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Antioxidant, Anti-Inflammatory, and Anticarcinogenic Efficacy of an Ayurvedic Formulation: Amritotharanam Kashyam

Antioxidant, Anti-Inflammatory, and Anticarcinogenic Efficacy of an Ayurvedic Formulation:... Hindawi Advances in Pharmacological and Pharmaceutical Sciences Volume 2023, Article ID 3387261, 13 pages https://doi.org/10.1155/2023/3387261 Research Article Antioxidant, Anti-Inflammatory, and Anticarcinogenic Efficacy of an Ayurvedic Formulation: Amritotharanam Kashyam 1 2 3 Lakshmi Sundaram Raman , Anitha Shanmuganathan , Sangeeta Chandrashekar , 4 5 6 Prabhu Kaliyaperumal , Elumalai Perumal , Ram Krishna Rao Mudiganti , 7 7 8 K. T. Nachammai , Langeswaran Kulanthaivel , Gowtham Kumar Subbaraj , 9 9 Kirubhanand Chandrasekaran , Bharat Ramrao Sontakke , and Senthilkumar Subramanian Faculty of Pharmacy, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, Tamil Nadu, India Department of Physics, Arulmigu Palaniandavar College of Arts and Science, Palani, Tamil Nadu, India Department of Physiology, Bharath Institute of Higher Education and Research, Chennai, Tamil Nadu, India Department of Anatomy, Sree Balaji Medical College and Hospital, Chrompet, Chennai, Tamil Nadu, India Centre for Transdisciplinary Research, Department of Pharmacology, Saveetha Dental College, Saveetha University, Chennai, Tamil Nadu, India M/s. Noahs Laboratories, No. 8/1, Old Mahabalipuram Road, Tiruporur, Tamil Nadu, India Molecular Cancer Biology Laboratory, Department of Biotechnology, Science Campus, Alagappa University, Karaikudi, Tamil Nadu, India Faculty of Allied Health Sciences, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelambakkam, Tamil Nadu, India Department of Anatomy, All India Institute of Medical Sciences, Nagpur, Maharashtra, India School of Medicine, College of Medicine and Health Science, Jigjiga University, Jigjiga, Somali Region, Ethiopia Correspondence should be addressed to Senthilkumar Subramanian; senthilkumarsubramanian@jju.edu.et Received 3 October 2022; Revised 9 March 2023; Accepted 16 March 2023; Published 28 April 2023 Academic Editor: Daniel Dias Rufno Arcanjo Copyright © 2023 Lakshmi Sundaram Raman et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Amritotharanam Kashyam, a specifc Ayurvedic drug, was the focus of the current inquiry to evaluate its efcacy. For liver and digestive-related issues, this medication is suggested. Tis was obtained from a standard Ayurvedic vendor in Chennai (India), and GC-MS analysis was carried out according to the standard procedure. A few critical biomolecules include benzoic acid, hex- adecanoic acid, 6,9-octadecadienoic acid, 9-octadecenoic acid, methyl ester (E)-, heptadecanoic acid, 16-methyl, methyl ester, methyl 18-methylnonadecanoate, tetracosanoic acid, distearin, hexadecanoic acid, and 1-(hydroxymethyl)-1,2-ethanediol ester. Te obtained biomolecules exhibited some signifcant therapeutic functions, including acidifcation, inhibition of arachidonic acid formation, increase in the aromatic amino acid decarboxylase, suppression of uric acid generation, inhibitors of catechol-O- methyltransferase, urine acidifers, etc. Te anticancer and antiviral potential of these phytocompounds were investigated using molecular docking and dynamics. Te phytocompounds pharmacokinetic characteristics were investigated using ADME analysis. Trough docking and dynamics simulation, in silico tests demonstrated the phytocompounds' inhibitory efciency against the target proteins. Tese functions reasonably relate to the medicinal function of Amritotharanam Kashyam. Te MTTassay fndings demonstrated this medication’s anticancer efects. Te ability to be an efective drug is demonstrated by its antioxidant, anti- infammatory, and membrane-stabilizing properties. 2 Advances in Pharmacological and Pharmaceutical Sciences infammatory, and membrane-stabilizing capacity of 1. Introduction Amritotharanam Kashyam studies prove its efcacy as Medicinal plants are currently gaining great attention as a potent medicine. Inhibitory efcacy of phytocompounds is possible sources of anticancer medicines and are frequently through Molecular docking and dynamics simulation. employed due to the availability of materials, afordability, relatively low cost, and few or no side efects, wide appli- 2. Materials and Methods cation, and therapeutic efcacy, which has expedited sci- 2.1. HPTLC Profle Chromatography Instrumentation. Te entifc study. Te World Health Organization (WHO) CAMAG (Muttenz, Switzerland) HPTLC unit is equipped encourages the use of traditional medicines because they are with Linomat 5 automatic sample applicator with a 100μL safe and efective for these reasons [1]. Amirtotharanam syringe, a TLC sample spotter, a twin trough glass chamber Kashyam is an efective Ayurvedic formulation to treat fu, for chromatogram plate development, and a TLC scanner 3 coughs, cold, and infammatory diseases and cures various liver disorders. It is efective in recovering liver cells after for densitometric evaluation of chromatograms, which is integrated winCATS 4 software (Version 1.4.3, Camag) for severe liver diseases such as cirrhosis or hepatitis. Tis also helps in improving appetite and digestion. Tis medicine is interpretation of data. prepared by adding the powders of Tinospora cordifolia (Guduchi), Zingiber ofcinale (Ginger), and Terminalia 2.2. Chromatography Conditions. Te ethyl acetate extract chebula (Haritaki) at the ratio of 2 parts: 6 parts: 4 parts and sample was chromatographed on aluminum-backed HPTLC boiling the mixture with 8 parts of water. Te boiling is plates (10 ×10cm), precoated with silica gel 60F (0.2mm th carried out till the quantity becomes 1/4 . It is then cooled thickness) (Merck). Before sample application, the plates and preserved in air-tight bottles for medicine. It is pre- were activated at 60 C for 5min after prewashing them with scribed at 12–24ml with an equal quantity of water and methanol. Te ethyl acetate extract sample (2, 4, 8, and 12 μl) a small spoon of sugar. Tis medicine references the was spotted as a band with 8mm width at diferent tracks at Ayurvedic treatise, Sahasra Yoga Kasaya Prakarana, Chi- a distance of 11mm from the bottom and 15mm from the kitamanjari. Te three substances are extensively utilized in side edges of HPTLC aluminum sheets with the help of home medicines, and substantial research has been carried 100μl syringe (Hamilton, Bonaduz, Switzerland) attached out on their therapeutic properties. withCamag semiautomatic Linomat 5 applicator (Camag, Ginger (Zingiber ofcinale): ginger is the most frequent Switzerland). Te nitrogen stream was passed simulta- home remedy for coughs, colds, and indigestion. Its ther- neously to dry the bands. Te plate development occurred in apeutic properties have been well-documented [2]. During the ascending mode in a twin-trough glass chamber pregnancy, [3] it has documented its antioxidant efects. (10 ×10cm), presaturated with 20ml of mobile phase for Ginger can help with nausea and vomiting [4]. By inhibiting 15min by sealing the chamber air-tight with paraflm. Te calcium channels, it lowers blood pressure [5]. Guduchi chromatogram was run up to 8cm from the sample ap- (Tinospora cordifolia): T. Chebula bark, rind, galls, and other plication point using n-hexane: ethyl acetate: formic acid: parts of the common Triphalachoornam have been found to acetic acid in diferent ratios (7.0:3.0:0.1:0.1) (v/v/v/v), as have antimicrobial, antioxidant, antidiabetic, anti- the mobile phase. Te solvent system was selected by trial infammatory, hepatoprotective, antiarthritic properties, as and error to elute the maximum number of compounds. well as antiproliferative, antimutagenic, radio and car- After the development, the solvent front was marked using dioprotective, antispasmodic, antiviral properties, immu- a pencil, and then the HPTLC plates were dried in an oven at nomodulatory, and hypolipidemic [6]. Haritaki (Terminalia 60 C for 5minutes to ensure that all mobile phase was re- chebula): antidiabetic, antispasmodic, antiperiodic, antiar- moved. Densitometric scanning was performed immediately thritic, antioxidant, anti-infammatory, antiallergic, anti- using Camag TLC scanner 3 with winCATS software leprotic, antistress, antimalarial, immune-modulatory, (Version 1.4.8) under 254nm with 5mm ×0.45mm slit antineoplastic, and hepatoprotective efects are some of the dimension for qualitative analysis of ethyl acetate extract therapeutic qualities of this plant [7]. Te present work deals −1 20mms scanning speed, 100μm/step data resolution. R with the efcacy determination of Amritotharanam values of the sample were marked and analyzed using the Kashyam using various parameters. Te computer-aided winCATS software. Te images were captured with visible drug design (CADD) method helps to identify prospective UV light at 366nm and 254nm by keeping the plate in the lead compounds and contribute to the development of photo-documentation chamber (CAMAG REPROSTAR 3). potential therapeutics for a wide variety of ailments. A low- Te peak numbers with their height and area, peak display, cost, time-saving, quick, and automated approach. It pro- and peak densitogram were identifed. vides information about the patterns of small molecules interacting with targeted protein molecules (drug-receptor) interactions [8]. In GC MS analysis to determine the mol- 2.3. GC MS Profle. Amritotharanam Kashyam was bought ecules present in medications and to try to comprehend the from a regular Ayurvedic seller in Chennai and submitted to types of molecules present and their potential medicinal conventional GC MS analysis. Instrument: Gas chroma- functions that