Antiviral compounds of Wrightia tinctoria(Roxb.) R.Br. against SARS CoV-2: An in vitro and in silico studies

Introduction

The exploration of natural sources for new safe and effective pharmaceutical broad spectrum antiviral candidates against widespread Severe Acute Respiratory Syndrome Coronavirus -SARS-CoV-2 is desirable. Currently, viral infection is the most serious health issue especially SARS-CoV-2 a β-Coronavirus, caused severe and life-threatening respiratory infections in humans which caused unexpected higher rate of death across the globe [1]. SARS-CoV-2, a zoonotic positive-sense single-stranded RNA (+ssRNA) enveloped virus invade the pulmonary epithelial cells of the Upper respiratory system (Shah et al., 2020) [2] . Its spike (S) protein is cleaved into S1 and S2 subunits, which attach to the receptors and facilitate membrane fusion. Viral genetic materials are then delivered to the host's cytoplasm through the membrane fusion pore so that they can multiply (Jackson et al., 2022) [3] . These viral proteins can be the primary targets of effective drugs to suppress viral entry and replication. Only a few antiviral therapeutic drugs may be helpful in controlling the spread of SARS-CoV-2 infection. Unfortunately, the toxicities, countless adverse effects, expenses and ineffective responses of emerging viral resistant strains to synthetic antiviral drugs have reinforced the search of effective and affordable alternative treatment options, such as plant-derived antiviral drug molecules (Kurokawa et al., 2010) [4] . The quest for novel antiviral phyto-compounds depends on the comprehensive screening of plant derived metabolites that comprises several functional bioactive compounds that can impede viral reproduction through modulating the immune responses of host against viral infections without affecting the host physiology with minimal side effect (Kurokawa et al., 2010; Hussain et al., 2017; Newton et al., 2016) [4,5,6] . Various literatures stated that W. tinctoria, an important medicinal plant with rich source of phytochemical constituents used for the treatment of variety of diseases and found effective against severe viral diseases (Devi et al., 2014; Vignesh et al., 2022) [7,8] . Considering the broad biological activity of W. tinctoria, the present study intended to explore the bioactive compounds of the W. tinctoria leaf, bark, and root extracts against SARS CoV-2, causative for pandemic COVID-19 through molecular docking via protein-ligand interactions analysis. SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic β-corona viruses that continue to have a significant impact on public health. Timely development and introduction of vaccines and antivirals against SARS-CoV-2 into the clinic have substantially mitigated the burden of COVID-19. However, rapidly arising SARS-CoV-2 variants calls for an expanded and diversified portfolio of antivirals, such as direct-acting antivirals (DAAs) against SARS-CoV-2 variants, the unprecedented pub lic health and economic impact of the COVID-19 pandemic caused by infection of SARS-CoV-2 has been met with an equally unprecedented scientific response.

Materials and Method

Collection and preparation of plant materials

The leaf, stem bark and root of W. tinctoria were collected from Periya Thadagam, Coimbatore district, Western Zone of Tamil Nadu situated between 11°03'32.2" N latitude and 76°50'59.8" E longitude. The collected plant samples were shade dried at room temperature, ground into fine powder and stored for further experimental analysis.

Preparation of the extracts

The powdered plant samples of W. tinctoria leaf, stem and root (20 g each) were subjected to hot extraction with 350 ml of solvents i.e. aqueous, methanol and petroleum ether using Soxhlet apparatus at 65-80 °C for 8-10 h. The solvents were evaporated in rotary vacuum evaporator until complete removal of solvents and stored at 4 °C for further use. The extracts were dissolved in respective solvents while experimentation to get the solution with a concentration of 10 mg/10 ml.

Proximate analysis

The analysis of the proximate composition of W. tinctoria leaf, stem bark and root was carried out using the standard method.

