Coronil biochemically inhibits the interaction of various clinically relevant mutants of SARS-CoV-2 Spike Proteins (Omicron Variants) with human ACE2 receptor

Coronil biochemically inhibits the interaction of various clinically relevant mutants of SARS-CoV-2 Spike Proteins (Omicron Variants) with human ACE2 receptor | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Coronil biochemically inhibits the interaction of various clinically relevant mutants of SARS-CoV-2 Spike Proteins (Omicron Variants) with human ACE2 receptor Acharya Balkrishna, Rishabh Dev, Sandeep Kumar, Anurag Varshney This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4805471/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Accumulating evidence suggests that the receptor binding domain (RBD) of the SARS-CoV-2 Omicron variant has several times more binding affinity to the human angiotensin-converting enzyme 2 (ACE2) receptor compared to the RBD of the original covid-19 strain This increased binding affinity of Omicron variant is responsible for its increased internalization and infectivity. Methods In the present study, the impact of Coronil, a tri-herbal formulation of extracts from Withania somnifera, Tinospora cordifolia, and Ocimum sanctum on the binding properties of Omicron SARS-CoV-2 variant spike proteins (S proteins) was investigated. Compositional analysis of Coronil was performed by the Prominence-XR UHPLC system. The ELISA-based ACE2 binding inhibition assay was performed to delineate the effect of Coronil on the interaction between human ACE2 receptor and different Omicron variant spike proteins such as BA.4/BA5, XBB, BA.2.75.2, BA4.6/BF.7, BA.2.75.2, BQ.1.1, and a recently found spike protein variant JN.1 which is thought to emerge from BA.2.86. Results Coronil showed a dose-dependent inhibitory effect on the interactions between ACE2 and receptor binding domains (RBD) of all variants of spike proteins evaluated in this study including the recently emerged, highly transmissible variant spike protein JN.1. Although, Coronil significantly reduced the binding percentage in almost all the variant spike proteins, the maximum inhibition was achieved against BA.4/BA.5 where it inhibited the S protein – ACE2 interaction even at a low concentration of 3 µg/ml (16.6%). This binding inhibition was further increased to 60.3 and 84.6% at 100 and 300 µg/ml respectively. Conclusions This capability of Coronil to inhibit the binding of spike protein variants with ACE2 receptor may interfere with viral binding and internalization resulting in reduced infectivity of these Omicron spike protein variants. Overall, our data underscores the potential of Coronil in combating the various newly emerged Omicron spike protein variants. These findings may provide a basis for further studies of Coronil for its clinical effectiveness against these Omicron variants. Coronil SARS-CoV-2 Omicron Variant Spike protein ACE2 RBD Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Several prominent variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) including Alpha, Beta, Delta, and Omicron have emerged since the beginning of the Covid-19 pandemic [ 1 ]. The potential of these emerging SARS-CoV-2 variants particularly the Omicron variants, to escape natural or vaccine-induced immunity is the major cause of concern [ 2 ]. The spike protein (S protein) plays an important role in the cellular invasion of viruses and mutations in the spike protein result in better immune escape [ 3 , 4 ]. Among all the variants, the Omicron variant was found to be less severe but highly transmissible [ 5 , 6 ]. As SARS-CoV-2 enters the host cells through a cell surface receptor known as Angiotensin-converting enzyme 2 (ACE2), the affinity of variant spike protein towards ACE2 plays a crucial role in the viral infectivity [ 7 , 8 ]. It is well documented that the affinity of the original SARS-CoV-2 strain towards ACE2 is several-fold less than the Omicron spike protein variants rendering it more infectious than the original strain [ 9 ]. The infectivity of Omicron variants is not only limited to their higher binding affinity for ACE2 but also because of their potential to escape the vaccine-induced immune response [ 10 ]. Among the Omicron sub-variants, B.1.1.529 was the first reported variant in November 2021 and several sub-variants of Omicron have emerged thereafter including BA.1, BA.2, BA.3, BA.4, and BA.5 [ 11 , 12 ]. Further, several other sub-variants emerged from these variants including BA4/5, XBB, BF.7, BA.2.75.2., BQ.1.1, and the most recent variant JN.1 which is thought to emerge from BA.2.86 (Fig. 1 ) [ 12 , 13 ]. These variants have evolved by multiple combinations of different mutations mainly in the spike protein (S1 and S2 subunits) of the virus [ 14 ]. The omicron sub-variants BA.4 and BA.5 first appeared in December 2021 and January 2022 respectively. As these two sub‐variants spread more rapidly than the earlier Omicron sub‐variants, they were reclassified as ‘variant of interest’ to ‘variant of concern by the European Centre for Disease Prevention and Control. Interestingly, BA.4 and BA.5 share the same amino acid mutation, F486V [ 15 ]. The other prevalent Omicron sub lineage among the omicron variant was XBB which is thought to originate from the recombination of BA.2 and BA.2.75 sub-lineages [ 16 ]. XBB variant was known to contain the maximum number of 14 mutations, 9 of which were found in the RBD region [ 17 ]. It was reported to be one of the most immune–evasive covid variants [ 18 ]. Like XBB, the Omicron sub-variant BA.2.75.2 was also derived from BA.2. It was one of the most predominant sub-variant circulating in the Indian population [ 19 ]. The Omicron variant BF.7 which is also known as BA.5.2.1.7 emerged from BA.5 and had a short incubation period responsible for its rapid transmissibility even in previously COVID-19-infected and vaccinated people [ 20 ]. BQ1.1 which is a sub lineage of BQ.1 shares a common space with other sub variant XBB in terms of their resistance towards the neutralizing antibodies [ 21 , 22 ]. Most recently, the Omicron variant JN.1 has caught attention due to its predominance over other circulating variants. It is different from its parent lineage BA.2.86 with a unique mutation L455S in its spike protein. Presently, the JN.1 variant is responsible for a growing proportion of infections throughout the world [ 23 ]. Although there is a comprehensive understanding of the infectivity, severity, and transmissibility of the Omicron sub-variants, protective measures against these variants are rather lacking due to their inherent tendency to escape immune response and resistance towards neutralizing antibodies. In the present study, we have investigated the effect of Coronil on the binding affinity of the Omicron sub-variants BA.4/BA.5, BF.7, BA.2.75.2., BQ.1.1, XBB, and JN.1 with ACE2. Coronil is a tri-herbal formulation containing the extracts of Withania somnifera (Ashwagandha), Tinospora cordifolia (Giloy), and Ocimum sanctum (Tulsi) [ 24 ]. These medicinal plants have been used effectively for the treatment of respiratory diseases and fever [ 25 ]. Withania somnifera contains Withanolides, which are known to reduce interactions with the host ACE2 receptor [ 26 ]. Withanone present in Withania somnifera has been predicted to destabilize the ACE2-RBD complex suggesting it as a potent inhibitor of SARS-CoV-2 coronavirus entry into the host cells [ 27 ]. Tinospora cordifolia is a well-recognized plant with antiviral properties and is also known to control SARS-CoV-2 replication. Interruption of electrostatic interactions between the RBD and ACE2 by Tinocordiside present in the extract of Tinospora cordifolia may interfere with SARS-CoV-2 entry [ 28 ]. Ocimum sanctum has been shown to possess immunomodulatory effects [ 29 – 31 ]. Coronil not only diminishes the Spike-protein-dependent SARS-CoV-2 viral entry into human alveolar epithelial cells but also inhibits the production of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α. Coronil has also been shown to interfere with the binding of wild type spike protein of SARS-CoV-2 and its variants spike proteins such as S D614G and S W436R [ 29 ]. These three ayurvedic medicinal plants are also predicted to contain six inhibitors against SARS-CoV-2 M pro (Main protease) which plays an important role in viral replication [ 25 ]. This study envisaged the potential of Coronil to inhibit the binding of Omicron variant spike proteins with ACE2 as these proteins are responsible for enhanced immune evasiveness and infectivity. Methods Individual standards of Palmatine hydrochloride (Sigma Aldrich, USA) (Cat # 361615), Cordifolioside A (Chem faces, China) (Cat # CFN95040), Magnoflorine (Sigma Aldrich, USA) (Cat. # 361615), Withaferine A (Natural remedies, India) (Cat # W003), Withanoside IV (Natural remedies, India) (Cat # W006),) Withanoside V (Natural remedies, India) (Cat. # W007), Withanone (Natural remedies, India) (Cat. # W005) Rosmarinic acid (Sigma Aldrich, USA) (Cat. # R4033), Palmaitine hydrochloride (Sigma Aldrich, USA) (Cat # 361615), Ursolic acid (Tokyo chemical industries, India) (Cat. # 102067769) and Betulinic acid (Natural remedies, India) (Cat. # B2836) were dissolved in methanol to prepare 1000 ppm standard solution. 