might contribute to Amritotharanam tography (Agilent: GC: (G3440A) 7890A. MS-MS: 7000 Kashyam’s medicinal role. MTT assays to understand the Triple Quad GCMS) was outftted with a mass spectrometry anticancer efcacy of this medicine. Te antioxidant, anti- detector. Advances in Pharmacological and Pharmaceutical Sciences 3 2.3.1. Sample Preparation. Samples of 100microliters and dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) 1mL of appropriate solvents were used to dissolve the into a dark blue/purple, water-insoluble formazan product. In compound. For 10seconds, the solution was forcefully ag- 96-well plates, the cells were seeded at a density of 5 ×10 cells/ itated using a vortex stirrer. Gas-chromatography was used well. Te media was changed after 24hours with 100 μl of to analyse the clear extract. medium with Amritotharanam Kashyam at various doses and cultured for another 24hours. Te medium from both the control and Amritotharanam Kashyam treated cells was 2.3.2. GC-MS Protocol. Te GC MS Column was con- withdrawn. Each well was given 50μl of MTT (5mg/ml), and structed of DB5 MS (30mm 0.25mm ID 0.25m, 5 percent again plate was keptin a CO incubator for 4hours at 37 C.Te phenyl, 95% methyl polysiloxane), Electron impact mode at MTTwas subsequently removed, and then 50μl of DMSO was 70eV, and Helium (99.999 percent) as a carrier gas at used to dissolve purple-colored formazan crystals. An ELISA a constant fow rate of 1ml/min. Te auxiliary temperature is reader (Bio-Rad) had been used to quantify the purple-blue ° ° 290 C, while the injector temperature is 280 C. Te ion formazan mixture at 570nm. Each sample’s optical density was source has a temperature of 280 C. For fragments ranging compared with the control, and the percentage of cell viability from 45 to 450Da, the oven temperature was set to climb was calculated and plotted in the graphs. ° ° from 50 C (isothermal for 1.0min) to 170 C (isothermal for ° ° 4.0min), then 10 C/min to 310 C (isothermal for 10min). Te GC’s overall running time is 32.02minutes. Te GC-MS 2.5. Determination of Nuclear Morphological Changes of Cells Library is employed (NIST and WILEY). (DAPI Staining). Te cell monolayer was washed with PBS and fxed in 3 percent paraformaldehyde for 10minutes at room temperature for the nuclear morphological analysis. Te 2.4.Chemicals. Te following chemicals were used for MTT frozen cells were permeabilized in PBS containing 0.2 percent Assay: DMEM, Antibiotics (Streptomycin, Penicillin), Triton X-100 for 10minutes at room temperature before being trypsin-EDTA, PBS, and FBS from Gibco (Invitrogen, USA). treated for 5minutes with 0.5g/ml DAPI. Te apoptotic nuclei MTT reagent and dimethyl sulfoxide (DMSO) are from were examined using a fuorescence microscope (intensely Sigma Aldrich Chemicals Pvt Ltd, USA. stained, shattered nuclei, and condensed chromatin). 2.4.1. Sample Preparation. 25ml of Amritotharanam 2.6. Antioxidant, Anti-Infammatory Assays, and Membrane Kashyam was taken in a Petri dish and kept in a lab water Stabilizing Assays bath at 75–85 C for 3-4hrs until a solid consistency of (a) Antioxidant assay - ABTS radical scavenging assay extract was obtained. Approximately 1000mg of the extract ferric reducing antioxidant potential (FRAP) assay was dissolved in 10ml of water and Dulbecco’s Modifed Eagle Medium (DMEM) for free radical scavenging and cell (b) Anti-infammatory assay - protein denaturation assay viability assay. (c) Membrane stabilisation assay 2.4.2. Cell Line and Culture. Te National Centre For Cell 2.6.1. ABTS (2,2′-Azino-bis (3-Ethylbenzothiazoline-6-sul- Science, Pune, India (NCCS) supplied the breast cancer cell phonic Acid)) Radical Scavenging Assay. Amrithotharam line (MCF-7). Dulbecco’s minimum essential medium Kashyam’s antioxidant capacity was investigated utilizing added with 10% FBS and 1% antibiotics were used to culture the ABTS (2,2′-azino-bis-3-ethyl benzothiazole-6-sulphonic the cells. At 37 C, the cells were maintained in a 5% CO acid) radical cation decolorization test. Te radical cations of environment. T-75 culture fasks were used to cultivate cells, ABTS (ABTS was created by reacting a 7mmol/L ABTS and the studies were conducted at a confuence of 70% to solution with 2.45mmol/L potassium persulphate) (K S O ) 2 2 8 80%. Once they had reached confuency, the cells were [9]. Before usage, the mixture was kept in the dark at room dissociated by 0.05% trypsin-EDTA enzyme. Cells were temperature for 12hours. Ethanol was used to dilute the 4 2 seeded at a density of 10 cells/cm and treated with various ABTS+solution. Diferent concentrations of test samples concentrations of Amritotharanam Kashyam. Te cells were (5–320g/ml) and standard, ascorbic acid (5–320g/ml) were harvested using trypsin after 24hours of treatment. Each mixed with the diluted ABTS solution (2.0ml). Te reaction sample was counted in triplicate under a Nikon (Japan) mixture was allowed to stand at room temperature for inverted microscope to assess the total cell numbers. 6minutes before measuring absorbance at 734nm with an ultraviolet-visible spectrophotometer. All determinations were made in triplicate. Te radical scavenging activity was 2.4.3. MTT Assay. Tis test was conducted to understand the denoted as ABTS, and the radical scavenging efect was anticancer activity of Amritotharanam Kashyam. To determine computed using the following equation: the cytotoxicity efect of Amritotharanam Kashyam in the breast cancer cell line, the MTT assay was used. Cell viability (A0 − A1) ABTSradicalscavengingeffect (%) � 􏼢 􏼣 × 100, (1) was measured by MTTassay, a sensitive, accurate, and reliable A0 colorimetric technique. Te assay is based on the ability of the cellular mitochondrial dehydrogenase enzyme in living cells to where A0 is the absorbance of control; A1 is the absorbance convert the yellow, water-soluble substrate 3-(4,5- of the test. 4 Advances in Pharmacological and Pharmaceutical Sciences 2.6.2. Ferric Reducing Potential Antioxidant Assay. Te solution in phosphate bufer and incubated in an incubator antioxidant ability of the test samples Amritotharanam at 37 C for 15minutes. Denaturation was achieved by im- Kashyam to convert Fe3+ TPTZ complex (colourless mersing the reaction mixture in a 60 C water bath for 2+ complex) to Fe tripyridyltriazine (blue coloured complex) 15minutes. After cooling, the turbidity was measured at generated by electron-donating antioxidants at low pH was 660nm. Te turbidity of the reference medication was calculated spectrophotometrically. It was calculated spec- measured at the same concentration. Te percentage of trophotometrically using a modifed approach developed by denaturation inhibition was estimated from control in which Benzie and Strain [10, 11]. Te change in absorbance at no medication was applied. Te typical medication was 593nm is used to monitor this process. At 37 C, 300mM diclofenac sodium. Te percentage inhibition of de- acetate bufer, 10ml TPTZ in 40mM HCl, and 20mM naturation was computed using the formula shown as FeCl .6H O were mixed to make the Ferric reducing anti- follows: 3 2 oxidant power (FRAP) reagent. 3.995ml of newly made (ODof test − ODof control) working FRAP reagent was pipetted and carefully mixed %Inhibition � 􏼢 􏼣x100. (2) ODof control with varying amounts (5–320g/ml) of test samples and standard, Ascorbic acid (5–320g/ml). When the ferric tri- 3+ pyridyl triazine (Fe TPTZ) complex was reduced to ferrous 2+ ° 2.6.4. Membrane Stabilisation Assay. Fresh whole human (Fe ) form after 30minutes at 37 C, a vivid blue colour blood (5ml) was collected and transferred to centrifuge complex was produced, and the absorbance at 593nm was tubes in a heparinized tube. It was centrifuged at 3000rpm measured against a reagent blank (3.995ml FRAP for 10minutes before being washed with normal saline of reagent+5l distilled water). Every judgment was made in equal volume three times. Te blood volume was measured triplicate. and reconstituted as a 40% v/v suspension with isotonic solution (10mM sodium phosphate bufer). Amritothar- 2.6.3. Anti-Infammatory Assay through Protein De- anam Kashyam and regular diclofenac sodium were com- naturation Assay. Amirthotharam Kashyam’s anti- bined in 0.1ml of 40% RBC solution at concentrations infammatory potential was determined using a protein ranging from 50–1600μg/ml. Te control was 0.1mL of RBC denaturation assay. Te experiment was carried out with mixed with an isotonic solution on its own. For 30minutes, minor modifcations by Gnana and colleagues in 2011 the reaction mixture was incubated in a water bath at 56 C. [12, 13]. Te reference medicine, diclofenac sodium, was Te tubes were cooled to room temperature after incubation. dissolved in dimethyl sulfoxide (DMSO), and the sample was Te reaction mixture was centrifuged for 5minutes at diluted using phosphate bufer (0.2M, PH 7.4). All solutions 2500rpm, and the supernatant absorbance was measured at had a fnal DMSO concentration of less than 2.5 percent. Te 560nm. To calculate the % membrane stabilizing activity, Test Solution (4ml) with varying drug concentrations use the formula as follows: (50–1600μg/ml) was mixed with 1ml of 1mM albumin (ODof test − ODof control) %Inhibitionof hemolysis � 􏼢 􏼣x100. (3) ODof control 2.7. Statistical Analysis. Te results will be presented as downloaded from Protein Data Bank (PDB Id: 4YOI, mean SEM. One-way ANOVA was used to establish sta- 6YB7, 6M17–SARS-Cov-2 and 1M17, 58XT–Breast can- tistical signifcance, followed by a Dunnett’s multiple- cer). Retrieved structures were imported into the Maestro comparison test with 95% confdence intervals. P values Schrodinger suite, and the receptors were optimized and less than 0.05 were regarded as signifcant. minimized using the force feld OPLS_2005 [8]. Deletion of water molecules and adding hydrogen were performed before grid generation. Te glide module generates the 2.8. Target and Lead Selection. Te resultant phyto- grid box of X, Y, and Z dimensions. After the grid gen- compounds from GC-MS analysis possess medicinal prop- eration, prepared ligands were docked with the target erties, therapeutic efcacies, and potential inhibitory actions receptors. against various tumours and some deadly diseases. Tis study focuses on the two deadliest diseases, COVID-19 and breast cancer. Te target receptors were examined with the selected 2.10. Ligands Preparation. Te reported phytocompounds phytocompounds to analyse their therapeutic efcacies. from GC-MS analysis were downloaded from the PubChem database. 