Bi-Digital O-Ring Test (BDORT), coupled with advanced bioinformatic and Artificial Intelligence (AI) tools of Molecular Docking, to create natural products consisted from cold pressed and etheric oils, for treatment of COVID-19 infection, from SARS-CoV-2;Natural products to bind molecular determinants in SARS-CoV-2

Introduction: Between the end of December 2019 and the beginning of January 2020, cases of an undetermined lung disease of viral origin began to appear, with an initial outbreak recorded in the Chinese city of Wuhan. It was of viral origin and was called SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus-2), to differentiate it from the previous SARS-CoV-1 (Severe Acute Respiratory Syndrome -Coronavirus-1), identified for the first time by the Italian infectious disease specialist Carlo Urbani and disappeared from the same pathology. This virus came out well taken from the Chinese borders, and, with an extremely high infectious rate (high R2), in a few months caused infectious people and deaths all over the world. Symptoms included: fever (38.5-39°C), joint pain, fatigue, loss of appetite, temporary loss of smell and ability to perceive aromas, dry cough, a feeling of closure of the respiratory tract and, in severe cases, dyspnea. Therapy included: steroidal and non-steroidal anti-inflammatory drugs (NSAIDs or FANS), antiepiretics, antivirals such as Ribavirin (usually used in the treatment of Flavivirus of Hepatitis C) and the application of the antimalarial drug Hydroxychloroquine (which has been shown to block replication of the virus at the level of cell vacuoles). For the most severe cases, with severe dyspnea, immediate hospitalization was required, with provision for intubation and induction of pharmacological coma. Concerning this virus, of which, at the current state of research, 17 different strains have differentiated, and the development of forms of cellular immunity and humoral, capable of counteracting this infection. One study evaluated the in vitro antiviral effect against influenza type A (H1N1) of commercial essential oils that included cinnamon (Cinnamomum zeylanicum), bergamot (Citrus bergamia), lemongrass (Cymbopogon flexuosus), thyme (Thymus vulgaris), and lavender (Lavandula angustifolia). The oils were tested in the liquid phase at a concentration of 0.3% and in the vapor phase. The oils of cinnamon, bergamot, thyme, and lemongrass displayed 100% inhibition of H1N1 in the liquid phase, while the inhibition for lavender essential oil was 85%. However, in the vapor phase, 100% inhibition was observed only for cinnamon leaf essential oil after 30 min of exposure. The bergamot, lemongrass, thyme, and lavender essential oils displayed inhibition rates of 95%, 90%, 70%, and 80%, respectively [1-2]. The research team responsible for the publication Essential Oils as Antiviral Agents, Potential of Essential Oils to Treat SARS-CoV-2 Infection: An In-Silico Investigation, reported the following conclusions: A molecular docking analysis was conducted, using Spartan 18 v 1.4.4, molecular docking software, using 171 components of essential oils with the following molecular species: 1) SARS-CoV-2 Main Protease (SARS-CoV-2 M pro) 2) SARS-CoV-2 endoribonuclease (SARS-CoV-2 Nsp15 / NendoU) 3) SARS- CoV-2 ADP-ribose-1 -phosphatase (SARS-CoV-2 ADRP) 4) SARS-CoV-2 RNA-dependent RNA polymerase (SARS-CoV-2 RdRp) 5) SARS-CoV-2 peak protein binding domain (SARS-CoV-2 rS) 6) Human angiotensin converting enzyme (hACE2) The best docking result, determined by Spartan 18 software, was between the aforementioned ligands before and the molecules (E, E)-α-farnesene, (E)-β-farnesene and (E, E)-farnesol. The docking energies determined from a bioinformatic point of view were relatively weak, and it is statistically unlikely that these molecules (Farnesene and Farnesol) interact with the virus targets (the molecules mentioned above). However, the components of the essential oil can act synergistically, in addition, they can be used in the form of integrated therapy (as a food supplement), to enhance the effect of antiviral agents and can provide some relief from the symptoms of the pathology COVID-19. Strengthened by these results and the possibility of being able to apply Bioinformatics to the BDORT Test [3-4], we began to verify the main chemical compounds present in the oils, prepared by us, whose proportions bet