Clinical and In-Silico study of COVID‑19 patients using Thymus capitatus extract as adjuvant Therapy

Thymus capitatus extract has been considered a promise as antiviral agent against COVID‑19 viruses. We hypothesized that Thymus capitatus components may interact with key protein targets of COVID‑19 (coronavirus 2 (SARS‑CoV‑2) causing severe acute respiratory syndromes. Molecular docking analysis was carried out using 31 components of Thymus capitatus with SARS‑CoV‑2 protease enzyme (6LU7) and spike glycoprotein (6VSB). The compounds with the best normalized docking scores to protease enzyme were allo‑Aromadendrene (‑6.3 kcal/mole), spathulenol (‑6.6 kcal/mole) and ledene (‑6.8 kcal/mole). The best docking ligands for spike glycoprotein were allo‑Aromadendrene (‑6.6 kcal/ mole), spathulenol (‑6.6 kcal/mole) and ledene (‑7.3 kcal/mole). All Thymus capitatus components may act synergistically to produce the therapeutic action. Thymus capitatus components may potentiate the effect of prednisolone, azithromycin and other medicines used to treat COVID‑19 patients.


Introduction
The outbreak of Coronavirus SARS-Cov-2 disease (COVID- 19) in Wuhan (China) in December 2019 and its worldwide spread has led in some cases to heavy disease symptoms finally causing deaths [1,2]. During the year 2020, the disease has affected almost all countries on the globe. The majority of people infected with the COVID-19 virus face mild medium respiratory symptoms and recover mostly without any treatment [3,4]. Elderly people and people with chronic medical conditions such as diabetes, chronic respiratory disease, cardiovascular disease, or cancer are expected to develop more serious symptoms [5,6]. Young people and adults who have no serious medical conditions are requested to self-manage their symptoms in isolation possibly taking a pain killer such as paracetamol or using hyperthermia and lifestyle modifications such as increased rest and taking good supply of water [7,8]. However, most of the present procedures do not provide adequate medical advice how to treat cough and other symptoms [9,10]. COVID-19 symptoms usually start with dry cough, fever, and dyspnea, which is similar to other viral respiratory diseases such as ordinary flu. Consequently, the diagnosis of COVID-19 becomes difficult. Generally, the incubation period of  is approximately a few to 15 days. SARS-Cov-2 has a dramatic transmission capability [9,10]. This could be related to the fact that who accidentally participate to the spread of the disease [11,12].
In COVID-19 the infection from mild upper respiratory tract problems to severe acute respiratory syndromes which may lead to respiratory failure and shock which in addition may cause multiple organ failure. Other accompanying features might arise such as fatigue, diarrhea, headache, or lymphopenia and cardiovascular symptoms [13,14].
Corticosteroids, as generally used immunosuppressants and anti-inflammatory agents play an significant role in the treatment of critical diseases [15,16]. Previous studies showed that corticosteroid was used to treat coronavirus diseases, including Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) [17]. Studies have demonstrated that cytokine reactions caused by the enormous release of cytokines were major cause of death in a number of cases of SARS and MER [18,19] and corticosteroids could have a positive antagonistic effect on the cytokine storm [20]. Azithromycin has immunomodulatory and antiviral properties that might be of attention in the treatment of COVID-19. The use of this macrolide has been connected with the progression of clinical therapy in other viral infections and community acquired pneumonia [21].
Azithromycin has been shown to exert in vitro activity against SARS-CoV-2 and may operate at many points of the viral cycle.
Additionally, the immunomodulation of Azithromycin improved clinical outcomes by its capability to preserve epithelial integrity, down regulate cytokine production and avoid lung fibrosis and hence could play a role in the hyperinflammatory stage during COVID-19 infection [22,23].
Current confirmation showed that nutritional supplementation might have a supportive role in COVID-19 patients. Administration of higher than recommended daily doses of nutrients such as vitamins C, D, E, omega-3 fatty acids and Zinc exert a valuable effect by significantly reducing SARS-CoV-2 viral load and the time of hospitalization [24,25]. These nutrients are known for their immunomodulatory and antioxidant ability. Deficiencies of such nutrients could lead to immune dysfunction and enhance vulnerability to pathological infection. Dietary deficiency of vitamins and minerals has been found in high-risk groups of COVID-19 patients, such as the elderly by increasing morbidity and risk of mortality [26]. Natural products and herbal medicine are employed to treat respiratory infection, and several have been approved as drugs, over-the-counter or food additives. The usual low to negligible toxicity makes herbal medicines and natural product reasonable prophylactic candidates for treatment. Using in silico technology to study natural products has been found utterly effective to find blockers of enzyme function and membrane receptors of human coronavirus [27,28]. The symptoms of COVID-19 are characteristic exhibiting uncontrolled inflammation, such cytokine release syndrome, and hence anti-inflammatory herbs are able to suppress such often deadly symptoms. Digestive enzymes, low pH in the gastric vicinity or the gut microbiome have less effect on the bioavailability of natural products and herbs in comparison to antibody and other prophylactics [29,30].
Thyme is a plant with a long history in traditional medicine in Libya that has antiseptic, antimicrobial, and antiviral properties.
It also has useful effects on the cardiovascular and respiratory system [31][32][33]. Thymus capitatus belongs to the thymus genus and the Lamiaceae family. This plant is small, perennial and grows in hot and dry climates like Libya, Tunisia, Algeria etc [34,35].
Thymus capitatus contains bioactive compounds such as phenols, phenolic acids, terpenes and flavonoids [36][37][38]. Thymus capitatus is containing a rich source of vitamins, particularly vitamin C, vitamin A, and vitamin B6. Furthermore, in the leaves of this plant, minerals such as calcium, iron, potassium, manganese, magnesium, and selenium are found in abundance [39].
Molecular modeling is a recognized computational tool to aid early drug discovery and development. It is used to generate ideas of a compounds or macromolecules 3D conformation, proteinligand interactions, and allows forecasts about biological activities.
The integration of molecular modeling in drug or vaccine design can help in early drug or vaccine discoveries [40][41][42].
The main aim of this study was to investigate the effects of Thymus Capitatus extract on COVID-19 patients clinically to provide information if it can be used as a clinically compatible option for adjuvant therapy. We also performed in-silico studies on two targets -protease enzyme and spike glycoprotein -to collect information if they are suited as targets by computational drug repurposing procedures to allocate appropriate inhibitory agents.

