Beta-Sitosterol: As Immunostimulant, Antioxidant and Inhibitor of SARS-CoV-2 Spike Glycoprotein

This article is an extension to our recently published article in Asian Journal of Pharmaceutical and Clinical Research, entitled “Β-Sitosterol: Isolation from Muntingia Calabura Linn. Bark Extract, Structural Elucidation, and Molecular Docking Studies as Potential Inhibitor of SARSCoV-2 Mpro (COVID-19)”[1]. The article describes detailed procedure for the isolation (by Column Chromatography) and structural characterization (by FTIR, UV-Visible Spectroscopy and HPTLC) of β-sitosterol from Muntingia Calabura bark. The β-sitosterol was docked on SARSCoV-2 Mpro to study the binding affinity (kcal/mol) in comparison with favipiravir. It has been found that favipiravir has a less binding affinity, i.e. 5.7 kcal/mol than β-sitosterol which has 6.9 kcal/mol. The number of hydrogen bonds formed by the favipiravir is much more, i.e., 4 than β-sitosterol which formed only 01 hydrogen bond with SARS-CoV-2 Mpro. As an extension to this published research work, we have investigated the role of β-sitosterol as immunostimulant, antioxidant and inhibitory potential against receptor binding domain (RBD) of SARS-CoV-2 Spike Glycoprotein with the aid of molecular docking. There are many studies which reveals the antioxidant and immune boosting role of β-sitosterol especially in viral infection including pneumoniae. This commentary emphasized on further potential of β-sitosterol in treatment of COVID-19 through molecular docking studies.


Introduction
This article is an extension to our recently published article in Asian Journal of Pharmaceutical and Clinical Research, entitled "Β-Sitosterol: Isolation from Muntingia Calabura Linn. Bark Extract, Structural Elucidation, and Molecular Docking Studies as Potential Inhibitor of SARS-CoV-2 M pro (COVID-19)" [1]. The article describes detailed procedure for the isolation (by Column Chromatography) and structural characterization (by FTIR, UV-Visible Spectroscopy and HPTLC) of β-sitosterol from Muntingia Calabura bark. The β-sitosterol was docked on SARS-CoV-2 M pro to study the binding affinity (kcal/mol) in comparison with favipiravir. It has been found that favipiravir has a less binding affinity, i.e. 5.7 kcal/mol than β-sitosterol which has 6.9 kcal/mol. The number of hydrogen bonds formed by the favipiravir is much more, i.e., 4 than β-sitosterol which formed only 01 hydrogen bond with SARS-CoV-2 M pro .
As an extension to this published research work, we have investigated the role of β-sitosterol as immunostimulant, antioxidant and inhibitory potential against receptor binding domain (RBD) of SARS-CoV-2 Spike Glycoprotein with the aid of molecular docking. There are many studies which reveals the antioxidant and immune boosting role of β-sitosterol especially in viral infection including pneumoniae. This commentary emphasized on further potential of β-sitosterol in treatment of COVID-19 through molecular docking studies.
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel, zoonotic, positive-sense [2], single-stranded RNA beta-coronavirus [3] (subgenus Sarbecovirus, sub-family Orthocoronaviridae) [4]. The disease caused due to SARS-CoV-2 is termed as COVID-19 [5]. Almost every country of the world is now affected by SARS-CoV-2 infection. The World Health Organization (WHO) declared it a Public Health Emergency of International Concern on January 30, 2020, and on February 28, it upraised the worldwide threat of COVID-19 to the utmost level [6]. A global pandemic was declared on March 11, 2020 [7]. The N, E, M, and S proteins are the four structural proteins encoded by SARS-CoV-2 [8]. This S protein of SARS-CoV-2 i.e. SARS-CoV-2 spike glycoprotein causes invasion to the host cell after binding with angiotensin converting enzyme-2 (ACE-2) [9]. The SARS-CoV-2 spike glycoprotein is cleaved into two subunits during entry [10]. The S1 subunit contains a receptor binding domain (RBD) and attaches to ACE-2 [11]. The S2 subunit then facilitates membrane fusion [12][13][14]. Therefore, we have targeted RBD of spike glycoprotein and performed molecular docking studies of β-sitosterol to find out its inhibitory potential of SARS-CoV-2.