0.05 mL of 1000 ppm standard stock solution was taken to prepare 50 ppm of standard working solution. Purified Omicron Spike (S) proteins and purified human ACE-2 protein were procured from Sino Biological (Beijing, China) (Cat # 10108-H08H-B). Coronil (Internal Batch No. CHIH/CORA/0122/2269) was obtained from Divya Pharmacy (Haridwar, India). Interactions between ACE-2 and seven different types of SARS-CoV-2 (Omicron) spike (S) proteins, namely, SARS-CoV-2(BA.4/BA.5) Spike RBD Protein (His Tag) (Cat # 40592-V08H130), SARS-CoV-2 BQ1.1 (Omicron) Spike RBD Protein (His Tag) (Cat # 40592-V08H143), SARS-CoV-2 XBB (Omicron) Spike RBD Protein (His Tag) (Cat # 40592-V08H144), SARS-CoV-2 BA.2.75.2 (Omicron) Spike RBD Protein (His Tag) (Cat # 40592-V08H141), SARS-CoV-2 (BA.4.6/BF.7) Spike RBD Protein (His Tag) (Cat # 40592-V08H140), SARS-CoV-2 JN.1 (omicron) Spike RBD Protein (aa319-529), His Tag (HPLC-verified) (Cat # 40592-V08H155), SARS-CoV-2 JN.1 (omicron) (Spike ECD His Tag) (Cat # 40589-V08H59) were studied. Bovine serum albumin (BSA) was purchased from Himedia (Thane, India) (Cat # TC194-500G). 3.3′,5,5′-Tetramethylbenzidine (TMB) was purchased from BD Bioscience, San Diego, USA (Cat # 555,214,) and Peroxidase Streptavidin was procured from Jackson ImmunoResearch (Cat # 016-030-084). Compositional Analysis of Coronil 0.5 gm powdered Coronil tablet sample was dissolved in 10 mL methanol: water (80:20) solution and sonicated for 30 min. The solution was centrifuged at 10000 rpm for 5 min and filtered using 0.45 µm nylon filters. The resulting filtered solution was used for the analysis. Compositional analysis of Coronil was performed by Prominence-XR UHPLC system (Shimadzu, Japan) equipped with Quaternary pump (NexeraXR LC-20AD XR), DAD detector (SPD-M20 A), Auto-sampler (Nexera XR SIL-20 AC XR), Degassing unit (DGU-20A 5R) and Column oven (CTO-10 AS VP). Separation was achieved using a Shimadzu Shim pack GIST-HP C18 (3µm, 3 X 100 mm) column subjected to binary gradient elution. The two solvents used for the analysis were water containing 0.1% orthophosphoric acid (pH 2.5 adjusted with diethyl amine (solvent A)) and Acetonitrile (solvent B). Gradient programming of the solvent system was done initially at 5% B for 0–10 min, 5–15% B from 10–20 min, 15–25% B from 20–40 min, 25–65% B from 40–60 min, 65–90% B from 60–65 min, 90 − 5%B from 65-66min, 5% B from 66–70 min with a flow rate of 0.7 ml/min. 10 µl of standard and test solution were injected and column temperature was maintained at 30°C. Wavelengths were set at 227 nm (for Withaferine A, Withanoside IV, Withanoside V, Withanone, Codifolioside A and Magnoflorine), 325 nm (for Rosmarinic acid and Palmatine) and 210 nm for Ursolic acid and Betulinic acid). ACE2 binding inhibition assay ACE2 binding inhibition assay was performed based on the published protocol with slight modifications [ 29 ]. Briefly, 100 µl of Spike protein (3 or 6 µg/ml) was coated in the 15 mM Sodium Carbonate, 35 mM Sodium Hydrogen Carbonate, and 7.7 mM Sodium Azide, pH 9.6 in Nunc Maxisorp plates (Denmark) (Cat # 442404.) for 16 hr at 4 o C. The plate was washed thrice with washing buffer (0.5% Tween 20 in PBS). 200 µl of the blocking buffer was added (2% BSA in the PBST) to each well followed by incubation at 37 o C for 1.5 hr. After washing thrice with washing buffer (0.5% Tween 20 in PBS), 100 µl of biotinylated ACE2 (2 µg/ml) was added to each well with different concentrations of Coronil (3.0, 10, 30, 100, and 300 µg/ml) and the plate was incubated at 37 o C for 1 hr. The wells were washed three times with washing buffer (0.1% Tween 20 in PBS) and 100 µl of Streptavidin-HRP solution (0.1 µg/ml) was added to each well and the plate was further incubated at 37 o C for 1 hr. This was followed by five washings with wash buffer (0.1% Tween 20 in PBS). Finally, 200 µl of TMB was added as substrate and the plate was further allowed for color development for up to 30 minutes at 37 0 C. The reaction was stopped by adding 50 µl of 2N H 2 SO 4 and absorbance was taken at 450 nm using Perkin Elmer Envision plate reader. Statistical Analysis All the statistical analyses were performed using GraphPad Prism software version 9.0 and the data was represented as mean ± SD. Multiple comparisons were done using one-way ANOVA (nonparametric) with a post hoc Tukey’s test. Each experiment was performed in atleast 3 replicates. Results Coronil is Enriched with Steroidal Lactones, Terpenoids, Alkaloids, and Furan Glycosides Coronil has been formulated using the aqueous extracts of Withania somnifera, Tinospora cordifolia , and Ocimum sanctum . The UHPLC pattern depicted that, Coronil is a rich blend of several phytochemicals; steroidal lactones, of which Withaferin A (1.752 µg/mg), Withanoside IV (2.673 µg/mg) and V (0.822 µg/mg) and Withanone (0.008 µg/mg) are the most predominant ones. All these steroidal lactones were mainly from W. somnifera . Among other phytochemicals, the major alkaloids like Magnoflorine (1.478 µg/mg), Palmatine (0.043 µg/mg) and furan glycoside, Cordifolioside A (0.181 µg/mg) were from T. cordifolia . Further, O. sanctum contributed polyphenolic compounds like Rosmarinic acid (0.091 µg/mg) and terpenoids like Betulinic (0.193 µg/mg) and Ursolic (0.046 µg/mg) acids (Fig. 2 ) and (Table 1 ). Table 1 Phytochemical Composition of Coronil S. N. Compounds Quantity (µg/mg) 1 Cordifolioside A 0.181 2 Magnoflorine 1.478 3 Withanoside IV 2.673 4 Withaferine A 1.752 5 Withanoside V 0.822 6 Withanone 0.008 7 Betulinic acid 0.193 8 Ursolic acid 0.046 9 Rosmarinic acid 0.091 10 Palmatine 0.043 Coronil inhibits the binding of Omicron variants spike proteins, BA4.6, BF.7, BA.2.75.2., BQ.1.1 and XBB with ACE2 In our previous computational data, we showed that the phytochemicals Withanone and Tinocordiside, present in Coronil inhibit the interactions between the human ACE-2 receptor and SARS-CoV-2 variants spike proteins [ 29 ]. Therefore, we asked the question of whether Coronil holds the potential to interfere with the binding of Omicron variants spike proteins such as BA4.6, XBB, BA.2.75.2, BF.7, and BQ.1.1. To address this, we evaluated the ability of Coronil to interfere with ACE-2-S protein interaction through an ELISA-based assay. Our data showed that Coronil has a dose-dependent inhibitory effect on the interactions between ACE2 and all variants of S proteins as evidenced by the decrease in the binding percentage (Fig. 3 A-E), although the extent of inhibition was different for variant spike proteins. Coronil was found to be most effective in the case of BA.4/BA.5 spike protein as it inhibited the protein binding to ACE2 even at low concentrations of 3 µg/ml (16.6% binding inhibition). This binding inhibition was increased to 60.3% ( p-value < 0.001 ) and 84.3% ( p-value < 0.0001 ) at 100 µg/ml and 300 µg/ml respectively. The effect of Coronil on the binding of other spike proteins BA4.6/BF.7, BA.2.75.2 and BQ.1.1 were similar with maximum inhibition at 300 µg/ml and minimum inhibition at 30 µg/ml. In the case of XBB, Coronil significantly diminished the binding of this spike with ACE2 even at 10 µg/ml ( p < 0.001 ). These results highlight the effectiveness of Coronil in preventing the binding of these Omicron spike protein variants. However, the extent of its effects differs slightly among variants. Coronil inhibits the binding of Omicron spike protein variants JN.1 (RBD and ECD) JN.1 variant of Omicron is now the most recent and prevalent variant circulating worldwide. Coronil showed remarkable inhibition of spike protein-ACE2 interaction for BA.4/BA.5, BA4.6, BF.7, BA.2.75.2., BQ.1.1, and XBB, we next tested if Coronil exerts the same potential for newly identified Omicron sub-variant JN.1. To achieve this, the effect of Coronil on JN.1 receptor binding domain (RBD) interaction with ACE2 was assessed. Coronil significantly repressed the binding of JN.1(RBD) with ACE2 at concentrations of 10, 30,100, and 300 µg/ml. The percent binding at these concentrations were 90.6, 77.0, 55.7, and 36.9% respectively (Fig. 4 A). During the development of the vaccine for SARS-CoV-2, the trimeric spike protein consisting of S1 and