GC/MS/MS analysis

The GC-MS/MS analysis was performed using a Varian 3800 coupled with a Mass spectrum 4000 GC-MS/MS system equipped with a fused silica capillary column (15m x 0.2 mm ID x 1μm) linked to an EI detector. Helium gas (99.99% purity) was used as a carrier gas at a constant flow rate of 1 ml/min and the sample injected was 1μl; the instrument was set to an initial temperature of 110 °C, and maintained at this temperature for 2 min. Then the oven temperature was rose up to 280 °C, at the rate of an increase of 5 °C/min, and maintained for 9 min. Injection port temperature was ensured as 250 °C. The ionization voltage was 70eV. The samples were injected in split mode as 10:1. Mass spectral scan range was set at 45-450 (m/z). Using computer searches on a NIST Ver.2.1 MS data library and comparing the spectrum obtained through GC-MS/MS, the bioactive compounds present in the plants sample were identified. The name, molecular weight and structure of the components were ascertained from these comparisons.

In silico docking for binding efficacy of SARS CoV-2 proteins

The three-dimensional (3D) structures of receptor proteins were retrieved from the RCSB Protein Data Bank (https://www.rcsb.org/) in pdb format. The molecular docking analysis was performed as previously described by Ganesan et al., 2020. The Schrodinger virtual screening tool software was used to predict the conformation of the target ligands within the appropriate target binding site of SARS-CoV-2 protein (3CLpro, ACE2, RdRp, TMPRSS2 and PLpro). The structures retrieved from the RCSB protein data bank was a monomer with co-crystallized ligand. The proteins were prepared by using the protein preparation wizard by refining bond orders, addition of hydrogens, and deleting water molecules beyond 5Å, and missing chains are included by using the prime module of Schrödinger suite 2019-4 (Kumar et al., 2022) [9] . Protein minimization was performed using optimized potentials for liquid simulations (OPLS3) molecular force field with RMSD of crystallographic heavy atoms kept at 0.30 Å. A grid box was generated to define the centroid of the active site. All the compounds were docked into the catalytic pocket of 3CLpro, ACE2, RdRp and PLpro by using the Glide module of Schrödinger suite 2019 in extra precision (XP) mode. The ligands with significant Glide scores ( < -4.0) were considered to have more binding affinity towards SARS-CoV-2 protein involved in the coronavirus disease -COVID 19 pathway (Vellanki et al., 2018) [10] .

Results and Discussion

Extract yield

The extraction yield of W. tinctoria was influenced by both the type of solvent and the plant part used. Methanol extracts showed the highest yields in all parts tested, followed by aqueous, while petroleum ether consistently produced the lowest values. In leaves, the yield was 0.76% in methanol, 0.32% in aqueous, and 0.11% in petroleum ether. Bark extracts recorded the maximum yield in methanol (0.82%), with comparatively lower values in aqueous (0.21%) and petroleum ether (0.10%). Similarly, root extracts gave 0.68% in methanol, 0.31% in aqueous, and 0.08% in petroleum ether. The consistently higher yield obtained with methanol can be attributed to its ability to solubilize a wide range of phytochemicals due to its polarity, whereas aqueous extraction recovered mainly hydrophilic constituents and petroleum ether extracted fewer non-polar compounds (Nagarajan et al., 2016) [11] . Among the plant parts, bark gave a slightly higher methanol yield compared to leaves and roots, which may reflect its richer composition of bioactive metabolites such as alkaloids and tannins. These findings suggest that methanol is the most effective solvent for recovering phytoconstituents from W. tinctoria and can be considered the preferred choice for further phytochemical and biological evaluations.

Proximate analysis

The phytochemical profiling of W. tinctoria extracts revealed the presence of key secondary metabolites such as flavonoids, phenols, sterols, saponins, tannins, and terpenoids, which are known for their broad-spectrum bioactivities and potential antiviral effects. Among the plant parts tested, root aqueous extract exhibited the highest diversity of compounds (seven classes), while bark methanolic extract was also rich in metabolites, highlighting the influence of solvent polarity on compound recovery. Flavonoids and phenolic compounds, consistently detected across different extracts, are of particular interest due to their reported ability to interfere with viral entry, replication, and protease activity in SARS-CoV-2. Terpenoids and sterols, detected mainly in methanolic and petroleum ether extracts, have also been linked to membrane stabilization and inhibition of viral enzymes. The presence of alkaloids and saponins further strengthens the antiviral potential, as these compounds are known to modulate immune responses and disrupt viral envelopes. The phytochemical diversity observed in W. tinctoria suggests its potential as a source of natural compounds that could be further explored for inhibitory effects against SARS-CoV-2 through in vitro and in silico approaches, emphasizing its relevance in the search for plant-based therapeutics during the COVID-19 pandemic.