Clinical evaluation
The study was performed between October 1st and November 30th, 2020, a total of 60 cases were investigated at Arada polyclinic, Tripoli Libya. All the patients were informed and consented. Sixty COVID-19 patients were enrolled in the study. The age group of patients was from 40-85 years. The patients were treated at the Out Patient Department (OPD) of Arada polyclinic, Arada, Tripoli Libya.
All patients were screened for COVID-19 symptoms by polymerase-chain reaction (PCR) as well IgM and IgG antibody tests within 5 days of the study. Additionally, a history of diabetes, chronic obstructive pulmonary disease COPD, coronary artery disease CAD, and use of immunosuppressants were recorded for risk evaluation.
All patients came to the clinic with pulmonary infiltrate bilateral atypical pneumonia (walking pneumonia). Patients diagnosed with clinical symptoms showed: sore throat (pharyngitis), felt tired (fatigue), had chest pain, mild to severe chills, high-grade fever, persistent cough that can be dry or produce colored mucus, sneezing dyspnea and severe headache. In addition, high-resolution Page 3 of 11 computed tomography HRCT, also called thin-section scan (CT scanning) was used to confirm pneumonia diagnosis.
The patients were divided into two groups: the first group consisted of 60 patients (45 males and 15 females) to whom Thymus capitatus (10 g/200 ml boiled water containing the 10g of stem and leaves of Thymus capitatus) was administered. In addition, patients were given prednisolon 40 mg daily for 7 days and then the dose gradually decreased by 10 mg every 7 days until the last dosage of 10 mg for 7 days. Azitromycine 500 mg tablets were used once a day for 6 days. Ceftriaxon (Rocephin 1g powder for injection solution) was given intramuscular twice a day for a week. Ipratropium (250 µg/1ml) and salbutamol (5mg/2.5ml) were inhaled using a nebulizer every 8 hours for two weeks. The patients were also treated with standard prophylactic LMWH Nadroparin calcium (Fraxiparine injection ® ) subcutaneously as a single daily dose of 0.3 ml (2,850 IU) or 0.6 ml (5700IU/0.6mL) for at least 7 days dosage. In addition, one tablet daily of Vitamin C (Ascorbic Acid) 500 mg, Vitamin D (from Cholecalciferol) 10 µg and zinc 15 mg were supplied for two months.