β-sitosterol as Immunostimulant, as Antiviral and as Antioxidant
Cheng et al. investigated the effects of dietary β-sitosterol at different levels on serum lipid levels, immune function, oxidative status, and intestinal morphology in broilers. They have concluded that dietary β-sitosterol supplementation could regulate serum cholesterol level, promote immune function, and improve intestinal oxidative status and morphology in broilers [15]. Fraile et al. reported that β-sitosterol can be considered an immunomodulator in pigs [16]. Bouic and Lamprecht reported that this phytosterol complex seems to target specific T-helper lymphocytes, the Th1 and Th2 cells, helping normalize their functioning and resulting in improved T-lymphocyte and natural killer cell activity. The re-establishment of these immune parameters may be of help in numerous disease processes relating to chronic immune-mediated abnormalities, including chronic viral infections, tuberculosis, rheumatoid arthritis, allergies, cancer, and autoimmune diseases [17]. Bouic et al. concluded that phytosterols could be used to prevent the subtle immunosuppression associated with excessive physical stress [18]. There are many studies which have reported the immunostimulant activity of the phytosterols [19][20][21][22][23][24].
Li et al. reported that β-sitosterol is a candidate for the development of anti-virulence agents against pathogens that rely on cholesterol-dependent toxins for successful infections [25]. Zhou et al. reported that β-sitosterol blocks the immune response mediated by RIG-I signaling and deleterious IFN production, providing a potential benefit for the treatment of influenza [26]. Parvez et. al. reported the antioxidative and hepatoprotective efficacy of of G. senegalensis leaves extract. HPTLC analysis of β-amyrin, β-sitosterol, lupeol and ursolic acid strongly supported the anti-HBV efficacy of GSLE via abating the cellular oxidative stress molecules [27]. There are many studies that have reported the antioxidant activity of β-sitosterol [27-34].

Molecular Docking Studies of β-sitosterol with RBD of SARS-CoV-2 Spike Glycoprotein
Autodock vina 1.1.2 in PyRx-Virtual Screening Tool 0.8 were used to perform the docking studies [35]. The active amino acid residues in the protein were identified and noted using BIOVIA Discovery Studio Visualizer (version-19.1.0.18287) [36]. The complete docking procedure along with ligand preparation and target preparation have been performed as described in the reference article [1]. The recently elucidated structure pre-fusion 2019-nCoV (SARS-CoV-2) spike glycoprotein with a single receptor-binding domain up was obtained from the RCSB Protein Data Bank (PDB ID: 6VSB) which was released on 26 February 2020 (https://www.rcsb. org/structure/6VSB) [37]. RBD from SARS-CoV-2 spike glycoprotein was identified from the official website of Protein Data Bank in Europe (EMBL-EBI) (https:// www.ebi.ac.uk/pdbe/entry/pdb/6vsb). There were three sequence domains in the 6VSB crystal structure; Spike receptor binding domain; Spike glycoprotein N-terminal domain; Coronavirus spike glycoprotein S1, C-terminal, along with three chains in the structure (Chain A, B, C). For molecular docking simulation, the three-dimensional grid box (size_x = 76.1623A o ; size_y = 84.3011A o ; size_z = 62.4413A o ) was designed (to define area for interactions) around the RBD (to occupy) using Autodock tool 1.5.6 with exhaustiveness value of 8 [35,36].

Results and Discussion
RBD comprises of amino acid residues from chain A, chain B, and chain C as well, therefore, the most potent inhibitor will be the one which interacts with amino acid residues from all the chains. β-sitosterol has showed a binding affinity of 7.

Conclusion
Currently, there is no specific treatment available for SARS-CoV-2 infection. Use of immunostimulants, antivirals, and antioxidants can help to reduce the risk of COVID-19. Literature supports the immunostimulant, antiviral, and antioxidant activity of the β-sitosterol.
Also, molecular docking studies have found very good binding affinity of β-sitosterol with RBD of SARS-CoV-2 spike glycoprotein which can restrict the viral invasion into the host cell. It has formed one hydrogen bond with Ala-B:419, which is good for better inhibition. From present commentary, we have concluded that β-sitosterol can be used to enhance immunity against the SARS-CoV-2 infection as well as to restrict the viral invasion into the host cell through angiotensin converting enzyme-2 (ACE-2) by inhibiting spike glycoprotein. If we can increase the dietary intake of β-sitosterol and other phytosterols it can modulate the immunity which is todays need to face COVID-19.  Table 1: Binding Affinity (kcal/mol), types of bond and active amino acid residues with bond length (A 0 ).