-2 in Egyptians patients, as it caused inhibition to approximately 100% for infected Vero E6 cell line, Carvacrol, Thymoquinone caused inhibition to about 98%, Chloroquine as a positive control caused inhibition to 98% and Thymol show the lowest inhibition percentage 96% for infected Vero E6 cell line.
The results showed significantly difference between natural products effect with (P-value < 0.0001) as shown in figure 5.
These results indicate that Hesperidine and other natural compounds as Carvacrol and Thymoquinone could be therapeutic agent against SARS-CoV-2 in Egypt as treatment resulted in the effective loss of essentially all viral material by time.
Eventually, development of an effective anti-viral for SARS-CoV-2, if given to patients early in infection, could help to limit the viral load, prevent severe disease progression and limit person-person transmission. 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.

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