Table 2. Phytochemical screening of the W. tinctoria extracts

S.

Plant parts used

Solvent system

Phytochemicals Alkaloids Flavonoids Tannins Saponins Sterols Phenols Glycosides Proteins Carbohydrates Terpenoids Total

The quantitative phytochemical evaluation of W. tinctoria revealed solvent-and tissue-specific variations that are relevant to antiviral applications, particularly against SARS-CoV-2. Leaf aqueous extracts showed the highest flavonoid (0.680 ± 0.002 mg/g) and phenolic (4.274 ± 0.002 mg/g) contents, while petroleum ether extracts contained notable levels of steroids (1.008 ± 0.01 mg/g), compounds reported to modulate viral replication. Bark extracts were especially rich in phenols when extracted with petroleum ether (6.952 ± 0.004 mg/g), whereas methanol favored flavonoid recovery (0.820 ± 0.002 mg/g). Root extracts exhibited remarkable levels of tannins (2.284 ± 0.002 mg/g) and phenols (5.129 ± 0.03 mg/g) under aqueous solvents, alongside moderate flavonoid recovery. The high content of flavonoids and phenols across leaf, bark, and root is of particular interest, as these metabolites have been widely associated with inhibition of SARS-CoV-2 main protease (Mpro) and spike-ACE2 interactions, thereby suppressing viral entry and replication. Similarly, tannins and terpenoids, especially abundant in roots and leaves, are known to disrupt viral proteins and enhance host immune defense. These findings suggest that W. tinctoria is a promising source of diverse phytoconstituents with potential inhibitory roles against SARS-CoV-2, and the observed solvent-dependent variation provides a scientific basis for selecting appropriate extraction systems for antiviral drug development.

GC-MS/MS analysis

The GC-MS/MS analysis of W. tinctoria methanolic extracts of leaf, bark, and root revealed a wide spectrum of bioactive compounds with potential antiviral implications against SARS-CoV-2 (Figure 2 & Table 4-

Molecular docking of W. tinctoria plant samples

GC-MS/MS analysis of various parts of the medicinal plant W. tinctoria revealed the presence of various compounds having biological activities such as antimicrobial, anti-inflammatory, anticancer, antibacterial, anthelmintic, hepatoprotective, immune potential, antipyretic, antioxidant, antifungal, anticancer, anti-HIV activity etc. Phytocompounds viz., Phytol, Gamma-sitosterol, Lupeol, Campesterol and Stigmasterol were reported to have antiviral activity. To confirm the viral inhibitory activity of these compounds, molecular docking study was conducted to evaluate their Binding affinity against the targeted proteins which somehow or other play's crucial role in reducing or blocking the activity of SARS-CoV-2. Molecular docking of the four targeted proteins viz., Angiotensin-converting enzyme 2 (ACE2), 3-chymotrypsin like protease (3CLpro), papain-like protease (PLpro) and RNA-dependent RNA polymerase (RdRp) which play crucial role in viral entry into the host cell and indispensable for the replication revealed that Stigmasterol exhibited a remarkable binding affinity towards all the four targeted protein selected for the study, followed by Lupeol. From the study it was found that the candidate drug compounds campesterol, γ-sitosterol, lupeol, phytol and stigmasterol were exhibited significant binding affinity and secured great gliding score energy against the protein target such as ACE2, 3CLpro, RdRp, TMPRSS2 and PLpro as antiviral agents and suppressed by inhibiting the viral functions such as facilitates the virus spike protein especially receptor binding unit of Spike protein to bind on the nasal epithelial cell wall which may be a foundation for discovering natural products-based drugs against COVID-19. They also significantly bounded with active sites of viral proteins and in turn which shall suppresses the viral metabolic pathways. All the an-tiviral compounds identified have rmsd/ub and rmsd/pb value < 2 Å.

W. tinctoria with five promising antiviral compounds can be considered as an effective source for healthcare products.

Figure 3. 3D and 2D interaction of Gamma-sitosterol with 3CLpro and TMPRSS2 protein

The ligand γ-sitosterol was reported in methanol extract of W. tinctoria leaf (46.056 %). γ-sitosterol was reported to be effective against 3CLpro Protein. The ligand gamma-sitosterol showed binding affinity of -7.4 kcal/mol with 3CLpro protein, -8.3 kcal/mol with ACE2 receptors and -6. 7). 8).