Molecular docking
The starting geometry of Thymus capitatus constituents was

Clinical spectrum
Patients who had recovered from COVID-19, 95.0% reported persistence of at least 1 symptom, particularly very mild dyspnea and mild fatigue. 5% (3 patients) died a 84 years old man with diabetes and hypertension, a lady 72 years old with myopathy and a 45 years man. Limitations of the study include the lack of information on symptom history before acute COVID-19 illness and the lack of details on symptom severity. Furthermore, this is a single-center study with a relatively small number of patients (60) and without a control group of patients who did not use Thymus capitatus.
Thymus capitatus contains a variety of functional chemical groups including phenols, phenolic acids, terpenes and flavonoids.
It is reported that in SARS-CoV-2 infection, the level of AngII increases [48]. The binding of AngII to AngII type 1 receptors (AT1R) is a vital mediator for majority of pro-inflammatory effects.
The AngII type 2 receptor (AT2R) is activated by AngII which activates some inflammatory cells. AT1R activation induces the overexpression of chemokines, adhesion molecules, and cytokines via special pathways such as NF-kB up-regulation. NF-kB plays an essential role in the regulation of numerous pro-inflammatory genes [49,50]. It has been reported that herbal thyme acts as a negative regulator of NF-kB. Furthermore, other studies showed that the plasma levels of most pro-inflammatory cytokines, such as TNF, IL1B, IL7, IL1RA, IL8, IL9, IL10, basic fibroblast growth factor (FGF), and other pro-inflammatory factors were increased in COVID-19. Consequently, thyme fits as an inhibitory agent of proinflammatory cytokines [51,52]. Hence, Thymus capitatus can be considered as adjuvant therapy for COVID-19 as described in the literature [53,54]. Thymol has antiaggregant ability and it is able to inhibit platelet aggregation induced by thrombin, collagen, adenosine diphosphate and arachidonic acid [62].
In Libya some people use Thymus capitatus extract as a remedy for hypertension and other cardiovascular diseases.
Thymus capitatus has effective components such as thymol and carvacrol against oxidative agents causing cardiovascular disease [63]. Cardiovascular damages contribute to the morbidity of COVID-19 patients. Patients with cardiovascular disease (CVDs) are in addition susceptible to develop severe COVID-19 symptoms, in particular acute myocardial injury [64,65]. SARS-COV-22 appears able to provoke myocardial injury via a cytokine storm connected with a rigorous response by type 1 and type 2 T helper cells [64,65]. Furthermore, COVID-19-induced hypoxemia [66] and respiratory collapse could lead to myocardial dysfunction [64,65]. Cytokine storms could provoke multiple organ failure and increase inflammatory chemokines such as IL-6, IL-2, IL-7, interferon-γ, (TNF)-α. Cardiovascular injury correlates with elevated inflammatory markers [64,65]. Moreover, the oxidative stress generated by SARS-CoV-2 might be a source of heart injures [64,65]. Thymus capitatus contains antioxidant components, and studies have shown that an appropriate dose of antioxidants can improve heart damage caused by COVID-19 infection [67]. It has been also demonstrated that thymol and carvacrol, derivatives of Thymus capitatus, have anti-inflammatory effects [68;69]. Taken together it appears that Thymus capitatus and its derivatives can protect cardiovascular damages induced by SARS-COV-22.   Table 1. The modeling studies indicate van der Waals, hydrogen bonding (Table 1)   The obtained results using computational drug repurposing offer an efficient way to find novel applications for already known drugs [75]. Molecular docking and binding free energy calculations for sesquiterpenes can be used to forecast drug-target interactions and binding affinity. The appearance of resistance to existing antiviral drugs or vaccines is a major challenge in antiviral drug development. The drug repurposing technique allows finding novel antiviral agents within a short period in order to overcome the challenges in antiviral therapy. Computational drug repurposing has previously been used to recognize drug candidates for viral infectious diseases like ZIKA, Ebola, influenza and dengue infections. These methods were also utilized to recognize possible drugs against MERS-CoV and SARS-CoV [76,77] and following the COVID-19 outbreak, computational repurposing can be used for finding COVID-19 antiviral agents. The Thymus capitatus sesquiterpenes are C15-terpenoids built from three isoprene units and are found mainly in higher plants as well as in fungi and marine organisms. Naturally, they are available as hydrocarbons or in oxygenated forms including alcohols, lactones, aldehydes, ketones and acids. Sesquiterpenes also include essential oils and aromatic constituents with several pharmacological activities. Studies report that sesquiterpenes have anti-inflammatory, anticancer, antidiabetic, antinociceptive and antimicrobial activities [78,79].