2D structures of selected compounds were pre- 2.9. Protein Preparation and Grid Generation. pared by the LigPrep module using the force feld OPLS_2005 [14]; the resultant prepared ligands were of high Tree-dimensional structures of the three target receptors of SARS-CoV-2 and two receptors of Breast cancer quality with accurate bond lengths and angles. Advances in Pharmacological and Pharmaceutical Sciences 5 ligand parameters. Cl− or Na+ was added to neutralize the 2.11. Molecular Docking. By computing the energy of the ligand binding to the protein, docking is a common mo- system, and the complex was placed in a cubic box of about 1.0A. To minimize the complex, NPT and NVT were per- lecular modelling approach for inserting ligands into the active site of a receptor molecule. Te value of this energy formed for 100ps at 300k, and MD was performed for 50ns. determines the biological activity of molecules; the greater the energy, the more efective the medicine based on the 3. Results and Discussion receptor will be assessed. Te G-score is calculated in Kcal/ mol and incorporates the energies of ligand-protein in- 3.1.HPTLC. In HPTLC observation, 12 peaks were observed teractions, hydrophobic interactions, hydrogen bonds, in- and two of them indicated the presence of two primary ternal energy, and pi-pi stacking interactions. Te XP’s compounds in the sample. Further analysis is required to GLIDE modules visualize the investigation of a specifc identify those two major components. Te peak at Rf 0.52 indicated 16.92% of compound A, Peak at Rf-0.71 indicated ligand-protein interaction [15]. 52.60% of compound B. Te results were illustrated in Figure 1. 2.12. Pharmacophore Hypothesis. Te pharmacophore hy- pothesis generated through the Phase module uses a recently built standard Pharmacophore perception algorithm that 3.2. GC MS Results of Amritotharanam Kashyam. reverses the conventional paradigm by detecting ligand Figure 2 depicts the GC MS profle of Amritotharanam alignments and then perceiving hypotheses [16]. Te Kashyam that displayed the retention values, categories of pharmacophore characteristics, such as Acceptor (A), Donor likely compounds, molecular formulas, molecular weight, (D), Hydrophobic (H), Negative (N), Positive (P), and peak area, and medicinal advantages of each chemical as Aromatic Rings (R), were determined by three chemical demonstrated in Amritotharanam Kashyam’s GC MS pro- structural patterns and were point, vector, and groups as fle. By comparing retention duration and fragmentation SMARTS inquiries. Tree diferent geometries, each of pattern with mass spectra in the NIST spectrum library which defned the physical attributes of the site, were recorded in the GC-MS computer program, metabolites assigned to these patterns. An aromatic ring’s directionality were identifed (version 1.10 beta, Shimadzu). Figure 2 is described by a vector that is normal to the ring’s plane in showed the pharmacological functions of each bio- this context. It swiftly develops high-quality hypotheses molecule based on Dr. Duke’s phytochemical and ethno- from a few to hundreds of known active ligands using botanical data source (National Agriculture Library, USA) pharmacophore-based shape alignments. and others. Benzoic acid, methyl ester, hexadecanoic acid, 6, 9-octadecadienoic acid, 9-octadecenoic acid, methyl ester, (E)-, heptadecanoic acid, 16-methyl-, methyl ester, methyl 2.13. MM-GBSA Calculation. Te energy of optimum-free 18-methylnonadecanoate, tetracosanoic acid. Tese com- receptors, free ligands, and ligand-receptor complexes is pounds have far-reaching biological functions, which bodes calculated using Prime MM-GBSA Molecular Mechan- well for Amritotharanam Kashyam’s position as a powerful ics–Generalized Born Surface Area [17]. It also calculates the treatment for which it is given. ligand strain energy by immersing the ligand in a solution generated automatically by the VSGB 2.0 suit. Te energy visualization was shown via the principal energy visualizer. 3.3. MTT Assay. Te MTT assay revealed that Amrito- tharanam Kashyam had good cytotoxic action against the development of nonsmall cell lung cancer cells in A549cell 2.14. ADME Properties. Te QikProp module was used to line. Amritotharanam Kashyam-treated cells exhibit de- determine absorption, distribution, metabolism, excretion, creased cell proliferation in MTT assays. Figures 3 and 4 and toxicity (ADME/T) characteristics for best-docked li- clearly showed that as the concentration of Amritotharanam gand molecules. Tis software predicts QP log Po/w, Kashayam increased, the cell viability reduced almost uni- QPlogBB, donor HB, acceptor HB, Caco, rule of 5, total formly. Tis clearly indicated the cytotoxicity of Amrito- percentage of human oral absorption, etc. Lipinski’s rule of tharanam Kashayam on cancer cells. fve evaluates the drug-likeness of a chemical molecule based on biological ingredients and pharmacological features to predict an orally active therapy [18]. 3.4. Nuclear Morphological Changes of Cells (DAPI Staining) Assay. Te microscopical observation displayed a decline in 2.15. Molecular Dynamics Simulation. Molecular dynamic the count of the cells and morphological alterations (Fig- simulations can be used to verify docking and understand ure 4). In this study, we used two diferent doses to treat the protein-ligand interactions. It enables the analysis of the sys- cell line. Te increase in the dose concentration declines the tem’s physical evolution through time and is a valuable tool for cell quantity compared to control cells. Fluorescence mi- interpolating between theory and experimentation. MD per- croscopic studies indicate abnormal cell boundaries, irreg- formed in GROMACS package with 50ns by using GRO- ular nucleus shape and enlargement of the cell nucleus. MOS96 43a1 force feld [19]. Te compound with the lowest Tese cell morphological changes and nucleus mutilation binding energy was selected for molecular dynamics (MD) have been recovered in the drug-treated cells compared to simulation. Te PRODRG web server was used to assess the control cells. 6 Advances in Pharmacological and Pharmaceutical Sciences All tracks @ 254 nm All tracks @ 254 nm 1000.0 (AU) 1000.0 800.0 1000.0 (AU) 1000.0 700.0 (AU) 800.0 (AU) 600.0 800.0 700.0 500.0 800.0 700.0 600.0 700.0 400.0 600.0 500.0 600.0 300.0 500.0 400.0 200.0 400.0 500.0 300.0 300.0 400.0 100.0 200.0 254 nm 366 nm 200.0 0.0 300.0 100.0 200.0 100.0 0.0 200.0 (mm) 200.0 100.0 0.0 (mm) 100.0 50.0 0.00 0.10 100.0 0.20 0.30 0.40 0.0 0.0 0.50 50.0 0.60 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 (RF) 1.00 0.70 0.80 (RF) 0.0 1.00 Track 1 , ID: Standard1 Track 2 , ID: Standard2 1000 1000 AU AU 900 900 800 800 700 700 600 600 500 500 400 400 300 300 6 7 200 200 3 1 9 2 5 3 1 100 100 0 0 0.00 0.20 0.40 0.60 0.80 1.00 0.00 0.20 0.40 0.60 0.80 1.00 Rf Rf Track 3 , ID: Standard3 Track 4 , ID: Standard4 1000 1000 AU AU 900 900 800 800 700 700 600 600 500 500 400 400 300 300 11 12 11 2 13 6 9 6 200 5 200 5 1 2 100 100 0 0 0.00 0.20 0.40 0.60 0.80 1.00 0.00 0.20 0.40 0.60 0.80 1.00 Rf Rf Figure 1: HPTLC-Profle data result. User Chromatogram +EI TIC Scan 220620018.D ×10 1 1 15.743 0.8 0.6 14.133 0.4 25.614 * 5.900 19.282 0.2 8.097 456789 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Counts vs. Acquisition Time (min) Figure 2: Indicates the GC MS profle of Amritothararam Kashyam. Advances in Pharmacological and Pharmaceutical Sciences 7 Amritotharanam Kashyam and diclofenac sodium, re- spectively. In membrane stabilisation activity standard is prone to show more activity than a sample. 