S2 subunits, was the main target as it mediates major entry steps such as receptor binding and membrane fusion [ 32 , 33 ]. Further, the ectodomain (ECD) region of the spike protein (S) is necessary for attachment and subsequent entry into the host cell and plays an important role in viral signaling pathways [ 34 ]. To test if Coronil could also interfere with the interaction of JN.1 (S1 + S2 trimer protein; ECD) with ACE2, we performed the assay using the JN.1 (S1 + S2) trimer protein. Coronil was found to be effective at 100 and 300 µg/ml concentrations (Fig. 4 B). These results highlight that Coronil may be effective in overcoming the neutralization resistance present in the recent Omicron variant. Discussion Several peaks of the SARS-CoV-2 infection were observed as a consequence of the continuous emergence of new variants. The Omicron variant was the most recent variant of concern due to its enhanced tendency to escape immune response and its reinfection potential. Many Omicron sub-variants appeared each with a unique combination of mutations in spike proteins making them more transmissible and immune evasive [ 12 ]. The resistance of these Omicron variants towards the current vaccines and the enhancement of its reinfection rate was the major cause of concern. In this study, the effect of the tri-herbal formulation, Coronil on the interaction between human ACE2 receptor and various Omicron variant spike proteins was studied. Coronil exerts an inhibitory effect on the binding of Omicron variant proteins with ACE2 regardless of variant subtype as evidenced by the ability of Coronil to inhibit Spike protein-ACE binding in all the variants of Omicron spike proteins studied. Coronil is rich in several phytochemicals such as steroidal lactones, alkaloids, and polyphenolic compounds. Steroidal lactones which include Withaferin A, Withanoside IV, and Withanoside V and Withanone are derived from W. somnifera. Alkaloids like Magnoflorine, Palmatine, furan glycoside, and Cordifolioside A are present in T. cordifolia . O. sanctum contributes to polyphenolic compounds like Rosmarinic acid and terpenoids like Betulinic and Ursolic acid. The steroidal lactones derived from W.somnifera reduce the levels of inflammatory cytokines such as IL-6, IL-1β, and TNF-α and have the potential to treat Covid-19 disease [ 35 ]. Withanone has also been found to destabilize the ACE2-RBD complex [ 27 ]. Magnoflorine, Palmatine, and Cordifolioside A derived from T. cordifolia . possess antioxidant and immunomodulatory activities [ 36 ]. Rosmarinic acid, Betulinic, and Ursolic acid derived from O. sanctum also exert anti-inflammatory properties. Rosmarinic protects against lethal H1N1 virus-mediated inflammation and lung injury [ 37 ]. Betulinic acid has also been shown to be effective against Zika virus and Chikungunya virus [ 38 ]. The other terpenoid, Ursolic acid exerts anti-inflammatory, antiviral, and antioxidant activity. It has been shown to suppress the release of pro-inflammatory cytokines and inhibit the production of reactive oxygen species [ 39 ]. Previously, Coronil was found to inhibit SARS-CoV-2 entry into the human alveolar epithelial cells and the interaction between the ACE2 receptor and spike protein [ 29 ]. The original SARS-CoV-2 strain kept on mutating with time leading to the emergence of new variants such as α, β, δ, and the highly transmissible and immune-evasive variant called Omicron [ 40 ]. It has not only spread and mutated rapidly but also affected the people who were vaccinated with the COVID-19 vaccines, highlighting the ability of this variant to escape the immune response [ 41 ]. The problem became more sophisticated as further sub-lineages like BA.1, BA.2 and BA.3 emerged from Omicron [ 42 ]. Omicron sub-variants BA.2 and BA.3 had higher transmission potential than BA.1 [ 43 ]. New BA.2 sub-lineages BA.2.74, BA.2.75, and BA.2.76 were initially identified in India with more BA.2.75 cases reported in September 2022 [ 44 ]. Further, BA.2.75 possesses a 3 to 6-fold higher binding affinity for human ACE2 than other Omicron variants [ 45 ]. The increased transmission of BA.2.75 can be attributed to the increased thermal stability of its spike trimer due to the N460K mutation [ 46 ]. BA.2.75 mutated further to sub-variant BA.2.75.2 which had increased neutralization resistance [ 47 ]. The ability of Coronil to effectively inhibit the binding of BA.2.75.2 variant spike protein with human ACE2 receptor suggests its possibility of being effective in this neutralization resistance variant. Another sub-variant, XBB emerged from the recombination between the second-generation BA.2 variants BJ.1 (BA.2.10.1.1) and BM.1.1.1 [ 48 ]. The main concern of the XBB variant was its exceptional immune evasion as 3-dose mRNA vaccination-induced antibodies did not neutralize XBB [ 16 ]. Intriguingly, Coronil significantly diminished the binding of human ACE2 with XBB variant spike protein pointing towards the potential of Coronil to prevent XBB sub-variant entry as it is the rate-limiting step in this process. The evolution of Omicron sub-variants continued and from BA.2, several other sub-variants emerged rapidly. These sub-variants included BA.4 and BA.5 and due to identical S proteins, were referred to as BA.4/BA.5 [ 49 ]. These BA.4/5 subvariants further diversified, with the emergence of several additional subvariants including the BA.4.6, BF.7, BQ.1, and BQ.1.1 [ 50 ]. These subvariants displayed the strongest immune evasion and neutralization resistance that was around 3–6 fold higher than the original D614G mutation [ 51 ]. Coronil not only reduced the binding of BA.4/BA.5 variant spike proteins but also inhibited BA.4.6, BF.7, BQ.1, and BQ.1.1 interaction with human ACE2 receptor. Recently, in December 2023, another variant of interest known as JN.1 was identified. This sub-variant is thought to have evolved from the BA.2.86 variant and was found alarming due to its higher immune-evasive capabilities as the existing vaccination regimen may not prove beneficial in this case [ 52 , 53 ]. However, Coronil may substantiate beneficial effects as it was found to inhibit the binding of both RBD and ECD domain of JN.1 variant spike proteins to human ACE2 receptor. Conclusion The intensive evolution of Omicron variants remains a tough challenge to global public health and raises several health concerns due to its uncertain mutable nature. Additionally, these variants are good at immune evasiveness and can reinfect individuals previously infected or vaccinated. The observations from this study indicate that Coronil can be effective in preventing the entry of Omicron variants including the recently emerged JN.1 variant, and prevent further infectivity possibly through inhibition of variant spike protein interaction with human ACE2 receptor (Fig. 5 ). Despite many therapeutic challenges such as the ability of these variants to escape immune response and increased neutralization resistance, Coronil seems to maintain its efficacy across different Omicron variants. Declarations Test article (Coronil) was provided by Divya Pharmacy, Haridwar, India. Acharya Balkrishna is the trustee in Divya Yog Mandir Trust, which governs Divya Pharmacy, Haridwar. In addition, he holds an honorary managerial position in Patanjali Ayurved Ltd, Haridwar, India. Divya Pharmacy, Haridwar, India, and Patanjali Ayurved Ltd, Haridwar, India manufacture and sell several herbal medicinal products. Other than providing the test formulation (Coronil), Divya Pharmacy was not involved in any aspect of the research reported in this study. All other authors have declared no conflict of interest. Funding The research work was funded internally by Patanjali Research Foundation Trust, Haridwar, India. Author Contribution A.B.: Conceptualization, Planning, Visualization, Supervision. R.D.: Methodology, Investigation, Writing – review & editing Methodology, Visualization, Project administration, Supervision, and Formal analysis. S.K.: Writing – original draft and formal nalysis. A.V.: Writing – review & editing, Project administration, Conceptualization, Visualization, and Supervision. Acknowledgement We thank Dr. Pradeep Nain, Dr. Jyotish Srivastava, and Ms. Meenu Tomer for their support in the phytochemical analysis of Coronil. 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NPJ Vaccines. 2023;8(1):38. Salazar E, Kuchipudi SV, Christensen PA, Eagar T, Yi X, Zhao P, Jin Z, Long SW, Olsen RJ, Chen J. Convalescent plasma anti–SARS-CoV-2 spike protein ectodomain and receptor-binding domain IgG correlate with virus neutralization. J Clin Investig. 