Molecular docking analysis
They also effect the central nervous system that make them possible targets for the development of novel therapeutics and of usage of medical intentions [78,79].
The results obtained by molecular modeling and by clinical use of Thymus capitatus extract are very promising also for fighting covid-19 viruses. It has been reported that sesquiterpenes directly inactivate herpes virus and might impede with virion envelope structures or mask viral structures that are essential for adsorption or entry into host cells. A virucidal activity of Melaleuca armillaris essential oil has been reported recently [80,81] and dissolution of the herpes simplex virus envelope by treatment with oregano essential oil has been described [82]. Thus, different mechanisms of antiviral activity of many essential oils and compounds of essential oils including sesquiterpenes seem to be available. It has been also reported that Santolina insularis essential oil causes inactivation of herpesviruses and prevention of cell-to-cell spread [83,84].
Sesquiterpenes, were found to inhibit the severe acute respiratory syndrome caused by coronavirus [85,86], cytomegalovirus [87], and rhinovirus [88]. Furthermore, it has been reported that the chemical composition Thymus capitatus essential oil has antioxidative and antimicrobial activities [68,89]. These herbal species were also found to be contained in thymol and its isomer carvacrol, followed by their biosynthetic precursors, terpinene and p-cymene as main compounds [68,89]. In addition, literature data indicate that the powerful bioactive potential of Thymus capitatus constituents is principally related to the high proportion of phenolic compounds such as thymol and carvacrol [90]. Anti-inflammatory, antibacterial, antifungal, cytotoxic, insecticidal and herbicidal properties of thymol and carvacrol were studied previously [90].
In addition, numerous vaccines were recently developed by aiming at the exploration of vaccines to prevent and treat 2019-nCoV. Computational-based technology has given the researchers the tools to explore and identify potentially effective drug and/ or vaccine candidates which can efficiently shorten the time and diminish cost.

Conclusion
Essential oils and their components are known to be active against a wide variety of microorganisms. Many chemical constituents present in Thymus capitatus might contribute to antiviral activity. COVID-19 increasing oxidative stress, inflammations, and disturbance of the renin-angiotensin system, lead to damage, particularly the respiratory and the cardiovascular system. Thymus capitatus and its bioactive components suppress the negative effects of the renin-angiotensin system in COVID-19 patients. Moreover, many components of Thymus capitatus decrease pro-inflammatory cytokines such as IL-6 and TNF-α. Hence, it can decrease the severity of acute lung injury and the acute respiratory distress syndrome. Thymus capitatus extract reduces oxidative stress due to its antioxidant properties that can reduce adverse cardiovascular effects during COVID-19 infection. Finally, we conclude that Thymus capitatus has antioxidant, anti-inflammatory, and immunomodulating properties and diminishes side effects in COVID-19 patients as adjuvant.