3.6. Glide Docking. XP Docking was performed using the Glide module, protein-ligand was docked using the XP module, and the conformers were evaluated by G score. Docking results declared that the phytocompound 1, 2, 3- benzenetriol exhibited the highest docking score among other phytocompound when examined with the SARS-CoV- 2 and breast cancer target proteins. Covid proteins such as 20 4YOI, 6YB7, and 6M17 showed docking scores of about −7.325, −5.621, and −6.814Kcal/Mol; furthermore, targets of breast cancer 1M17 and 5DXTshowed the docking score of Control DMSO 1 μL 2 μL 5 μL 10 μL 15 μL 20 μL about −6.840 and −6.932Kcal/Mol respectively. Te inter- Concentration (μL/mL) acting residues interacted with 1, 2, 3-benzenetriol was GLN_167, GLU_166, LEU_52, TYR_128, GLU_738, 24 hours THR_766, GLU_849, and VAL_851 respectively. Interacting 48 hours residue between lead compound 1, 2, 3-benzenetriol and Figure 3: MTT assay comparative chart for 24hr and 48hr for 4YOI is GLN amino acid at the position of 167. Interacting Amritotharanam Kashyam. residue between is 1, 2, 3-benzenetriol and 6M17 leucine amino acid at the position of 52 and tyrosine amino acid at the position of 128. Interacting residue between is 1, 2, 3- 3.5. Antioxidant, Anti-Infammatory, and Membrane Stabi- benzenetriol and 6YB7 glutamic acid amino acid at the lizing Potential of Amritotharanam Kashyam. Te standard position of 166. Interacting residue between is 1, 2, 3- drug ascorbic acid was used as a standard for ABTS and benzenetriol and 1M17 glutamic acid amino acid at the FRAP antioxidant assay. In the case of anti-infammatory position of 738 and threonine amino acid at the position of assays such as protein denaturation assay and membrane 766. Interaction profle between 1, 2, 3-benzenetriol and stabilisation assay; diclofenac sodium is used as standard. It targeted receptor 5DXT shows the interacting residue VAL was conducted to have a better comparative study with the 851 and GLU 849. Te results were illustrated in Figure 9. test sample, Amritotharanam Kashyam. Te sample Amri- totharanam Kashyam and standard concentrations are 5, 10, 20, 40, 80,160, and 320μg/ml for all antioxidant assays, 3.7. Binding-Free Energy. Te binding afnity of ligands to where 50, 100, 200, 400, 800, and 1600μg/ml concentration the receptor was estimated using postdocking binding free was taken for anti-infammatory assay. Te average value of energy. Te results of the prime MM-GBSA analysis revealed the reactions performed in triplicate was obtained and that 1, 2, 3-Benzenetriol had ΔGbind of 35.231 (4YOI), plotted against the diferent concentrations of Amrito- 33.214 (6YB7), 34.127 (6M17), 34.563 (1M17), and tharanam Kashyam and its standards. Te IC 32.501Kcal/Mol, respectively (Figure 9). According to the value that is half-maximal inhibitory concentration was calculated from binding-free energy, the substances may frmly engage in the active site residues of the target proteins to block enzy- the R equation obtained from a linear thread line from the respective graph of concentration of Amritotharanam matically, stop COVID-19 infection, and inhibit breast Kashyam/standard against % inhibition and activity values. cancer activity. Figure 5 clearly showed that the IC value for the ABTS radical scavenging efect of Amritotharanam Kashyam was 3.8. ADME Properties. Te ADME properties and pharma- 58.27μg/ml, and ascorbic acid was 33.43μg/ml. Amrito- cokinetics of phytocompounds play a signifcant role in their tharanam Kashyam showed more than four times less efcacy. Figure 10 reveals the ADME properties of phyto- radical scavenging efect than standard. compounds. Te Lipinski principles of fve were followed for Te FRAP assay result was presented in Figure 6. An the phytocompounds, which included 500 molecular weight, increase in value was noted in an increase in concentrations 10 hydrogen bond acceptors, 5 hydrogen bond donors, and of both test and standard. Te results indicate that Amri- logP values of 5. Also acceptable were physicochemical pa- totharanam Kashyam showed comparatively more FRAP rameters such as human oral absorption, partition coefcient activity than standard ascorbic acid. Te IC value for (QPlogPo/w), and (QPlogBB) values. As a result, the phy- protein denaturation activity of Amritotharanam Kashyam tocompounds reported values could be deemed powerfully and standard, diclofenac sodium was 160.7μg/ml and inhibits the COVID-19 infection and Breast Cancer. 221.23μg/ml, respectively, which was obtained from Fig- ure 7. Here the Amritotharanam Kashyam shows better protein denaturation activity than standard. Te IC value 3.9. Pharmacophore Hypothesis. Pharmacophore generated for membrane stabilisation activity was obtained from through Phase module and the features of lead compound 1, Figure 8, where 525.87μg/ml and 378.83μg/ml are IC 2, 3-benzenetriol was illustrated in Figure 10. Cell Viability (%) 8 Advances in Pharmacological and Pharmaceutical Sciences Light Microscope DAPI Staining Control 5 μl/ml (Conc.) 5 μl/ml (Conc.) 10 μl/ml (Conc.) 10 μl/ml (Conc.) Figure 4: Cell morphology details of the MTT assay for Amritotharanam Kashyam Cell morphology—24h. Pharmacophore hypothesis reveals the acceptor, donor and score, the interaction residues, and the binding afnity aromatic ring present in the compound. In this lead com- between the desired proteins and the lead compound, pound three acceptors, three donors and one aromatic ring molecular dynamics simulations were run for 50ns. Te present in the active compound. behaviour of macromolecules is typically predicted using MD simulation, which utilizes Newton’s equation of motion and classical mechanics to determine the speed and site of 3.10.MDSimulation. Targeted protein-ligand complex were each atom in the system under study. Te least binding score examined for stability using molecular dynamics simula- complex was subjected to MD simulation. RMSD (root tions from the Gromacs package. According to the docking mean square deviation), RMSF (root mean square Advances in Pharmacological and Pharmaceutical Sciences 9 ABTS Radical Scavenging activity 5 10 20 40 80 160 320 Con (μg/ml) Amruthotharam Kashayam Ascorbic acid Figure 5: Comparative graphical representation of ABTS radical scavenging activity of Amritotharanam Kashyam and ascorbic acid. FRAP assay 5 10 20 40 80 160 320 Conc (μg/ml) Amruthotharam Kashayam Ascorbic acid Figure 6: Comparative graphical representation of FRAP assay of Amritotharanam Kashyam and ascorbic acid. fuctuation), and hydrogen bond plots were generated to numerous powerful biomolecules. According to scientists, evaluate the structural changes or stability in the complex. these powerful chemical components from nature are RMSD plot showed a slight deviation with the stability employed to cure various illnesses with fewer adverse efects observed from 30ns throughout the simulation period; the [20]. Plant extracts may contribute to the performance and complex attained a slight deviation at 0.4nm. RMSF plot general well-being of the fowl. Te enhancement of en- showed less fuctuation in the loop or disorder region at the dogenous digestive enzyme production, activation of the initial simulation period and 0.35nm. Tese deviations do immunological response, and antibacterial, antiviral, anti- not afect the structural changes or stability of the complex. oxidant, and antihelminthic properties are just a few of the Hydrogen bond interaction between the protein and the positive efects of herbal extracts or active substances on ligand was noted as fve. Te overall result (Figure 11) poultry nutrition. Computational developments signif- concluded that the complex showed better stability without cantly infuenced the process of developing new drugs. changes in structural confrmations. Virtual screening methods are often and widely used to reduce the price and duration of drug development. A key component of structure-based drug design is the discovery 4. Discussion of new ligands for protein structures using the molecular Phytochemical substances found in medicinal plants are docking technique [21]. 1, 2, 3-benzenetriol and 4YOI a plentiful source of treatment for several chronic disorders. interacted with GLN 167 with docking score of −7.325. In recent years, many therapeutic plants have produced Interaction profle between the 1, 2, 3-benzenetriol and % of inhibition % of inhibition 10 Advances in Pharmacological and Pharmaceutical Sciences Protein denaturation activity 50 100 200 400 800 1600 Conc (μg/ml) Amruthotharam Kashayam Diclofenac sodium Figure 7: Comparative graphical representation of protein denaturation activity of Amritotharanam Kashyam and diclofenac sodium. Membrane Stabilisation Activity 50 100 200 400 800 1600 Conc (μg/ml) Amruthotharam Kashayam Diclofena sodium Figure 8: Comparative graphical representation of the membrane stabilisation activity of Amritotharanam Kashyam and diclofenac sodium. 6YB7 show the docking score of −5.621and interacting with reveals the interacting residue between the ligand and GLU 166 amino acid. Interaction profle between the 1, 2, 3- protein receptor such as VAL 851 and GLU 849. In silico benzenetriol and 6M17 show the docking score of −6.814 approaches revealed the potential efcacy of the phyto- and interacting with LEU 52 and TYR128 amino acid. compounds against the target receptors of breast cancer and Targeted receptor 1M17 docked against the 1, 2, 3- SARS-CoV 2. Te resultant phytocompounds have potential benzenetriol and the interacting residues are GLU 738 therapeutic efcacy, which was analyzed in vitro and in silico and THR 766. 5DXT docked against 1, 2, 3-Benzenetriol examinations. Activity (%) activity (%) Advances in Pharmacological and Pharmaceutical Sciences 11 1,2,3-Benzenetriol_4YOI 1,2,3-Benzenetriol_6M17 1,2,3-Benzenetriol_6YB7 (a) 1,2,3-Benzenetriol_1M17 1,2,3-Benzenetriol_5DXT (b) Figure 9: (a) Docking interactions of lead compound 1, 2, 3-Benzenetriol with the SARS-CoV-2 target proteins; (b) docking interactions of lead compound 1, 2, 3-Benzenetriol with the breast cancer target proteins. Figure 10: Pharmacophore hypothesis of 1, 2, 3-Benzenetriol. 12 Advances in Pharmacological and Pharmaceutical Sciences RMSD Backbone after 1sq fit to Backbone RMS fluctuation 0.5 0.6 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 10000 20000 30000 40000 50000 0 50 100 150 200 250 300 Time (ps) Residue Hydrogen Bonds 0 10000 20000 30000 40000 50000 Time (ps) Hydrogen bonds Figure 11: Molecular dynamics simulation on the lead complex 1, 2, 3-Benzenetriol_4YOI. 5. Conclusion Data Availability Overall, based on the fndings of the tests mentioned above, All data used to support the fndings of this study are it was evident that the compounds identifed in the GC MS available from the corresponding author upon reasonable profle may aid in the function of this drug as a potent request. antioxidant and anticancer agent. Molecular docking and dynamics, ADME prediction, and pharmacophore hy- Conflicts of Interest pothesis are some of the computational methods used in the current work to inhibit expression. According to a molecular Te authors declare that they have no conficts of interest. dynamics simulation study, reveals the lead docked complex showed improved stability and reduced fuctuation with Authors’ Contributions a signifcant number of hydrogen bond interactions. Te phytocompounds’ potential efectiveness against the SARS- LSR, PK, EP, and RKRM conceptualized the study. NKT, KL, CoV 2 and breast cancer target receptors was discovered and GS wrote the original draft. AS, SC, KC, and BRS using in silico approaches. Tus, these results indicate the supported in revision and editing the manuscript. SSK su- genius of the complementary and alternative medicine pervised the study. All authors have read and agreed to the practitioners. published version of the manuscript. Langeswaran RMSD (nm) Number (nm) Advances in Pharmacological and Pharmaceutical Sciences 13 cancer cell death via triggering apoptotic pathway,” Bio- Kulanthaivel, Anitha Shanmuganathan, Kirubhanand medicine and Pharmacotherapy, vol. 103, pp. 562–573, 2018. Chandrasekaran, Bharat Ramrao Sontakke and Gowtham [13] S. Kumari, P. B. Katare, R. 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Antioxidant, Anti-Inflammatory, and Anticarcinogenic Efficacy of an Ayurvedic Formulation: Amritotharanam Kashyam