2020;130(12):6728–38. Rizvi ZA, Babele P, Madan U, Sadhu S, Tripathy MR, Goswami S, Mani S, Dikshit M, Awasthi A. Pharmacological potential of Withania somnifera (L.) Dunal and Tinospora cordifolia (Willd.) Miers on the experimental models of COVID-19, T cell differentiation, and neutrophil functions. Front Immunol. 2023;14:1138215. Singh D, Chaudhuri PK. Chemistry and Pharmacology of Tinospora cordifolia. Nat Prod Commun. 2017;12(2):299–308. Zhou B, Wang L, Yang S, Liang Y, Zhang Y, Pan X, Li J. Rosmarinic acid treatment protects against lethal H1N1 virus-mediated inflammation and lung injury by promoting activation of the h-PGDS-PGD(2)-HO-1 signal axis. Chin Med. 2023;18(1):139. Loe MWC, Hao E, Chen M, Li C, Lee RCH, Zhu IXY, Teo ZY, Chin WX, Hou X, Deng J, et al. Betulinic acid exhibits antiviral effects against dengue virus infection. Antiviral Res. 2020;184:104954. Al-Kuraishy HM, Al-Gareeb AI, Negm WA, Alexiou A, Batiha GE. Ursolic acid and SARS-CoV-2 infection: a new horizon and perspective. Inflammopharmacology. 2022;30(5):1493–501. Singh H, Dahiya N, Yadav M, Sehrawat N. Emergence of SARS-CoV-2 New Variants and Their Clinical Significance. Can J Infect Dis Med Microbiol 2022, 2022:7336309. Wei J, Stoesser N, Matthews PC, Khera T, Gethings O, Diamond I, Studley R, Taylor N, Peto TEA, Walker AS, et al. Risk of SARS-CoV-2 reinfection during multiple Omicron variant waves in the UK general population. Nat Commun. 2024;15(1):1008. Kumar S, Karuppanan K, Subramaniam G. Omicron (BA.1) and sub-variants (BA.1.1, BA.2, and BA.3) of SARS-CoV-2 spike infectivity and pathogenicity: A comparative sequence and structural-based computational assessment. J Med Virol. 2022;94(10):4780–91. Kumar S, Karuppanan K, Subramaniam G. Omicron (BA.1) and sub-variants (BA.1.1, BA.2, and BA.3) of SARS-CoV-2 spike infectivity and pathogenicity: A comparative sequence and structural-based computational assessment. J Med Virol. 2022;94(10):4780–91. Singh UB, Deb S, Rani L, Bhardwaj D, Gupta R, Kabra M, Bala K, Kumari L, Perumalla S, Shukla J, et al. Genomic surveillance of SARS-CoV-2 upsurge in India due to Omicron sub-lineages BA.2.74, BA.2.75 and BA.2.76. Lancet Reg Health Southeast Asia. 2023;11:100148. Cao Y, Song W, Wang L, Liu P, Yue C, Jian F, Yu Y, Yisimayi A, Wang P, Wang Y et al. Characterizations of enhanced infectivity and antibody evasion of Omicron BA.2.75. bioRxiv 2022:2022.2007.2018.500332.. Izumi H, Aoki H, Nafie LA, Dukor RK. Effect of Conformational Variability on Seasonable Thermal Stability and Cell Entry of Omicron Variants. ACS Omega. 2023;8(7):7111–8. Kurhade C, Zou J, Xia H, Liu M, Chang HC, Ren P, Xie X, Shi PY. Low neutralization of SARS-CoV-2 Omicron BA.2.75.2, BQ.1.1 and XBB.1 by parental mRNA vaccine or a BA.5 bivalent booster. Nat Med. 2023;29(2):344–7. Tamura T, Ito J, Uriu K, Zahradnik J, Kida I, Anraku Y, Nasser H, Shofa M, Oda Y, Lytras S, et al. Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants. Nat Commun. 2023;14(1):2800. Tuekprakhon A, Nutalai R, Dijokaite-Guraliuc A, Zhou D, Ginn HM, Selvaraj M, Liu C, Mentzer AJ, Supasa P, Duyvesteyn HME, et al. Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum. Cell. 2022;185(14):2422–e24332413. Varghese R, Kumar D, Sharma R. Global threat from novel SARS-CoV-2 variants, BF.7, XBB.1.5, BQ.1, and BQ.1.1: variants of concern? Hum Cell. 2023;36(3):1218–21. Qu P, Evans JP, Faraone JN, Zheng YM, Carlin C, Anghelina M, Stevens P, Fernandez S, Jones D, Lozanski G, et al. Enhanced neutralization resistance of SARS-CoV-2 Omicron subvariants BQ.1, BQ.1.1, BA.4.6, BF.7, and BA.2.75.2. Cell Host Microbe. 2023;31(1):9–e1713. Satapathy P, Kumar P, Mehta V, Suresh V, Khare A, Rustagi S, Daulati MN, Neyazi M, Najafi E, Neyazi A. Global spread of COVID-19's JN.1 variant: Implications and public health responses. New Microbes New Infect. 2024;57:101225. Ou G, Yang Y, Zhang S, Niu S, Cai Q, Liu Y, Lu H. Evolving immune evasion and transmissibility of SARS-CoV-2: The emergence of JN.1 variant and its global impact. Drug Discov Ther. 2024;18(1):67–70. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4805471","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":341078128,"identity":"51d65b3e-490c-4d19-ab0a-cc044f45774b","order_by":0,"name":"Acharya Balkrishna","email":"","orcid":"","institution":"Drug Discovery and Development Division, Patanjali Research Foundation, NH-58, Haridwar-249405, Uttarakhand","correspondingAuthor":false,"prefix":"","firstName":"Acharya","middleName":"","lastName":"Balkrishna","suffix":""},{"id":341078129,"identity":"62a0bf4f-073b-4573-8d3e-ca766f96562d","order_by":1,"name":"Rishabh Dev","email":"","orcid":"","institution":"Drug Discovery and Development Division, Patanjali Research Foundation, NH-58, Haridwar-249405, Uttarakhand","correspondingAuthor":false,"prefix":"","firstName":"Rishabh","middleName":"","lastName":"Dev","suffix":""},{"id":341078130,"identity":"d8ce1521-8721-4cb6-aee9-191c051c7edb","order_by":2,"name":"Sandeep Kumar","email":"","orcid":"","institution":"Drug Discovery and Development Division, Patanjali Research Foundation, NH-58, Haridwar-249405, Uttarakhand","correspondingAuthor":false,"prefix":"","firstName":"Sandeep","middleName":"","lastName":"Kumar","suffix":""},{"id":341078131,"identity":"92c9dfa7-eaee-412f-9a13-36d2720b9e6e","order_by":3,"name":"Anurag Varshney","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYPACGwglwWDBA2EZENSSxsDABtYiQbSWwxAtIE0EAf/sHrMPP36dz+Of3/zsgUWNhAyD9OHDHxgK7uDUInHnjPHM3r7bxRLH2MwNJI4BHcaXlibBYPAMtzU3cowZeHtuJzYcYzCTkGADauHhMQP65TBOHfJALYx/e84lzj/G/k1C4h9IC//nD/i0GAC1MPP8OJC44RiPmYRkG9gWYCDg0WJ4I62YWbYhudjwWE6ZhGSfBA8bD5uZRAIeLXI3kjczvvljlyd3+Pg2aYlvNvb8PMyPP3z4g1sLGDC2MSSAaGZQrIDjJwG/BiD4A1HD+IGgylEwCkbBKBiJAAD7Tkq0sVjJ5QAAAABJRU5ErkJggg==","orcid":"","institution":"Drug Discovery and Development Division, Patanjali Research Foundation, NH-58, Haridwar-249405, Uttarakhand","correspondingAuthor":true,"prefix":"","firstName":"Anurag","middleName":"","lastName":"Varshney","suffix":""}],"badges":[],"createdAt":"2024-07-26 05:23:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4805471/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4805471/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62747065,"identity":"d4932a5f-19c4-4fe6-85e3-2dd5cd84999f","added_by":"auto","created_at":"2024-08-19 04:30:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5645818,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of\u003cstrong\u003e \u003c/strong\u003eOmicron sub-variants lineage map.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4805471/v1/646dba34887921c889a5acc7.png"},{"id":62747520,"identity":"02a5cf52-248d-486e-bcbe-99e76b64bb7c","added_by":"auto","created_at":"2024-08-19 04:38:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4737570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCompositional analysis of Coronil\u003c/strong\u003e. Overlap UHPLC chromatogram of standard mix (black line) and Coronil (blue line). Cordifolioside A, Magnoflorine, Withanoside IV, Withaferin A, Withanoside V and Withanone were quantified at 227 nm, Rosmarinic acid and Palmatine at 325 nm, and Betulinic and Ursolic acids at 210 nm wavelength.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4805471/v1/3e2ac811e23bfd2e31f434c8.png"},{"id":62746797,"identity":"c0a90f2d-3571-433d-8386-58a63ff3cc3c","added_by":"auto","created_at":"2024-08-19 04:22:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":738086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Coronil on interactions between human ACE-2 receptor and Omicron variant S proteins \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e–\u003cstrong\u003eE\u003c/strong\u003e). The dose-dependent effect of Coronil on the interactions between human ACE-2 receptor and different types of Omicron variant spike proteins namely (A) BA.4/BA.5, (B) XBB, (C) BA.2.75.2, (D) BA.4.6/BF.7 and (E) BQ1.1 was evaluated and represented as percent (%) binding. Data are represented as mean ± SD. Statistics for A-E, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ANOVA with Tukey’s multiple comparison test.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4805471/v1/bb7562ef8d4988d6247f61da.png"},{"id":62747064,"identity":"a0d7e6a4-edcf-4707-acc4-bb31d154673a","added_by":"auto","created_at":"2024-08-19 04:30:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":297038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Coronil on interactions between human ACE-2 receptor and JN. 1 (RBD) and JN.1 (ECD) spike proteins.\u003c/strong\u003e Dose-dependent effect of Coronil on the interactions between human ACE-2 receptor and Omicron variant spike protein (A) JN.1 (RBD) and (B) JN.1 spike trimer protein (ECD) (A). Data are represented as mean ± SD. Statistics for A-B, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ANOVA with Tukey’s multiple comparison test.