een the individual components were determined by means of the BDORT Test Test itself and, through Molecular Docking software, we verified the binding capacity and strength of the main molecules present in the oils, with the 6 main antigens, mentioned above, expressed by SARSCoV- 2. Materials and Methods: By using indirect BDORT, we detected effective anti-COVID-19 oils and BDORT was applied to determine the proportion between the various components present in the mixed oils that have been prepared. Three tables are shown below in which they are present: Common Name of the vegetable from which the oil was obtained, Scientific Name of the aforementioned vegetable and Type of Oil, that is, Cold Pressed Oil or Etheric Oil. The tables are indicated for three types of oils, the composition of which, as mentioned above, determined by BDORT, should be effective in countering not only the symptomatology of the COVID-19 pathology, but acting directly against the specific viral particle SARS-CoV-2. The primary purpose of this research work is not only to verify the effectiveness of the components present through the use of Molecular Docking software (Swiss Dock, from Swiss Bioinformatic Institute http://www.swissdock.ch/docking), but to demonstrate the effective effectiveness of the BDORT Test, by comparing it with accredited scientific method of Molecular Docking (Figure 1) and, finally, the possibility of comparing methods such as the BDORT test and the modern advanced Bioinformatics technique of Molecular Docking. Bond energy is calculated as Gibbs Free Energy (ΔG). In Thermochemistry and, in this case, in Thermodynamics, Gibbs Free Energy, defined as the difference between the Enthalpy (ΔH) and Entropy (ΔS) variation of a thermodynamic system, in which the temperature of the system (which is multiplied by the variation of Entropy (ΔS), is a representation of the spontaneity of a given chemical reaction variation of Entropy (ΔS), is a representation of the spontaneity of a given chemical reaction. Its formula is represented as ΔG = ΔH - TΔS. If the value of ΔG is negative, then the reaction will be spontaneous and exergonic (the reaction occurs with a release of energy into the environment outside the reaction system), vice versa if the ΔG value is positive and the reaction will be non-spontaneous and endergonic (the reaction requires and calls energy from the external environment to the reaction system.) Regarding Molecular Docking, if the binding ΔG between ligand and protein will be negative, then it means that the affinity will be high and the ligand will take naturally contact with the protein. The higher the negative coefficient, the higher the affinity. In order to better understand the type and strength of the bond, the following division has been prepared: • Positive: When the ΔG value is between -1 KCal / mol and -6 KCal / mol and there is at least one unity difference between the maximum and minimum G value (e.g. between -6 KCal / mol and - 7 KCal / mol there is a difference of one unit). • Positive / Discreet: When the ΔG value is between -6 KCal / mol and -7 KCal / mol and there is a difference of at least one unit between the maximum and the minimum ΔG value. • Positive / Strong: When the ΔG value is between -7 KCal / mol and -8 KCal / mol or less than -7 KCal / mol (-8 KCal / mol for example) and there is a difference of at least one unity between the maximum and the minimum ΔG value. Could be also Positive / Strong when value of ΔG is between -6 KCal / mol and -8 KCal / mol, but with a difference less than one unity. • Strong: When the ΔG value is between -8 KCal / mol and -9 KCal / mol and there is a difference of less than one unit between the maximum and minimum ΔG value, (an example, -9,54 KCal / mol and -9,30 KCal / mol). In this distinction, no reference is made to the fact that the bond occurs outside or inside the acceptor molecule, which differ from the temporal point of view of the bond. To give an example, a strong bond outside the molecule will still have a shorter time dura ion than a strong bond inside the molecule. In the same way a weak bond behaves. As initially mentioned in the Introduction, 6 molecular determinants were considered as targets, of which 5 belonging to the viral particle SARS-CoV-2 and, the last, the hACE2 (Angiotensin Converting Enzyme-2), molecule having a role in SARS-CoV-2 infection.