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10.1155/2023/3387261
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Hindawi Advances in Pharmacological and Pharmaceutical Sciences Volume 2023, Article ID 3387261, 13 pages https://doi.org/10.1155/2023/3387261 Research Article Antioxidant, Anti-Inflammatory, and Anticarcinogenic Efficacy of an Ayurvedic Formulation: Amritotharanam Kashyam 1 2 3 Lakshmi Sundaram Raman , Anitha Shanmuganathan , Sangeeta Chandrashekar , 4 5 6 Prabhu Kaliyaperumal , Elumalai Perumal , Ram Krishna Rao Mudiganti , 7 7 8 K. T. Nachammai , Langeswaran Kulanthaivel , Gowtham Kumar Subbaraj , 9 9 Kirubhanand Chandrasekaran , Bharat Ramrao Sontakke , and Senthilkumar Subramanian Faculty of Pharmacy, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, Tamil Nadu, India Department of Physics, Arulmigu Palaniandavar College of Arts and Science, Palani, Tamil Nadu, India Department of Physiology, Bharath Institute of Higher Education and Research, Chennai, Tamil Nadu, India Department of Anatomy, Sree Balaji Medical College and Hospital, Chrompet, Chennai, Tamil Nadu, India Centre for Transdisciplinary Research, Department of Pharmacology, Saveetha Dental College, Saveetha University, Chennai, Tamil Nadu, India M/s. Noahs Laboratories, No. 8/1, Old Mahabalipuram Road, Tiruporur, Tamil Nadu, India Molecular Cancer Biology Laboratory, Department of Biotechnology, Science Campus, Alagappa University, Karaikudi, Tamil Nadu, India Faculty of Allied Health Sciences, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelambakkam, Tamil Nadu, India Department of Anatomy, All India Institute of Medical Sciences, Nagpur, Maharashtra, India School of Medicine, College of Medicine and Health Science, Jigjiga University, Jigjiga, Somali Region, Ethiopia Correspondence should be addressed to Senthilkumar Subramanian; senthilkumarsubramanian@jju.edu.et Received 3 October 2022; Revised 9 March 2023; Accepted 16 March 2023; Published 28 April 2023 Academic Editor: Daniel Dias Rufno Arcanjo Copyright © 2023 Lakshmi Sundaram Raman et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Amritotharanam Kashyam, a specifc Ayurvedic drug, was the focus of the current inquiry to evaluate its efcacy. For liver and digestive-related issues, this medication is suggested. Tis was obtained from a standard Ayurvedic vendor in Chennai (India), and GC-MS analysis was carried out according to the standard procedure. A few critical biomolecules include benzoic acid, hex- adecanoic acid, 6,9-octadecadienoic acid, 9-octadecenoic acid, methyl ester (E)-, heptadecanoic acid, 16-methyl, methyl ester, methyl 18-methylnonadecanoate, tetracosanoic acid, distearin, hexadecanoic acid, and 1-(hydroxymethyl)-1,2-ethanediol ester. Te obtained biomolecules exhibited some signifcant therapeutic functions, including acidifcation, inhibition of arachidonic acid formation, increase in the aromatic amino acid decarboxylase, suppression of uric acid generation, inhibitors of catechol-O- methyltransferase, urine acidifers, etc. Te anticancer and antiviral potential of these phytocompounds were investigated using molecular docking and dynamics. Te phytocompounds pharmacokinetic characteristics were investigated using ADME analysis. Trough docking and dynamics simulation, in silico tests demonstrated the phytocompounds' inhibitory efciency against the target proteins. Tese functions reasonably relate to the medicinal function of Amritotharanam Kashyam. Te MTTassay fndings demonstrated this medication’s anticancer efects. Te ability to be an efective drug is demonstrated by its antioxidant, anti- infammatory, and membrane-stabilizing properties. 2 Advances in Pharmacological and Pharmaceutical Sciences infammatory, and membrane-stabilizing capacity of 1. Introduction Amritotharanam Kashyam studies prove its efcacy as Medicinal plants are currently gaining great attention as a potent medicine. Inhibitory efcacy of phytocompounds is possible sources of anticancer medicines and are frequently through Molecular docking and dynamics simulation. employed due to the availability of materials, afordability, relatively low cost, and few or no side efects, wide appli- 2. Materials and Methods cation, and therapeutic efcacy, which has expedited sci- 2.1. HPTLC Profle Chromatography Instrumentation. Te entifc study. Te World Health Organization (WHO) CAMAG (Muttenz, Switzerland) HPTLC unit is equipped encourages the use of traditional medicines because they are with Linomat 5 automatic sample applicator with a 100μL safe and efective for these reasons [1]. Amirtotharanam syringe, a TLC sample spotter, a twin trough glass chamber Kashyam is an efective Ayurvedic formulation to treat fu, for chromatogram plate development, and a TLC scanner 3 coughs, cold, and infammatory diseases and cures various liver disorders. It is efective in recovering liver cells after for densitometric evaluation of chromatograms, which is integrated winCATS 4 software (Version 1.4.3, Camag) for severe liver diseases such as cirrhosis or hepatitis. Tis also helps in improving appetite and digestion. Tis medicine is interpretation of data. prepared by adding the powders of Tinospora cordifolia (Guduchi), Zingiber ofcinale (Ginger), and Terminalia 2.2. Chromatography Conditions. Te ethyl acetate extract chebula (Haritaki) at the ratio of 2 parts: 6 parts: 4 parts and sample was chromatographed on aluminum-backed HPTLC boiling the mixture with 8 parts of water. Te boiling is plates (10 ×10cm), precoated with silica gel 60F (0.2mm th carried out till the quantity becomes 1/4 . It is then cooled thickness) (Merck). Before sample application, the plates and preserved in air-tight bottles for medicine. It is pre- were activated at 60 C for 5min after prewashing them with scribed at 12–24ml with an equal quantity of water and methanol. Te ethyl acetate extract sample (2, 4, 8, and 12 μl) a small spoon of sugar. Tis medicine references the was spotted as a band with 8mm width at diferent tracks at Ayurvedic treatise, Sahasra Yoga Kasaya Prakarana, Chi- a distance of 11mm from the bottom and 15mm from the kitamanjari. Te three substances are extensively utilized in side edges of HPTLC aluminum sheets with the help of home medicines, and substantial research has been carried 100μl syringe (Hamilton, Bonaduz, Switzerland) attached out on their therapeutic properties. withCamag semiautomatic Linomat 5 applicator (Camag, Ginger (Zingiber ofcinale): ginger is the most frequent Switzerland). Te nitrogen stream was passed simulta- home remedy for coughs, colds, and indigestion. Its ther- neously to dry the bands. Te plate development occurred in apeutic properties have been well-documented [2]. During the ascending mode in a twin-trough glass chamber pregnancy, [3] it has documented its antioxidant efects. (10 ×10cm), presaturated with 20ml of mobile phase for Ginger can help with nausea and vomiting [4]. By inhibiting 15min by sealing the chamber air-tight with paraflm. Te calcium channels, it lowers blood pressure [5]. Guduchi chromatogram was run up to 8cm from the sample ap- (Tinospora cordifolia): T. Chebula bark, rind, galls, and other plication point using n-hexane: ethyl acetate: formic acid: parts of the common Triphalachoornam have been found to acetic acid in diferent ratios (7.0:3.0:0.1:0.1) (v/v/v/v), as have antimicrobial, antioxidant, antidiabetic, anti- the mobile phase. Te solvent system was selected by trial infammatory, hepatoprotective, antiarthritic properties, as and error to elute the maximum number of compounds. well as antiproliferative, antimutagenic, radio and car- After the development, the solvent front was marked using dioprotective, antispasmodic, antiviral properties, immu- a pencil, and then the HPTLC plates were dried in an oven at nomodulatory, and hypolipidemic [6]. Haritaki (Terminalia 60 C for 5minutes to ensure that all mobile phase was re- chebula): antidiabetic, antispasmodic, antiperiodic, antiar- moved. Densitometric scanning was performed immediately thritic, antioxidant, anti-infammatory, antiallergic, anti- using Camag TLC scanner 3 with winCATS software leprotic, antistress, antimalarial, immune-modulatory, (Version 1.4.8) under 254nm with 5mm ×0.45mm slit antineoplastic, and hepatoprotective efects are some of the