The study was compared with the studies conducted by several research about the antiviral and biological properties of lupeol provides significant activities against some harmful virus was reported to decrease the ROS level and restore the antioxidant enzyme activities in mouse liver against chemical-induced oxidative stress [12]. Also, lupeol treatment is shown to induce growth inhibition and apoptosis in HCC cell line, SMMC7721 (Maurya et al., 2020) [13] . Though lupeol has shown weak antiviral activities in several studies, it has served as a lead drug for the generation of more effective compounds against Influenza A and HSV [14]. On the contrary, lupeol isolated from Strobilanthes cusia root has demonstrated a robust anti-HSV activity, in vitro (Flekhter et al., 2004) [15] . Notably, to the best of our knowledge, the antiviral activity of lupeol against hepatitis viruses has not been reported, so far. Nevertheless, lupeol (6.72 μg/mg) identified in the antioxidant GSLE, could have a direct or indirect role in the plant's anti-HBV activity. [16] . Very recently, antiviral effect of β-sitosterol isolated from cottonseed oil against tobacco mosaic virus (TMV) is reported (Donald et al., 1997) [17] . Interestingly, β-sitosterol has been also shown to attenuate in vitro chemical-induced hepatotoxicity [18] and cardiotoxicity by enhancing mitochondrial glutathione redox mechanism (Zhao et al., 2018) [19] . In line with this, our identification of stigmasterol (l6.72 μg/mg) as the abundant compound strongly supports its in vitro antioxidative, hepatoprotective and anti-HBV activities, indepen Molecular docking studies of the identified antiviral compounds Phytol, Gamma-sitosterol, Lupeol, Campesterol and Stigmasterol against the target protein viz., ACE2, TMPRSS2, 3CLpro, PLpro, RdRp revealed that among the 5 antiviral compounds, stigmasterol has significant docking score against 4 target proteins viz., ACE2, 3CLpro, PLpro, RdRp and TMPRSS2 (Table 12). All the 5 antiviral compounds have rmsd/ub and rmsd/pb value < 2 Å. The antiviral compounds selected for molecular docking study showed significant binding affinity with ACE2 and TMPRSS2 proved that the ligands strongly inhibit the entry of SARS-CoV 2 entry into the host and strong binding affinity with 3Clpro proved the inhibition of further replication the virus in the host cell. The amino acid residues Cys 145, His 41 and Glu 166 are essential for the enzymatic activity of 3CLpro. Stigmasterol with significant Binding affinity towards 4 target proteins may be considered as a promising lead compound with antiviral activity.

Conclusion

The findings provide biochemical evidence of the presence of five major antiviral compounds such as γ-sitosterol, stigmasterol, campesterol, lupeol, and phytol in W. tinctoria. Molecular docking analysis as computational evidence, confirmed the strong binding affinities of these antiviral compounds with key SARS-CoV-2 targets including ACE2, TMPRSS2, 3CLpro, PLpro, and RdRp receptors, thereby indicating their potential to inhibit viral entry, replication, and pathogenesis. Notably, stigmasterol displayed the most promising multi-target activity, suggesting its role as a lead molecule for further development. These data recognizes the potential of W. tinctoria and its antiviral compounds against SARS-CoV-2. Future validation through in vivo efficacy studies, and formulation development will be essential to translate these insights into therapeutic applications.

References

1. Docea AO, Tsatsakis A, Albulescu D, Cristea O, Zlatian O, Vinceti M, Calina D. A new threat from an old enemy: Re-emergence of coronavirus. International Journal of Molecular Medicine. 2020;45:1631-1643.
2. Shah A, Kashyap R, Tosh P, Sampathkumar P, O'Horo JC. Guide to understanding the 2019 novel coronavirus. Mayo Clinic Proceedings. 2020;95:646-652.
3. Jackson CB, Farzan M, Chen B, Choe H. Mechanisms of SARSCoV-2 entry into cells. Nature Reviews Molecular Cell Biology. 2022;23:3-20.
4. Kurokawa M, Shimizu T, Watanabe W, Shiraki K. Development of new antiviral agents from natural products. Open Antimicrobial Agents Journal. 2010;2:49-57.
5. Hussain M, Farooq M, Nawaz A, Al-Sadi AM, Solaiman ZM, Alghamdi SS, Siddique KH. Biochar for crop production: potential benefits and risks. Journal of Soils and Sediments. 2017;17:685-716.
6. Newton AH, Cardani A, Braciale TJ. The host immune response in respiratory virus infection: balancing virus clearance and immunopathology. Seminars in Immunopathology. 2016;38:471482.
7. Devi SL, Divakar MC. Wrightia tinctoria (Roxb.) R.Br.-An updated review. Hygeia: Journal for Drugs and Medicines. 2014;6:95-105.
8. Vignesh A, Sivalingam R, Selvakumar S, Vasanth K. A review on ethnomedicinal and phytopharmacological potential of Berberis tinctoria Lesch. Thai Journal of Pharmaceutical Sciences. 2022;46:137-148.
9. Kumar BK, Faheem N, Sekhar KVGC, Ojha R, Prajapati VK, Pai A, Murugesan S. Pharmacophore-based virtual screening, molecular docking, molecular dynamics and MM-GBSA approach for identification of prospective SARS-CoV-2 inhibitor from natural product databases. Journal of Biomolecular Structure and Dynamics. 2022;40:1363-1386.
10. Vellanki K, Hou S. Menopause in CKD. American Journal of Kidney Diseases. 2018;71:710-719.
11. Nagarajan J, Wah Heng W, Galanakis CM, Nagasundara Ramanan R, Raghunandan ME, Sun J, Prasad KN. Extraction of phytochemicals using hydrotropic solvents. Separation Science and Technology. 2016;51:1151-1165.
12. Mohammad T, Siddiqui S, Shamsi A, Alajmi MF, Hussain A, Islam A, Hassan MI. Virtual screening approach to identify highaffinity inhibitors of serum-and glucocorticoid-regulated kinase 1 among bioactive natural products: combined molecular docking and simulation studies. Molecules. 2020;25:823.
13. Maurya SK, Shadab GGHA, Siddique HR. Chemosensitization of therapy-resistant tumors: targeting multiple cell signaling pathways by lupeol, a pentacyclic triterpene. Current Pharmaceutical Design. 2020;26:455-465.
14. Akram M, Tahir IM, Shah SMA, Mahmood Z, Altaf A, Ahmad K, Mehboob H. Antiviral potential of medicinal plants against HIV, HSV, influenza, hepatitis and coxsackievirus: a systematic review. Phytotherapy Research. 2018;32:811-822.
15. Flekhter OB, Boreko EI, Nigmatullina LR, Pavlova NI, Medvedeva NI, Nikolaeva SN, Tolstikov GA. Synthesis and antiviral activity of lupane triterpenoids and their derivatives. Pharmaceutical Chemistry Journal. 2004;38:355-358.
16. Tanaka T, Kanda T, Takahashi T, Saegusa S, Moriya J, Kurabayashi M. Interleukin-6-induced reciprocal expression of SERCA and natriuretic peptides mRNA in cultured rat ventricular myocytes. Journal of International Medical Research. 2004;32:57-61.
17. Donald PR, Lamprecht JH, Freestone M, Albrecht CF, Bouic PJD, Kotze D, Van Jaarsveld PP. A randomised placebo-controlled trial of the efficacy of beta-sitosterol and its glucoside as adjuvants in the treatment of pulmonary tuberculosis. International Journal of Tuberculosis and Lung Disease. 1997;1:518-522.
18. Parvez MK, Alam P, Arbab AH, Al-Dosari MS, Alhowiriny TA, Alqasoumi SI. Analysis of antioxidative and antiviral biomarkers β-amyrin, β-sitosterol, lupeol, ursolic acid in Guiera senegalensis leaves extract by validated HPTLC methods. Saudi Pharmaceutical Journal. 2018;26:685-693.
19. Zhao L, Qi Y, Xu L, Tao X, Han X, Yin L, Peng J. MicroRNA140-5p aggravates doxorubicin-induced cardiotoxicity by promoting myocardial oxidative stress via targeting Nrf2 and Sirt2. Redox Biology. 2018;15:284-296.