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4805471/v1/61b034608656ffdce00cc9b4.png"},{"id":62746801,"identity":"8f6acf60-3cb3-4137-8aff-e04c42ebde2d","added_by":"auto","created_at":"2024-08-19 04:22:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11113842,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram summarizing the effect of Coronil on the interactions between human ACE-2 receptor and Omicron variant spike proteins.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4805471/v1/36419a889b609b6034b1e216.png"},{"id":62747523,"identity":"0d28db47-8a1b-4496-a641-f8c2c35689d4","added_by":"auto","created_at":"2024-08-19 04:38:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25265258,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4805471/v1/744a7f3e-7e49-47a5-bbea-36e6e714e713.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Coronil biochemically inhibits the interaction of various clinically relevant mutants of SARS-CoV-2 Spike Proteins (Omicron Variants) with human ACE2 receptor","fulltext":[{"header":"Background","content":"\u003cp\u003eSeveral prominent variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) including Alpha, Beta, Delta, and Omicron have emerged since the beginning of the Covid-19 pandemic [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The potential of these emerging SARS-CoV-2 variants particularly the Omicron variants, to escape natural or vaccine-induced immunity is the major cause of concern [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The spike protein (S protein) plays an important role in the cellular invasion of viruses and mutations in the spike protein result in better immune escape [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among all the variants, the Omicron variant was found to be less severe but highly transmissible [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. As SARS-CoV-2 enters the host cells through a cell surface receptor known as Angiotensin-converting enzyme 2 (ACE2), the affinity of variant spike protein towards ACE2 plays a crucial role in the viral infectivity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It is well documented that the affinity of the original SARS-CoV-2 strain towards ACE2 is several-fold less than the Omicron spike protein variants rendering it more infectious than the original strain [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The infectivity of Omicron variants is not only limited to their higher binding affinity for ACE2 but also because of their potential to escape the vaccine-induced immune response [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among the Omicron sub-variants, B.1.1.529 was the first reported variant in November 2021 and several sub-variants of Omicron have emerged thereafter including BA.1, BA.2, BA.3, BA.4, and BA.5 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Further, several other sub-variants emerged from these variants including BA4/5, XBB, BF.7, BA.2.75.2., BQ.1.1, and the most recent variant JN.1 which is thought to emerge from BA.2.86 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These variants have evolved by multiple combinations of different mutations mainly in the spike protein (S1 and S2 subunits) of the virus [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The omicron sub-variants BA.4 and BA.5 first appeared in December 2021 and January 2022 respectively. As these two sub‐variants spread more rapidly than the earlier Omicron sub‐variants, they were reclassified as \u0026lsquo;variant of interest\u0026rsquo; to \u0026lsquo;variant of concern by the European Centre for Disease Prevention and Control. Interestingly, BA.4 and BA.5 share the same amino acid mutation, F486V [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The other prevalent Omicron sub lineage among the omicron variant was XBB which is thought to originate from the recombination of BA.2 and BA.2.75 sub-lineages [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. XBB variant was known to contain the maximum number of 14 mutations, 9 of which were found in the RBD region [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It was reported to be one of the most immune\u0026ndash;evasive covid variants [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Like XBB, the Omicron sub-variant BA.2.75.2 was also derived from BA.2. It was one of the most predominant sub-variant circulating in the Indian population [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The Omicron variant BF.7 which is also known as BA.5.2.1.7 emerged from BA.5 and had a short incubation period responsible for its rapid transmissibility even in previously COVID-19-infected and vaccinated people [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. BQ1.1 which is a sub lineage of BQ.1 shares a common space with other sub variant XBB in terms of their resistance towards the neutralizing antibodies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Most recently, the Omicron variant JN.1 has caught attention due to its predominance over other circulating variants. It is different from its parent lineage BA.2.86 with a unique mutation L455S in its spike protein. Presently, the JN.1 variant is responsible for a growing proportion of infections throughout the world [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough there is a comprehensive understanding of the infectivity, severity, and transmissibility of the Omicron sub-variants, protective measures against these variants are rather lacking due to their inherent tendency to escape immune response and resistance towards neutralizing antibodies. In the present study, we have investigated the effect of Coronil on the binding affinity of the Omicron sub-variants BA.4/BA.5, BF.7, BA.2.75.2., BQ.1.1, XBB, and JN.1 with ACE2. Coronil is a tri-herbal formulation containing the extracts of \u003cem\u003eWithania somnifera\u003c/em\u003e (Ashwagandha), \u003cem\u003eTinospora cordifolia\u003c/em\u003e (Giloy), and \u003cem\u003eOcimum sanctum\u003c/em\u003e (Tulsi) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These medicinal plants have been used effectively for the treatment of respiratory diseases and fever [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. \u003cem\u003eWithania somnifera contains\u003c/em\u003e Withanolides, which are known to reduce interactions with the host ACE2 receptor [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Withanone present in \u003cem\u003eWithania somnifera\u003c/em\u003e has been predicted to destabilize the ACE2-RBD complex suggesting it as a potent inhibitor of SARS-CoV-2 coronavirus entry into the host cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. \u003cem\u003eTinospora cordifolia\u003c/em\u003e is a well-recognized plant with antiviral properties and is also known to control SARS-CoV-2 replication. Interruption of electrostatic interactions between the RBD and ACE2 by Tinocordiside present in the extract of \u003cem\u003eTinospora cordifolia\u003c/em\u003e may interfere with SARS-CoV-2 entry [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. \u003cem\u003eOcimum sanctum\u003c/em\u003e has been shown to possess immunomodulatory effects [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Coronil not only diminishes the Spike-protein-dependent SARS-CoV-2 viral entry into human alveolar epithelial cells but also inhibits the production of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α. Coronil has also been shown to interfere with the binding of wild type spike protein of SARS-CoV-2 and its variants spike proteins such as S\u003csup\u003eD614G\u003c/sup\u003e and S\u003csup\u003eW436R\u003c/sup\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These three ayurvedic medicinal plants are also predicted to contain six inhibitors against SARS-CoV-2 M\u003csup\u003epro\u003c/sup\u003e (Main protease) which plays an important role in viral replication [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study envisaged the potential of Coronil to inhibit the binding of Omicron variant spike proteins with ACE2 as these proteins are responsible for enhanced immune evasiveness and infectivity.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eIndividual standards of Palmatine hydrochloride (Sigma Aldrich, USA) (Cat # 361615), Cordifolioside A (Chem faces, China) (Cat # CFN95040), Magnoflorine (Sigma Aldrich, USA) (Cat. # 361615), Withaferine A (Natural remedies, India) (Cat # W003), Withanoside IV (Natural remedies, India) (Cat # W006),) Withanoside V (Natural remedies, India) (Cat. # W007), Withanone (Natural remedies, India) (Cat. # W005) Rosmarinic acid (Sigma Aldrich, USA) (Cat. # R4033), Palmaitine hydrochloride (Sigma Aldrich, USA) (Cat # 361615), Ursolic acid (Tokyo chemical industries, India) (Cat. # 102067769) and Betulinic acid (Natural remedies, India) (Cat. # B2836) were dissolved in methanol to prepare 1000 ppm standard solution. 0.05 mL of 1000 ppm standard stock solution was taken to prepare 50 ppm of standard working solution.\u003c/p\u003e \u003cp\u003ePurified Omicron Spike (S) proteins and purified human ACE-2 protein were procured from Sino Biological (Beijing, China) (Cat # 10108-H08H-B). Coronil (Internal Batch No. CHIH/CORA/0122/2269) was obtained from Divya Pharmacy (Haridwar, India). Interactions between ACE-2 and seven different types of SARS-CoV-2 (Omicron) spike (S) proteins, namely, SARS-CoV-2(BA.4/BA.5) Spike RBD Protein (His Tag) (Cat # 40592-V08H130), SARS-CoV-2 BQ1.1 (Omicron) Spike RBD Protein (His Tag) (Cat # 40592-V08H143), SARS-CoV-2 XBB (Omicron) Spike RBD Protein (His Tag) (Cat # 40592-V08H144), SARS-CoV-2 BA.2.75.2 (Omicron) Spike RBD Protein (His Tag) (Cat # 40592-V08H141), SARS-CoV-2 (BA.4.6/BF.7) Spike RBD Protein (His Tag) (Cat # 40592-V08H140), SARS-CoV-2 JN.1 (omicron) Spike RBD Protein (aa319-529), His Tag (HPLC-verified) (Cat # 40592-V08H155), SARS-CoV-2 JN.1 (omicron) (Spike ECD His Tag) (Cat # 40589-V08H59) were studied. Bovine serum albumin (BSA) was purchased from Himedia (Thane, India) (Cat # TC194-500G). 3.3\u0026prime;,5,5\u0026prime;-Tetramethylbenzidine (TMB) was purchased from BD Bioscience, San Diego, USA (Cat # 555,214,) and Peroxidase Streptavidin was procured from Jackson ImmunoResearch (Cat # 016-030-084).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCompositional Analysis of Coronil\u003c/h2\u003e \u003cp\u003e0.5 gm powdered Coronil tablet sample was dissolved in 10 mL methanol: water (80:20) solution and sonicated for 30 min. The solution was centrifuged at 10000 rpm for 5 min and filtered using 0.45 \u0026micro;m nylon filters. The resulting filtered solution was used for the analysis. Compositional analysis of Coronil was performed by Prominence-XR UHPLC system (Shimadzu, Japan) equipped with Quaternary pump (NexeraXR LC-20AD XR), DAD detector (SPD-M20 A), Auto-sampler (Nexera XR SIL-20 AC XR), Degassing unit (DGU-20A 5R) and Column oven (CTO-10 AS VP). Separation was achieved using a Shimadzu Shim pack GIST-HP C18 (3\u0026micro;m, 3 X 100 mm) column subjected to binary gradient elution. The two solvents used for the analysis were water containing 0.1% orthophosphoric acid (pH 2.5 adjusted with diethyl amine (solvent A)) and Acetonitrile (solvent B). Gradient programming of the solvent system was done initially at 5% B for 0\u0026ndash;10 min, 5\u0026ndash;15% B from 10\u0026ndash;20 min, 15\u0026ndash;25% B from 20\u0026ndash;40 min, 25\u0026ndash;65% B from 40\u0026ndash;60 min, 65\u0026ndash;90% B from 60\u0026ndash;65 min, 90\u0026thinsp;\u0026minus;\u0026thinsp;5%B from 65-66min, 5% B from 66\u0026ndash;70 min with a flow rate of 0.7 ml/min. 10 \u0026micro;l of standard and test solution were injected and column temperature was maintained at 30\u0026deg;C. Wavelengths were set at 227 nm (for Withaferine A, Withanoside IV, Withanoside V, Withanone, Codifolioside A and Magnoflorine), 325 nm (for Rosmarinic acid and Palmatine) and 210 nm for Ursolic acid and Betulinic acid).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eACE2 binding inhibition assay\u003c/h2\u003e \u003cp\u003eACE2 binding inhibition assay was performed based on the published protocol with slight modifications [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Briefly, 100 \u0026micro;l of Spike protein (3 or 6 \u0026micro;g/ml) was coated in the 15 mM Sodium Carbonate, 35 mM Sodium Hydrogen Carbonate, and 7.7 mM Sodium Azide, pH 9.6 in Nunc Maxisorp plates (Denmark) (Cat # 442404.) for 16 hr at 4 \u003csup\u003eo\u003c/sup\u003eC. The plate was washed thrice with washing buffer (0.5% Tween 20 in PBS). 200 \u0026micro;l of the blocking buffer was added (2% BSA in the PBST) to each well followed by incubation at 37 \u003csup\u003eo\u003c/sup\u003eC for 1.5 hr. After washing thrice with washing buffer (0.5% Tween 20 in PBS), 100 \u0026micro;l of biotinylated ACE2 (2 \u0026micro;g/ml) was added to each well with different concentrations of Coronil (3.0, 10, 30, 100, and 300 \u0026micro;g/ml) and the plate was incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 1 hr. The wells were washed three times with washing buffer (0.1% Tween 20 in PBS) and 100 \u0026micro;l of Streptavidin-HRP solution (0.1 \u0026micro;g/ml) was added to each well and the plate was further incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 1 hr. This was followed by five washings with wash buffer (0.1% Tween 20 in PBS). Finally, 200 \u0026micro;l of TMB was added as substrate and the plate was further allowed for color development for up to 30 minutes at 37 \u003csup\u003e0\u003c/sup\u003eC. The reaction was stopped by adding 50 \u0026micro;l of 2N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and absorbance was taken at 450 nm using Perkin Elmer Envision plate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll the statistical analyses were performed using GraphPad Prism software version 9.0 and the data was represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Multiple comparisons were done using one-way ANOVA (nonparametric) with a \u003cem\u003epost hoc\u003c/em\u003e Tukey\u0026rsquo;s test. Each experiment was performed in atleast 3 replicates.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCoronil is Enriched with Steroidal Lactones, Terpenoids, Alkaloids, and Furan Glycosides\u003c/h2\u003e \u003cp\u003eCoronil has been formulated using the aqueous extracts of \u003cem\u003eWithania somnifera, Tinospora cordifolia\u003c/em\u003e, and \u003cem\u003eOcimum sanctum\u003c/em\u003e. The UHPLC pattern depicted that, Coronil is a rich blend of several phytochemicals; steroidal lactones, of which Withaferin A (1.752 \u0026micro;g/mg), Withanoside IV (2.673 \u0026micro;g/mg) and V (0.822 \u0026micro;g/mg) and Withanone (0.008 \u0026micro;g/mg) are the most predominant ones. All these steroidal lactones were mainly from \u003cem\u003eW. somnifera\u003c/em\u003e. Among other phytochemicals, the major alkaloids like Magnoflorine (1.478 \u0026micro;g/mg), Palmatine (0.043 \u0026micro;g/mg) and furan glycoside, Cordifolioside A (0.181 \u0026micro;g/mg) were from \u003cem\u003eT. cordifolia\u003c/em\u003e. Further, \u003cem\u003eO. sanctum\u003c/em\u003e contributed polyphenolic compounds like Rosmarinic acid (0.091 \u0026micro;g/mg) and terpenoids like Betulinic (0.193 \u0026micro;g/mg) and Ursolic (0.046 \u0026micro;g/mg) acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhytochemical Composition of Coronil\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS. N.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQuantity (\u0026micro;g/mg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCordifolioside A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.181\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMagnoflorine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.478\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWithanoside IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.673\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWithaferine A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.752\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWithanoside V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.822\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWithanone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBetulinic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.193\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUrsolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.046\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRosmarinic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.091\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePalmatine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.043\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCoronil inhibits the binding of Omicron variants spike proteins, BA4.6, BF.7, BA.2.75.2., BQ.1.1 and XBB with ACE2\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn our previous computational data, we showed that the phytochemicals Withanone and Tinocordiside, present in Coronil inhibit the interactions between the human ACE-2 receptor and SARS-CoV-2 variants spike proteins [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, we asked the question of whether Coronil holds the potential to interfere with the binding of Omicron variants spike proteins such as BA4.6, XBB, BA.2.75.2, BF.7, and BQ.1.1. To address