dimension for qualitative analysis of ethyl acetate extract therapeutic qualities of this plant [7]. Te present work deals −1 20mms scanning speed, 100μm/step data resolution. R with the efcacy determination of Amritotharanam values of the sample were marked and analyzed using the Kashyam using various parameters. Te computer-aided winCATS software. Te images were captured with visible drug design (CADD) method helps to identify prospective UV light at 366nm and 254nm by keeping the plate in the lead compounds and contribute to the development of photo-documentation chamber (CAMAG REPROSTAR 3). potential therapeutics for a wide variety of ailments. A low- Te peak numbers with their height and area, peak display, cost, time-saving, quick, and automated approach. It pro- and peak densitogram were identifed. vides information about the patterns of small molecules interacting with targeted protein molecules (drug-receptor) interactions [8]. In GC MS analysis to determine the mol- 2.3. GC MS Profle. Amritotharanam Kashyam was bought ecules present in medications and to try to comprehend the from a regular Ayurvedic seller in Chennai and submitted to types of molecules present and their potential medicinal conventional GC MS analysis. Instrument: Gas chroma- functions that might contribute to Amritotharanam tography (Agilent: GC: (G3440A) 7890A. MS-MS: 7000 Kashyam’s medicinal role. MTT assays to understand the Triple Quad GCMS) was outftted with a mass spectrometry anticancer efcacy of this medicine. Te antioxidant, anti- detector. Advances in Pharmacological and Pharmaceutical Sciences 3 2.3.1. Sample Preparation. Samples of 100microliters and dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) 1mL of appropriate solvents were used to dissolve the into a dark blue/purple, water-insoluble formazan product. In compound. For 10seconds, the solution was forcefully ag- 96-well plates, the cells were seeded at a density of 5 ×10 cells/ itated using a vortex stirrer. Gas-chromatography was used well. Te media was changed after 24hours with 100 μl of to analyse the clear extract. medium with Amritotharanam Kashyam at various doses and cultured for another 24hours. Te medium from both the control and Amritotharanam Kashyam treated cells was 2.3.2. GC-MS Protocol. Te GC MS Column was con- withdrawn. Each well was given 50μl of MTT (5mg/ml), and structed of DB5 MS (30mm 0.25mm ID 0.25m, 5 percent again plate was keptin a CO incubator for 4hours at 37 C.Te phenyl, 95% methyl polysiloxane), Electron impact mode at MTTwas subsequently removed, and then 50μl of DMSO was 70eV, and Helium (99.999 percent) as a carrier gas at used to dissolve purple-colored formazan crystals. An ELISA a constant fow rate of 1ml/min. Te auxiliary temperature is reader (Bio-Rad) had been used to quantify the purple-blue ° ° 290 C, while the injector temperature is 280 C. Te ion formazan mixture at 570nm. Each sample’s optical density was source has a temperature of 280 C. For fragments ranging compared with the control, and the percentage of cell viability from 45 to 450Da, the oven temperature was set to climb was calculated and plotted in the graphs. ° ° from 50 C (isothermal for 1.0min) to 170 C (isothermal for ° ° 4.0min), then 10 C/min to 310 C (isothermal for 10min). Te GC’s overall running time is 32.02minutes. Te GC-MS 2.5. Determination of Nuclear Morphological Changes of Cells Library is employed (NIST and WILEY). (DAPI Staining). Te cell monolayer was washed with PBS and fxed in 3 percent paraformaldehyde for 10minutes at room temperature for the nuclear morphological analysis. Te 2.4.Chemicals. Te following chemicals were used for MTT frozen cells were permeabilized in PBS containing 0.2 percent Assay: DMEM, Antibiotics (Streptomycin, Penicillin), Triton X-100 for 10minutes at room temperature before being trypsin-EDTA, PBS, and FBS from Gibco (Invitrogen, USA). treated for 5minutes with 0.5g/ml DAPI. Te apoptotic nuclei MTT reagent and dimethyl sulfoxide (DMSO) are from were examined using a fuorescence microscope (intensely Sigma Aldrich Chemicals Pvt Ltd, USA. stained, shattered nuclei, and condensed chromatin). 2.4.1. Sample Preparation. 25ml of Amritotharanam 2.6. Antioxidant, Anti-Infammatory Assays, and Membrane Kashyam was taken in a Petri dish and kept in a lab water Stabilizing Assays bath at 75–85 C for 3-4hrs until a solid consistency of (a) Antioxidant assay - ABTS radical scavenging assay extract was obtained. Approximately 1000mg of the extract ferric reducing antioxidant potential (FRAP) assay was dissolved in 10ml of water and Dulbecco’s Modifed Eagle Medium (DMEM) for free radical scavenging and cell (b) Anti-infammatory assay - protein denaturation assay viability assay. (c) Membrane stabilisation assay 2.4.2. Cell Line and Culture. Te National Centre For Cell 2.6.1. ABTS (2,2′-Azino-bis (3-Ethylbenzothiazoline-6-sul- Science, Pune, India (NCCS) supplied the breast cancer cell phonic Acid)) Radical Scavenging Assay. Amrithotharam line (MCF-7). Dulbecco’s minimum essential medium Kashyam’s antioxidant capacity was investigated utilizing added with 10% FBS and 1% antibiotics were used to culture the ABTS (2,2′-azino-bis-3-ethyl benzothiazole-6-sulphonic the cells. At 37 C, the cells were maintained in a 5% CO acid) radical cation decolorization test. Te radical cations of environment. T-75 culture fasks were used to cultivate cells, ABTS (ABTS was created by reacting a 7mmol/L ABTS and the studies were conducted at a confuence of 70% to solution with 2.45mmol/L potassium persulphate) (K S O ) 2 2 8 80%. Once they had reached confuency, the cells were [9]. Before usage, the mixture was kept in the dark at room dissociated by 0.05% trypsin-EDTA enzyme. Cells were temperature for 12hours. Ethanol was used to dilute the 4 2 seeded at a density of 10 cells/cm and treated with various ABTS+solution. Diferent concentrations of test samples concentrations of Amritotharanam Kashyam. Te cells were (5–320g/ml) and standard, ascorbic acid (5–320g/ml) were harvested using trypsin after 24hours of treatment. Each mixed with the diluted ABTS solution (2.0ml). Te reaction sample was counted in triplicate under a Nikon (Japan) mixture was allowed to stand at room temperature for inverted microscope to assess the total cell numbers. 6minutes before measuring absorbance at 734nm with an ultraviolet-visible spectrophotometer. All determinations were made in triplicate. Te radical scavenging activity was 2.4.3. MTT Assay. Tis test was conducted to understand the denoted as ABTS, and the radical scavenging efect was anticancer activity of Amritotharanam Kashyam. To determine computed using the following equation: the cytotoxicity efect of Amritotharanam Kashyam in the breast cancer cell line, the MTT assay was used. Cell viability (A0 − A1) ABTSradicalscavengingeffect (%) � 􏼢 􏼣 × 100, (1) was measured by MTTassay, a sensitive, accurate, and reliable A0 colorimetric technique. Te assay is based on the ability of the cellular mitochondrial dehydrogenase enzyme in living cells to where A0 is the absorbance of control; A1 is the absorbance convert the yellow, water-soluble substrate 3-(4,5- of the test. 4 Advances in Pharmacological and Pharmaceutical Sciences 2.6.2. Ferric Reducing Potential Antioxidant Assay. Te solution in phosphate bufer and incubated in an incubator antioxidant ability of the test samples Amritotharanam at 37 C for 15minutes. Denaturation was achieved by im- Kashyam to convert Fe3+ TPTZ complex (colourless mersing the reaction mixture in a 60 C water bath for 2+ complex) to Fe tripyridyltriazine (blue coloured complex) 15minutes. After cooling, the turbidity was measured at generated by electron-donating antioxidants at low pH was 660nm. Te turbidity of the reference medication was calculated spectrophotometrically. It was calculated spec- measured at the same concentration. Te percentage of trophotometrically using a modifed approach developed by denaturation inhibition was estimated from control in which Benzie and Strain [10, 11]. Te change in absorbance at no medication was applied. Te typical medication was 593nm is used to monitor this process. At 37 C, 300mM diclofenac sodium. Te percentage inhibition of de- acetate bufer, 10ml TPTZ in 40mM HCl, and 20mM naturation was computed using the formula shown as FeCl .6H O were mixed to make the Ferric reducing anti- follows: 3 2 oxidant power (FRAP) reagent. 3.995ml of newly made (ODof test − ODof control) working FRAP reagent was pipetted and carefully mixed %Inhibition � 􏼢 􏼣x100. (2) ODof control with varying amounts (5–320g/ml) of test samples and standard, Ascorbic acid (5–320g/ml). When the ferric tri- 3+ pyridyl triazine (Fe TPTZ) complex was reduced to ferrous 2+ ° 2.6.4. Membrane Stabilisation Assay. Fresh whole human (Fe ) form after 30minutes at 37 C, a vivid blue colour blood (5ml) was collected and transferred to centrifuge complex was produced, and the absorbance at 593nm was tubes in a heparinized tube. It was centrifuged at 3000rpm measured against a reagent blank (3.995ml FRAP for 10minutes before being washed with normal saline of reagent+5l distilled water). Every judgment was made in equal volume three times. Te blood volume was measured triplicate. and reconstituted as a 40% v/v suspension with isotonic solution (10mM sodium phosphate bufer). Amritothar- 2.6.3. Anti-Infammatory Assay through Protein De- anam Kashyam and regular diclofenac sodium were com- naturation Assay. Amirthotharam Kashyam’s anti- bined in 0.1ml of 40% RBC solution at concentrations infammatory potential was determined using a protein ranging from 50–1600μg/ml. Te control was 0.1mL of RBC denaturation assay. Te experiment was carried out with mixed with an isotonic solution on its own. For 30minutes, minor modifcations by Gnana and colleagues in 2011 the reaction mixture was incubated in a water bath at 56 C. [12, 13]. Te reference medicine, diclofenac sodium, was Te tubes were cooled to room temperature after incubation. dissolved in dimethyl sulfoxide (DMSO), and the sample was Te reaction mixture was centrifuged for 5minutes at diluted using phosphate bufer (0.2M, PH 7.4). All solutions 2500rpm, and the supernatant absorbance was measured at had a fnal DMSO concentration of less than 2.5 percent. Te 560nm. To calculate the % membrane stabilizing activity, Test Solution (4ml) with varying drug concentrations use the formula as follows: (50–1600μg/ml) was mixed with 1ml of 1mM albumin (ODof test − ODof control) %Inhibitionof hemolysis � 􏼢 􏼣x100. (3) ODof control 2.7. Statistical Analysis. Te results will be presented as downloaded from Protein Data Bank (PDB Id: 4YOI, mean SEM. One-way ANOVA was used to establish sta- 6YB7, 6M17–SARS-Cov-2 and 1M17, 58XT–Breast can- tistical signifcance, followed by a Dunnett’s multiple- cer). Retrieved structures were imported into the Maestro comparison test with 95% confdence intervals. P values Schrodinger suite, and the receptors were optimized and less than 0.05 were regarded as signifcant. minimized using the force feld OPLS_2005 [8]. Deletion of water molecules and adding hydrogen were performed before grid generation. Te glide module generates the 2.8. Target and Lead Selection. Te resultant phyto- grid box of X, Y, and Z dimensions. After the grid gen- compounds from GC-MS analysis possess medicinal prop- eration, prepared ligands were docked with the target erties, therapeutic efcacies, and potential inhibitory actions receptors. against various tumours and some deadly diseases. Tis study focuses on the two deadliest diseases, COVID-19 and breast cancer. Te target receptors were examined with the selected 2.10. Ligands Preparation. Te reported phytocompounds phytocompounds to analyse their therapeutic efcacies. from GC-MS analysis were downloaded from the PubChem database. 2D structures of selected compounds were pre- 2.9. Protein Preparation and Grid Generation. pared by the LigPrep module using the force feld OPLS_2005 [14]; the resultant prepared ligands were of high Tree-dimensional structures of the three target receptors of SARS-CoV-2 and two receptors of Breast cancer quality with accurate bond lengths and angles. Advances in Pharmacological and Pharmaceutical Sciences 5 ligand parameters. Cl− or Na+ was added to neutralize the 2.11. Molecular Docking. By computing the energy of the ligand binding to the protein, docking is a common mo- system, and the complex was placed in a cubic box of about 1.0A. To minimize the complex, NPT and NVT were per- lecular modelling approach for inserting ligands into the active site of a receptor molecule. Te value of this energy formed for 100ps at 300k, and MD was performed for 50ns. determines the biological activity of molecules; the greater the energy, the more efective the medicine based on the 3. Results and Discussion receptor will be assessed. Te G-score is calculated in Kcal/ mol and incorporates the energies of ligand-protein in- 3.1.HPTLC. In HPTLC observation, 12 peaks were observed teractions, hydrophobic interactions, hydrogen bonds, in- and two of them indicated the presence of two primary ternal energy, and pi-pi stacking interactions. Te XP’s compounds in the sample. Further analysis is required to GLIDE modules visualize the investigation of a specifc identify those two major components. Te peak at Rf 0.52 indicated 16.92% of compound A, Peak at Rf-0.71 indicated ligand-protein interaction [15]. 52.60% of compound B. Te results were illustrated in Figure 1. 2.12. Pharmacophore Hypothesis. Te pharmacophore hy- pothesis generated through the Phase module uses a recently built standard Pharmacophore perception algorithm that 3.2. GC MS Results of Amritotharanam Kashyam. reverses the conventional paradigm by detecting ligand Figure 2 depicts the GC MS profle of Amritotharanam alignments and then perceiving hypotheses [16]. Te Kashyam that displayed the retention values, categories of pharmacophore characteristics, such as Acceptor (A), Donor likely compounds, molecular formulas, molecular weight, (D), Hydrophobic (H), Negative (N), Positive (P), and peak area, and medicinal advantages of each chemical as Aromatic Rings (R), were determined by three chemical demonstrated in Amritotharanam Kashyam’s GC MS pro- structural patterns and were point, vector, and groups as fle. By comparing retention duration and fragmentation SMARTS inquiries. Tree diferent geometries, each of pattern with mass spectra in the NIST spectrum library which defned the physical attributes of the site, were recorded in the GC-MS computer program, metabolites assigned to these patterns. An aromatic ring’s directionality were identifed (version 1.10 beta, Shimadzu). Figure 2 is described by a vector that is normal to the ring’s plane in showed the pharmacological functions of each bio- this context. It swiftly develops high-quality hypotheses molecule based on Dr. Duke’s phytochemical and ethno- from a few to hundreds of known active ligands using botanical data source (National Agriculture Library, USA) pharmacophore-based shape alignments. and others. Benzoic acid, methyl ester, hexadecanoic acid, 6, 9-octadecadienoic acid, 9-octadecenoic acid, methyl ester, (E)-, heptadecanoic acid, 16-methyl-, methyl ester, methyl 2.13. MM-GBSA Calculation. Te energy of optimum-free 18-methylnonadecanoate, tetracosanoic acid. Tese com- receptors, free ligands, and ligand-receptor complexes is pounds have far-reaching biological functions, which bodes calculated using Prime MM-GBSA Molecular Mechan- well for Amritotharanam Kashyam’s position as a powerful ics–Generalized Born Surface Area [17]. It also calculates the treatment for which it is given. ligand strain energy by immersing the ligand in a solution generated automatically by the VSGB 2.0 suit. Te energy visualization was shown via the principal energy visualizer. 3.3. MTT Assay. Te MTT assay revealed that Amrito- tharanam Kashyam had good cytotoxic action against the development of nonsmall cell lung cancer cells in A549cell 2.14. ADME Properties. Te QikProp module was used to line. Amritotharanam Kashyam-treated cells exhibit de- determine absorption, distribution, metabolism, excretion, creased cell proliferation in MTT assays. Figures 3 and 4 and toxicity (ADME/T) characteristics for best-docked li- clearly showed that as the concentration of Amritotharanam gand molecules. Tis software predicts QP log Po/w, Kashayam increased, the cell viability reduced almost uni- QPlogBB, donor HB, acceptor HB, Caco, rule of 5, total formly. Tis clearly indicated the cytotoxicity of Amrito- percentage of human oral absorption, etc. Lipinski’s rule of tharanam Kashayam on cancer cells. fve evaluates the drug-likeness of a chemical molecule based on biological ingredients and pharmacological features to predict an orally active therapy [18]. 3.4. Nuclear Morphological Changes of Cells (DAPI Staining) Assay. Te microscopical observation displayed a decline in 2.15. Molecular Dynamics Simulation. Molecular dynamic the count of the cells and morphological alterations (Fig- simulations can be used to verify docking and understand ure 4). In this study, we used two diferent doses to treat the protein-ligand interactions. It enables the analysis of the sys- cell line. Te increase in the dose concentration declines the tem’s physical evolution through time and is a valuable tool for cell quantity compared to control cells. Fluorescence mi- interpolating between theory and experimentation. MD per- croscopic studies indicate abnormal cell boundaries, irreg- formed in GROMACS package with 50ns by using GRO- ular nucleus shape and enlargement of the cell nucleus. MOS96 43a1 force feld [19]. Te compound with the lowest Tese cell morphological changes and nucleus mutilation binding energy was selected for molecular dynamics (MD) have been recovered in the drug-treated cells compared to simulation. Te PRODRG web server was used to assess the control cells. 6 Advances in Pharmacological and Pharmaceutical Sciences All tracks @ 254 nm All tracks @ 254 nm 1000.0 (AU) 1000.0 800.0 1000.0 (AU) 1000.0 700.0 (AU) 800.0 (AU) 600.0 800.0 700.0 500.0 800.0 700.0 600.0 700.0 400.0 600.0 500.0 600.0 300.0 500.0 400.0 200.0 400.0 500.0 300.0 300.0 400.0 100.0 200.0 254 nm 366 nm 200.0 0.0 300.0 100.0 200.0 100.0 0.0 200.0 (mm) 200.0 100.0 0.0 (mm) 100.0 50.0 0.00 0.10 100.0 0.20 0.30 0.40 0.0 0.0 0.50 50.0 0.60 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 (RF) 1.00 0.70 0.80 (RF) 0.0 1.00 Track 1 , ID: Standard1 Track 2 , ID: Standard2 1000 1000 AU AU 900 900 800 800 700 700 600 600 500 500 400 400 300 300 6 7 200 200 3 1 9 2 5 3 1 100 100 0 0 0.00 0.20 0.40 0.60 0.80 1.00 0.00 0.20 0.40 0.60 0.80 1.00 Rf Rf Track 3 , ID: Standard3 Track 4 , ID: Standard4 1000 1000 AU AU 900 900 800 800 700 700 600 600 500 500 400 400 300 300 11 12 11 2 13 6 9 6 200 5 200 5 1 2 100 100 0 0 0.00 0.20 0.40 0.60 0.80 1.00 0.00 0.20 0.40 0.60 0.80 1.00 Rf Rf Figure 1: HPTLC-Profle data result. User Chromatogram +EI TIC Scan 220620018.D ×10 1 1 15.743 0.8 0.6 14.133 0.4 25.614 * 5.900 19.282 0.2 8.097 456789 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Counts vs. Acquisition Time (min) Figure 2: Indicates the GC MS profle of Amritothararam Kashyam. Advances in Pharmacological and Pharmaceutical Sciences 7 Amritotharanam Kashyam and diclofenac sodium, re- spectively. In membrane stabilisation activity standard is prone to show more activity than a sample. 3.6. Glide Docking. XP Docking was performed using the Glide module, protein-ligand was docked using the XP module, and the conformers were evaluated by G score. Docking results declared that the phytocompound 1, 2, 3- benzenetriol exhibited the highest docking score among other phytocompound when examined with the SARS-CoV- 2 and breast cancer target proteins. Covid proteins such as 20 4YOI, 6YB7, and 6M17 showed docking scores of about −7.325, −5.621, and −6.814Kcal/Mol; furthermore, targets of breast cancer 1M17 and 5DXTshowed the docking score of Control DMSO 1 μL 2 μL 5 μL 10 μL 15 μL 20 μL about −6.840 and −6.932Kcal/Mol respectively. Te inter- Concentration (μL/mL) acting residues interacted with 1, 2, 3-benzenetriol was GLN_167, GLU_166, LEU_52, TYR_128, GLU_738, 24 hours THR_766, GLU_849, and VAL_851 respectively. Interacting 48 hours residue between lead compound 1, 2, 3-benzenetriol and Figure 3: MTT assay comparative chart for 24hr and 48hr for 4YOI is GLN amino acid at the position of 167. Interacting Amritotharanam Kashyam. residue between is 1, 2, 3-benzenetriol and 6M17 leucine amino acid at the position of 52 and tyrosine amino acid at the position of 128. Interacting residue between is 1, 2, 3- 3.5. Antioxidant, Anti-Infammatory, and Membrane Stabi- benzenetriol and 6YB7 glutamic acid amino acid at the lizing Potential of Amritotharanam Kashyam. Te standard position of 166. Interacting residue between is 1, 2, 3- drug ascorbic acid was used as a standard for ABTS and benzenetriol and 1M17 glutamic acid amino acid at the FRAP antioxidant assay. In the case of anti-infammatory position of 738 and threonine amino acid at the position of assays such as protein denaturation assay and membrane 766. Interaction profle between 1, 2, 3-benzenetriol and stabilisation assay; diclofenac sodium is used as standard. It targeted receptor 5DXT shows the interacting residue VAL was conducted to have a better comparative study with the 851 and GLU 849. Te results were illustrated in Figure 9. test sample, Amritotharanam Kashyam. Te sample Amri- totharanam Kashyam and standard concentrations are 5, 10, 20, 40, 80,160, and 320μg/ml for all antioxidant assays, 3.7. Binding-Free Energy. Te binding afnity of ligands to where 50, 100, 200, 400, 800, and 1600μg/ml concentration the receptor was estimated using postdocking binding free was taken for anti-infammatory assay. Te average value of energy. Te results of the prime MM-GBSA analysis revealed the reactions performed in triplicate was obtained and that 1, 2, 3-Benzenetriol had ΔGbind of 35.231 (4YOI), plotted against the diferent concentrations of Amrito- 33.214 (6YB7), 34.127 (6M17), 34.563 (1M17), and tharanam Kashyam and its standards. Te IC 32.501Kcal/Mol, respectively (Figure 9). According to the value that is half-maximal inhibitory concentration was calculated from binding-free energy, the substances may frmly engage in the active site residues of the target proteins to block enzy- the R equation obtained from a linear thread line from the respective graph of concentration of Amritotharanam matically, stop COVID-19 infection, and inhibit breast Kashyam/standard against % inhibition and activity values. cancer activity. Figure 5 clearly showed that the IC value for the ABTS radical scavenging efect of Amritotharanam Kashyam was 3.8. ADME Properties. Te ADME properties and pharma- 58.27μg/ml, and ascorbic acid was 33.43μg/ml. Amrito- cokinetics of phytocompounds play a signifcant role in their tharanam Kashyam showed more than four times less efcacy. Figure 10 reveals the ADME properties of phyto- radical scavenging efect than standard. compounds. Te Lipinski principles of fve were followed for Te FRAP assay result was presented in Figure 6. An the phytocompounds, which included 500 molecular weight, increase in value was noted in an increase in concentrations 10 hydrogen bond acceptors, 5 hydrogen bond donors, and of both test and standard. Te results indicate that Amri- logP values of 5. Also acceptable were physicochemical pa- totharanam Kashyam showed comparatively more FRAP rameters such as human oral absorption, partition coefcient activity than standard ascorbic acid. Te IC value for (QPlogPo/w), and (QPlogBB) values. As a result, the phy- protein denaturation activity of Amritotharanam Kashyam tocompounds reported values could be deemed powerfully and standard, diclofenac sodium was 160.7μg/ml and inhibits the COVID-19 infection and Breast Cancer. 221.23μg/ml, respectively, which was obtained from Fig- ure 7. Here the Amritotharanam Kashyam shows better protein denaturation activity than standard. Te IC value 3.9. Pharmacophore Hypothesis. Pharmacophore generated for membrane stabilisation activity was obtained from through Phase module and the features of lead compound 1, Figure 8, where 525.87μg/ml and 378.83μg/ml are IC 2, 3-benzenetriol was illustrated in Figure 10. Cell Viability (%) 8 Advances in Pharmacological and Pharmaceutical Sciences Light Microscope DAPI Staining Control 5 μl/ml (Conc.) 5 μl/ml (Conc.) 10 μl/ml (Conc.) 10 μl/ml (Conc.) Figure 4: Cell morphology details of the MTT assay for Amritotharanam Kashyam Cell morphology—24h. Pharmacophore hypothesis reveals the acceptor, donor and score, the interaction residues, and the binding afnity aromatic ring present in the compound. In this lead com- between the desired proteins and the lead compound, pound three acceptors, three donors and one aromatic ring molecular dynamics simulations were run for 50ns. Te present in the active compound. behaviour of macromolecules is typically predicted using MD simulation, which utilizes Newton’s equation of motion and classical mechanics to determine the speed and site of 3.10.MDSimulation. Targeted protein-ligand complex were each atom in the system under study. Te least binding score examined for stability using molecular dynamics simula- complex was subjected to MD simulation. RMSD (root tions from the Gromacs package. According to the docking mean square deviation), RMSF (root mean square Advances in Pharmacological and Pharmaceutical Sciences 9 ABTS Radical Scavenging activity 5 10 20 40 80 160 320 Con (μg/ml) Amruthotharam Kashayam Ascorbic acid Figure 5: Comparative graphical representation of ABTS radical scavenging activity of Amritotharanam Kashyam and ascorbic acid. FRAP assay 5 10 20 40 80 160 320 Conc (μg/ml) Amruthotharam Kashayam Ascorbic acid Figure 6: Comparative graphical representation of FRAP assay of Amritotharanam Kashyam and ascorbic acid. fuctuation), and hydrogen bond plots were generated to numerous powerful biomolecules. According to scientists, evaluate the structural changes or stability in the complex. these powerful chemical components from nature are RMSD plot showed a slight deviation with the stability employed to cure various illnesses with fewer adverse efects observed from 30ns throughout the simulation period; the [20]. Plant extracts may contribute to the performance and complex attained a slight deviation at 0.4nm. RMSF plot general well-being of the fowl. Te enhancement of en- showed less fuctuation in the loop or disorder region at the dogenous digestive enzyme production, activation of the initial simulation period and 0.35nm. Tese deviations do immunological response, and antibacterial, antiviral, anti- not afect the structural changes or stability of the complex. oxidant, and antihelminthic properties are just a few of the Hydrogen bond interaction between the protein and the positive efects of herbal extracts or active substances on ligand was noted as fve. Te overall result (Figure 11) poultry nutrition. Computational developments signif- concluded that the complex showed better stability without cantly infuenced the process of developing new drugs. changes in structural confrmations. Virtual screening methods are often and widely used to reduce the price and duration of drug development. A key component of structure-based drug design is the discovery 4. Discussion of new ligands for protein structures using the molecular Phytochemical substances found in medicinal plants are docking technique [21]. 1, 2, 3-benzenetriol and 4YOI a plentiful source of treatment for several chronic disorders. interacted with GLN 167 with docking score of −7.325. In recent years, many therapeutic plants have produced Interaction profle between the 1, 2, 3-benzenetriol and % of inhibition % of inhibition 10 Advances in Pharmacological and Pharmaceutical Sciences Protein denaturation activity 50 100 200 400 800 1600 Conc (μg/ml) Amruthotharam Kashayam Diclofenac sodium Figure 7: Comparative graphical representation of protein denaturation activity of Amritotharanam Kashyam and diclofenac sodium. Membrane Stabilisation Activity 50 100 200 400 800 1600 Conc (μg/ml) Amruthotharam Kashayam Diclofena sodium Figure 8: Comparative graphical representation of the membrane stabilisation activity of Amritotharanam Kashyam and diclofenac sodium. 6YB7 show the docking score of −5.621and interacting with reveals the interacting residue between the ligand and GLU 166 amino acid. Interaction profle between the 1, 2, 3- protein receptor such as VAL 851 and GLU 849. In silico benzenetriol and 6M17 show the docking score of −6.814 approaches revealed the potential efcacy of the phyto- and interacting with LEU 52 and TYR128 amino acid. compounds against the target receptors of breast cancer and Targeted receptor 1M17 docked against the 1, 2, 3- SARS-CoV 2. Te resultant phytocompounds have potential benzenetriol and the interacting residues are GLU 738 therapeutic efcacy, which was analyzed in vitro and in silico and THR 766. 5DXT docked against 1, 2, 3-Benzenetriol examinations. Activity (%) activity (%) