Exploring the Potential of Siddha Formulation MilagaiKudineer-Derived Phytotherapeutics Against SARS-CoV-2: An In-Silico Investigation for Antiviral Intervention

Baskar Rajamanickam^{*, 1}, Ramesh Balasubramanian², Manekshah Yovas Rajammal³, Bharathkumar Govindaraju⁴, Selvapriya Sikkal Selvaraaju⁵, Hemadevi Thangarasu⁶, Kanakavalli Kadarkarai⁷

¹Lecturer II, Department of Gunapadam (Pharmacology), Government Siddha Medical College, The Tamil Nadu Dr. MGR Medical University, Chennai, Tamil Nadu, India. http://orcid. org/0009-0002-0662-4837.

²Research Department of Biotechnology, Sri Sankara Arts and Science College, University of Madras, Kanchipuram, Tamil Nadu, India.http://orcid.org/0000-0003-0890-3178.

³State Licencing Authority (Indian Medicine), Chennai, Tamilnadu, India. http://orcid.org/0009-0008-8431-1880.

⁴Assistant Professor, Department of Siddha, The Tamil Nadu Dr.MGR Medical University, Chennai, Tamil Nadu, India.http://orcid.org/0009-002-1917-4775.

⁵PG Scholar, Department of Siddhar Yoga Maruthuvam, Government Siddha Medical College, The Tamil Nadu Dr. MGR Medical University, Chennai, Tamil Nadu, India.http://orcid.org/0009-0004-6101-8623

⁶Assistant Medical Officer, Arignar Anna Government Hospital of Indian Medicine, Chennai, Tamil Nadu, India.http://orcid.org/0009-0006-5771-759X.

⁷Principal, Government Siddha Medical College, The Tamil Nadu Dr.MGR Medical University, Chennai, Tamil Nadu, India.

^*Corresponding author: Baskar Rajamanickam, Lecturer II, Department of Gunapadam (Pharmacology), Government Siddha Medical College, The Tamil Nadu Dr. MGR Medical University, Chennai, Tamil Nadu, India. 

Received: 07 January 2025; Accepted: 10 February 2025; Published: 20 February 2025

Introduction

The Siddha system of medicine, deeply rooted in South India, particularly in Tamil Nadu, has garnered renown for its holistic approach to healthcare. During challenging times such as the outbreaks of diseases like Dengue and the COVID-19 pandemic, Siddha medicines have played a vital role in saving lives. Among these remedies, MilagaiKudineer, a formulation attributed to the Tiruvanamalai Guru Parambariyam, has emerged as particularly noteworthy during the COVID-19 crisis. This herbal concoction has demonstrated significant efficacy in combating the coronavirus, making it a crucial asset in the fight against the pandemic. The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to significant global health and economic challenges and underscores the need for effective antiviral interventions. The search for effective therapeutic agents to combat this virus is of paramount importance. The spike glycoprotein of SARS-CoV-2 plays a crucial role in viral entry by binding to host cell receptors. Targeting the receptor-binding domain of the spike protein presents a promising approach for therapeutic development. Siddha medicine offers a repertoire of natural compounds with potential antiviral properties, including those found in MilagaiKudineer. Traditional medicinal systems, such as Siddha medicine, offer a repertoire of natural compounds with potential pharmacological activities against viral infections.MilagaiKudineer is a traditional Siddha formulation comprising herbs like Cuminum cyminum, Curcuma longa, and Capsicum annuum, which are known for their bioactive constituents with diverse medicinalproperties. The main protease of SARS-CoV-2, 3-chymotrypsin-like protease (3CLpro), plays a crucial role in viral replication by cleaving the polyproteins into functional non-structural proteins.

Targeting 3CL pro presents a promising strategy for developing antiviral therapeutics. Siddha medicine, an ancient traditional medicinal system, offers potential sources of bioactive compounds for combating viral infections. MilagaiKudineer, a Siddha formulation, comprises various herbs known for their pharmacological properties. This study was envisaged with objectives such as i)to assess the inhibitory potential of bioactive compounds from MilagaiKudineer against the COVID-19 spike glycoprotein receptor-binding domain through computational docking analysis, ii)to investigate the potential of bioactive compounds derived from MilagaiKudineer against the SARS-CoV2 RNA-dependent RNA polymerase through computational methods and iii) to investigate the inhibitory potential of bioactive compounds  from MilagaiKudineer against the SARS-CoV2 main protease, 3CL pro, through computational docking analysis.