this, we evaluated the ability of Coronil to interfere with ACE-2-S protein interaction through an ELISA-based assay. Our data showed that Coronil has a dose-dependent inhibitory effect on the interactions between ACE2 and all variants of S proteins as evidenced by the decrease in the binding percentage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-E), although the extent of inhibition was different for variant spike proteins. Coronil was found to be most effective in the case of BA.4/BA.5 spike protein as it inhibited the protein binding to ACE2 even at low concentrations of 3 \u0026micro;g/ml (16.6% binding inhibition). This binding inhibition was increased to 60.3% (\u003cem\u003ep-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) and 84.3% (\u003cem\u003ep-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/em\u003e) at 100 \u0026micro;g/ml and 300 \u0026micro;g/ml respectively. The effect of Coronil on the binding of other spike proteins BA4.6/BF.7, BA.2.75.2 and BQ.1.1 were similar with maximum inhibition at 300 \u0026micro;g/ml and minimum inhibition at 30 \u0026micro;g/ml. In the case of XBB, Coronil significantly diminished the binding of this spike with ACE2 even at 10 \u0026micro;g/ml (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). These results highlight the effectiveness of Coronil in preventing the binding of these Omicron spike protein variants. However, the extent of its effects differs slightly among variants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCoronil inhibits the binding of Omicron spike protein variants JN.1 (RBD and ECD)\u003c/h2\u003e \u003cp\u003eJN.1 variant of Omicron is now the most recent and prevalent variant circulating worldwide. Coronil showed remarkable inhibition of spike protein-ACE2 interaction for BA.4/BA.5, BA4.6, BF.7, BA.2.75.2., BQ.1.1, and XBB, we next tested if Coronil exerts the same potential for newly identified Omicron sub-variant JN.1. To achieve this, the effect of Coronil on JN.1 receptor binding domain (RBD) interaction with ACE2 was assessed. Coronil significantly repressed the binding of JN.1(RBD) with ACE2 at concentrations of 10, 30,100, and 300 \u0026micro;g/ml. The percent binding at these concentrations were 90.6, 77.0, 55.7, and 36.9% respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). During the development of the vaccine for SARS-CoV-2, the trimeric spike protein consisting of S1 and S2 subunits, was the main target as it mediates major entry steps such as receptor binding and membrane fusion [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Further, the ectodomain (ECD) region of the spike protein (S) is necessary for attachment and subsequent entry into the host cell and plays an important role in viral signaling pathways [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To test if Coronil could also interfere with the interaction of JN.1 (S1\u0026thinsp;+\u0026thinsp;S2 trimer protein; ECD) with ACE2, we performed the assay using the JN.1 (S1\u0026thinsp;+\u0026thinsp;S2) trimer protein. Coronil was found to be effective at 100 and 300 \u0026micro;g/ml concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results highlight that Coronil may be effective in overcoming the neutralization resistance present in the recent Omicron variant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSeveral peaks of the SARS-CoV-2 infection were observed as a consequence of the continuous emergence of new variants. The Omicron variant was the most recent variant of concern due to its enhanced tendency to escape immune response and its reinfection potential. Many Omicron sub-variants appeared each with a unique combination of mutations in spike proteins making them more transmissible and immune evasive [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The resistance of these Omicron variants towards the current vaccines and the enhancement of its reinfection rate was the major cause of concern.\u003c/p\u003e \u003cp\u003eIn this study, the effect of the tri-herbal formulation, Coronil on the interaction between human ACE2 receptor and various Omicron variant spike proteins was studied. Coronil exerts an inhibitory effect on the binding of Omicron variant proteins with ACE2 regardless of variant subtype as evidenced by the ability of Coronil to inhibit Spike protein-ACE binding in all the variants of Omicron spike proteins studied.\u003c/p\u003e \u003cp\u003eCoronil is rich in several phytochemicals such as steroidal lactones, alkaloids, and polyphenolic compounds. Steroidal lactones which include Withaferin A, Withanoside IV, and Withanoside V and Withanone are derived from \u003cem\u003eW. somnifera.\u003c/em\u003e Alkaloids like Magnoflorine, Palmatine, furan glycoside, and Cordifolioside A are present in \u003cem\u003eT. cordifolia\u003c/em\u003e. \u003cem\u003eO. sanctum\u003c/em\u003e contributes to polyphenolic compounds like Rosmarinic acid and terpenoids like Betulinic and Ursolic acid. The steroidal lactones derived from \u003cem\u003eW.somnifera\u003c/em\u003e reduce the levels of inflammatory cytokines such as IL-6, IL-1β, and TNF-α and have the potential to treat Covid-19 disease [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Withanone has also been found to destabilize the ACE2-RBD complex [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Magnoflorine, Palmatine, and Cordifolioside A derived from \u003cem\u003eT. cordifolia\u003c/em\u003e. possess antioxidant and immunomodulatory activities [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Rosmarinic acid, Betulinic, and Ursolic acid derived from \u003cem\u003eO. sanctum\u003c/em\u003e also exert anti-inflammatory properties. Rosmarinic protects against lethal H1N1 virus-mediated inflammation and lung injury [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Betulinic acid has also been shown to be effective against Zika virus and Chikungunya virus [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The other terpenoid, Ursolic acid exerts anti-inflammatory, antiviral, and antioxidant activity. It has been shown to suppress the release of pro-inflammatory cytokines and inhibit the production of reactive oxygen species [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePreviously, Coronil was found to inhibit SARS-CoV-2 entry into the human alveolar epithelial cells and the interaction between the ACE2 receptor and spike protein [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The original SARS-CoV-2 strain kept on mutating with time leading to the emergence of new variants such as α, β, δ, and the highly transmissible and immune-evasive variant called Omicron [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. It has not only spread and mutated rapidly but also affected the people who were vaccinated with the COVID-19 vaccines, highlighting the ability of this variant to escape the immune response [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The problem became more sophisticated as further sub-lineages like BA.1, BA.2 and BA.3 emerged from Omicron [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Omicron sub-variants BA.2 and BA.3 had higher transmission potential than BA.1 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. New BA.2 sub-lineages BA.2.74, BA.2.75, and BA.2.76 were initially identified in India with more BA.2.75 cases reported in September 2022 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Further, BA.2.75 possesses a 3 to 6-fold higher binding affinity for human ACE2 than other Omicron variants [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The increased transmission of BA.2.75 can be attributed to the increased thermal stability of its spike trimer due to the N460K mutation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. BA.2.75 mutated further to sub-variant BA.2.75.2 which had increased neutralization resistance [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The ability of Coronil to effectively inhibit the binding of BA.2.75.2 variant spike protein with human ACE2 receptor suggests its possibility of being effective in this neutralization resistance variant. Another sub-variant, XBB emerged from the recombination between the second-generation BA.2 variants BJ.1 (BA.2.10.1.1) and BM.1.1.1 [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The main concern of the XBB variant was its exceptional immune evasion as 3-dose mRNA vaccination-induced antibodies did not neutralize XBB [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Intriguingly, Coronil significantly diminished the binding of human ACE2 with XBB variant spike protein pointing towards the potential of Coronil to prevent XBB sub-variant entry as it is the rate-limiting step in this process. The evolution of Omicron sub-variants continued and from BA.2, several other sub-variants emerged rapidly. These sub-variants included BA.4 and BA.5 and due to identical S proteins, were referred to as BA.4/BA.5 [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. These BA.4/5 subvariants further diversified, with the emergence of several additional subvariants including the BA.4.6, BF.7, BQ.1, and BQ.1.1 [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. These subvariants displayed the strongest immune evasion and neutralization resistance that was around 3\u0026ndash;6 fold higher than the original D614G mutation [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Coronil not only reduced the binding of BA.4/BA.5 variant spike proteins but also inhibited BA.4.6, BF.7, BQ.1, and BQ.1.1 interaction with human ACE2 receptor. Recently, in December 2023, another variant of interest known as JN.1 was identified. This sub-variant is thought to have evolved from the BA.2.86 variant and was found alarming due to its higher immune-evasive capabilities as the existing vaccination regimen may not prove beneficial in this case [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. However, Coronil may substantiate beneficial effects as it was found to inhibit the binding of both RBD and ECD domain of JN.1 variant spike proteins to human ACE2 receptor.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe intensive evolution of Omicron variants remains a tough challenge to global public health and raises several health concerns due to its uncertain mutable nature. Additionally, these variants are good at immune evasiveness and can reinfect individuals previously infected or vaccinated. The observations from this study indicate that Coronil can be effective in preventing the entry of Omicron variants including the recently emerged JN.1 variant, and prevent further infectivity possibly through inhibition of variant spike protein interaction with human ACE2 receptor (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Despite many therapeutic challenges such as the ability of these variants to escape immune response and increased neutralization resistance, Coronil seems to maintain its efficacy across different Omicron variants.\u003c/p\u003e "},{"header":"Declarations","content":" \u003cp\u003eTest article (Coronil) was provided by Divya Pharmacy, Haridwar, India. Acharya Balkrishna is the trustee in Divya Yog Mandir Trust, which governs Divya Pharmacy, Haridwar. In addition, he holds an honorary managerial position in Patanjali Ayurved Ltd, Haridwar, India. Divya Pharmacy, Haridwar, India, and Patanjali Ayurved Ltd, Haridwar, India manufacture and sell several herbal medicinal products. Other than providing the test formulation (Coronil), Divya Pharmacy was not involved in any aspect of the research reported in this study. All other authors have declared no conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe research work was funded internally by Patanjali Research Foundation Trust, Haridwar, India.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.B.: Conceptualization, Planning, Visualization, Supervision. R.D.: Methodology, Investigation, Writing \u0026ndash; review \u0026amp; editing Methodology, Visualization, Project administration, Supervision, and Formal analysis. S.K.: Writing \u0026ndash; original draft and formal nalysis. A.V.: Writing \u0026ndash; review \u0026amp; editing, Project administration, Conceptualization, Visualization, and Supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Dr. Pradeep Nain, Dr. Jyotish Srivastava, and Ms. Meenu Tomer for their support in the phytochemical analysis of Coronil. We also thank Mr. Devendra Kumawat for helping with the graphics. We extend our gratitude to Mr. Tarun Rajput and Mr. Gagan Kumar for their swift administrative support.\u003c/p\u003e\n\u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data generated or analyzed in this study has been incorporated in this published article.\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLaine L, Sk\u0026ouml;n M, V\u0026auml;is\u0026auml;nen E, Julkunen I, \u0026Ouml;sterlund P. 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Emergence of SARS-CoV-2 New Variants and Their Clinical Significance. \u003cem\u003eCan J Infect Dis Med Microbiol\u003c/em\u003e 2022, 2022:7336309.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei J, Stoesser N, Matthews PC, Khera T, Gethings O, Diamond I, Studley R, Taylor N, Peto TEA, Walker AS, et al. Risk of SARS-CoV-2 reinfection during multiple Omicron variant waves in the UK general population. Nat Commun. 2024;15(1):1008.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Karuppanan K, Subramaniam G. Omicron (BA.1) and sub-variants (BA.1.1, BA.2, and BA.3) of SARS-CoV-2 spike infectivity and pathogenicity: A comparative sequence and structural-based computational assessment. J Med Virol. 2022;94(10):4780\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Karuppanan K, Subramaniam G. 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New Microbes New Infect. 2024;57:101225.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOu G, Yang Y, Zhang S, Niu S, Cai Q, Liu Y, Lu H. Evolving immune evasion and transmissibility of SARS-CoV-2: The emergence of JN.1 variant and its global impact. Drug Discov Ther. 2024;18(1):67\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Coronil, SARS-CoV-2, Omicron, Variant Spike protein, ACE2, RBD","lastPublishedDoi":"10.21203/rs.3.rs-4805471/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4805471/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAccumulating evidence suggests that the receptor binding domain (RBD) of the SARS-CoV-2 Omicron variant has several times more binding affinity to the human angiotensin-converting enzyme 2 (ACE2) receptor compared to the RBD of the original covid-19 strain This increased binding affinity of Omicron variant is responsible for its increased internalization and infectivity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the present study, the impact of Coronil, a tri-herbal formulation of extracts from \u003cem\u003eWithania somnifera, Tinospora cordifolia, and Ocimum sanctum\u003c/em\u003e on the binding properties of Omicron SARS-CoV-2 variant spike proteins (S proteins) was investigated. Compositional analysis of Coronil was performed by the Prominence-XR UHPLC system. The ELISA-based ACE2 binding inhibition assay was performed to delineate the effect of Coronil on the interaction between human ACE2 receptor and different Omicron variant spike proteins such as BA.4/BA5, XBB, BA.2.75.2, BA4.6/BF.7, BA.2.75.2, BQ.1.1, and a recently found spike protein variant JN.1 which is thought to emerge from BA.2.86.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCoronil showed a dose-dependent inhibitory effect on the interactions between ACE2 and receptor binding domains (RBD) of all variants of spike proteins evaluated in this study including the recently emerged, highly transmissible variant spike protein JN.1. Although, Coronil significantly reduced the binding percentage in almost all the variant spike proteins, the maximum inhibition was achieved against BA.4/BA.5 where it inhibited the S protein \u0026ndash; ACE2 interaction even at a low concentration of 3 \u0026micro;g/ml (16.6%). This binding inhibition was further increased to 60.3 and 84.6% at 100 and 300 \u0026micro;g/ml respectively.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis capability of Coronil to inhibit the binding of spike protein variants with ACE2 receptor may interfere with viral binding and internalization resulting in reduced infectivity of these Omicron spike protein variants. Overall, our data underscores the potential of Coronil in combating the various newly emerged Omicron spike protein variants. These findings may provide a basis for further studies of Coronil for its clinical effectiveness against these Omicron variants.\u003c/p\u003e","manuscriptTitle":"Coronil biochemically inhibits the interaction of various clinically relevant mutants of SARS-CoV-2 Spike Proteins (Omicron Variants) with human ACE2 receptor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-19 04:22:26","doi":"10.21203/rs.3.rs-4805471/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"822d30b0-9b21-4ab5-addd-9844b6c1091b","owner":[],"postedDate":"August 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-19T04:22:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-19 04:22:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4805471","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4805471","identity":"rs-4805471","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}