Materials and Methods

Milagaikudineer, a kudineer formulation of Tiruvanamalai Guru Parambariyam is routinely prepared as described (Asan, 2023). All the ingredients in the proportion given in Table 1 are taken, and 200 ml of water is added and boiled for 2 minutes. After filtration 50      ml dose can be given onlyin the morning for 3 consecutive days. For children, 5-25 ml can be given as per their age.

Phytocompounds Selected for docking:

Totally11 compounds, 4 phytocompounds from Cuminum cyminum viz. Carvacrol Cuminaldehyde, Linalool, Coumaric acid (Ali, 2016), 4 from Curcuma longa viz. Curcumin, Quercetin, Sabinene, Cineol (Hewlingset al., 2017; Zhang and Kitts, 2021) and 3 compounds, Dihydrocapsaicin, Capsaicin, Ascorbic acid from capsicum annuum (Hamed et al., 2019) were selected for this study.

The three-dimensional structures of the SARS-CoV-2 viral spike glycoprotein receptor-binding domain (PDBID:6VSB),SARS-CoV2RNA-dependentRNApolymerase (PDB ID:6NUR)and SARS-CoV-2 viral main protease (3-chymotrypsin-like protease (3CL pro) (PDB ID: 6LU7) were retrieved from RCSB-PDB (Goodsell et al.,2020).

The crystalline structure of the SARS-CoV-2 viral spike glycoprotein receptor-binding domain (PDB ID: 6VSB), SARS-CoV2 RNA-dependent RNA polymerase (PDB ID: 6NUR) and SARS- CoV-2 viral main protease (3-chymotrypsin-like protease (3CL pro) (PDB ID: 6LU7) were retrieved from RCSB-PDB and protein clean-up process was done and essential missing hydrogen atom were added. Watermolecules and cocrystallized ligands wereremoved. Different orientation of the lead molecules with respect to the target protein was evaluated by theAutodock program and the best dock pose was selected based on the interaction study analysis.

All the 11 compounds, 4 phytocompounds from Cuminum cyminum viz. Carvacrol Cumin aldehyde, Linalool, Coumaric acid, 4 from Curcuma longa viz. Curcumin, Quercetin, Sabinene, Cineol and 3 compounds, Dihydrocapsaicin, Capsaicin, and Ascorbic acid from capsicum annuum were built using Chem Draw prof online tool version 12.0. Ligands prepared through geometry optimization method (MMFF94).

Docking calculations were carried out for retrieved phytocomponents against all three target proteins. Essential hydrogen atoms, Kollman united atom type charges, and solvation parameters were added with the aid of AutoDock tools (Morris, Goodsell et al., 1998). Affinity (grid) maps of 25 Å grid points and 0.375 Å spacing were generated using the Autogrid program (Morris, Goodsell et al., 1998). AutoDock parameter set- and distance-dependent dielectric functionswere used in the calculation of the van der Waals and the electrostatic terms, respectively. Docking simulations were performed using the Lamarckian genetic algorithm (LGA) and the Solis & Wets local search method (Solis and Wets, 1981). The initial position, orientation, and torsions of the ligand molecules were set randomly. All rotatable torsions were released during docking. Each docking experiment was derived from 2 different runs that were set to terminate after a maximum of 250000 energy evaluations. The population size was set to 150. During the search, a translational step of 0.2 Å, and quaternion and torsion steps of 5 were applied.

Results and Discussion

Totally11 compounds were selected for this study and their structures are shown in Figure-1. These were four phytocompounds from Cuminum cyminum viz. Carvacrol Cumin aldehyde, Linalool,Coumaric acid, 4from Curcumalonga viz.Curcumin,Quercetin,Sabinene,Cineoland 3 compounds, Dihydrocapsaicin, Capsaicin, and
Ascorbic acid from capsicum annuum.

Figure-1: 2 D Structure of Phytocomponents from MilagaiKudineer

Docking studies of phytochemicals from MilagaiKudineer against SARS-CoV-2spike glycoprotein receptor-binding domain:

The Summary of the molecular docking studies of compounds against SARS-CoV-2 spike glycoprotein receptor-binding – PDB 6VSB is shown in Table-2. The docked pose of the top molecule Carvacrol with SARS-CoV-2 spike glycoprotein receptor is shown in Figure-2.

Figure-2:DockingPoseofCarvacrolwithSARS-Co 2spikeglycoproteinreceptor binding Domain

Outof11compounds, the lead molecules such as Carvacrol,Cuminald eyde,Linalool, Curcumin, Quercetin, Cineol, Dihydrocapsaicin, Capsaicin and ascorbic acid reveals a maximum of 3-5 interactions with the core active amino acid residues present on the target SARS-CoV-2 spike glycoprotein receptor-binding domain. the interaction between lead molecules and with target protein are shown in Table-3

Based on the results of the computational analysis it was concluded that the bio-active compounds such as Carvacrol, Cuminaldeyde, Linalool, Curcumin, Quercetin, Cineol, Dihydrocapsaicin, Capsaicin and ascorbic acid present in the Siddha formulation MilagaiKudineer reveals significant binding against the target proteinSARS-CoV-2 spike glycoprotein receptor-binding domain.

The Summary of the molecular docking studies of compounds against SARS-CoV-2 RNA- dependent RNA polymerase (PDB)-6NUR is shown in Table-4. The docked pose of the top molecule Carvacrol with SARS-CoV-2 RNA-dependent RNA polymerase is shown in Figure-3.

A total of 11 bioactive lead compounds were retrieved from the Siddha formulation Milagaikudineer and were subject edtoin-silicoinvestigationofwhichleadcompoundssuchasLinalool, Coumaric acid, Curcumin, Quercetin, Carvacrol, Cineol, Dihydrocapsaicin and Capsaicin reveals maximum interactions (2-3) with the binding sites on the core active amino acid residues present on the target receptor RdRp. The interaction between lead molecules and with target protein is shown in Table-5.

Based on the results of the computational analysis it was concluded that compounds such as Linalool, Coumaric acid, Curcumin, Quercetin, Carvacrol, Cineol, Dihydrocapsaicin and Capsaicin present in the Siddha formulation Milagaikudineer revels significant binding efficacy against active aminoacid present on the target enzyme and it was concluded that these compounds exert promising inhibiting against RdRp enzyme and thereby halt the viral replication.

The Summary of the molecular docking studies of compounds against SARS-CoV-2 Main protease PDB 6LU7 is shown in Table-6. The docked pose of the top molecule Carvacrol with SARS-CoV-2 spike glycoprotein receptor is shown in Figure-4.

Figure-4:DockingPoseofCarvacrolwithSARS-CoV-2MainproteasePDB6LU7

Out of eleven compounds, the lead compounds such as Curcumin, Quercetin, Sabinene, Cineol, Dihydrocapsaicin and Capsaicin reveal a maximum of 3 to 5 interactions with the core active amino acid residues present on the target 3CLpro.The interaction between the lead molecule and with target protein is shown in Table-7.

Based on the results of the computational analysis it was concluded that the bio-active compounds such as Curcumin, Quercetin, Sabinene, Cineol, Dihydrocapsaicin and Capsaicin present in the Siddha formulation reveal significant binding against the target protein3CLpro thereby it was concluded that these compounds may exert promising inhibiting against 3CLpro enzyme and hereby halt the formation of 16 non-structural proteins (nsp1-nsp16) that are highly essential for viral replication and thereby prevents the viral survival in the host environment.

Conclusion

MilagaiKudineer exhibits promising antiviral properties attributed to its constituents. Capsicumannuum Linn (Green chilly) contains capsaicin, which possesses antiviral effects. Cuminum cyminum Linn (Cumin) and Curcuma longa Linn (Turmeric) are known for theirimmunomodulatory and anti-inflammatory properties, which can help combat viral infections. Computational analysis revealed the potential of bioactive compounds from MilagaiKudineer to bind effectively to the receptor-binding domain of the SARS-CoV-2 spike glycoprotein, the SARS-CoV-2 RNA-dependent RNA polymerase and the SARS-CoV-2 Main protease. Compounds such as Carvacrol, Cuminaldehyde, Linalool, Curcumin, Quercetin, Cineol, Dihydrocapsaicin, Capsaicin, and Ascorbic acid hold promise as inhibitors of viral entry and replication, highlighting their potential as therapeutic agents against COVID-19. Furtherexperimental studies are warranted to validate these findings and explore their clinical applications.

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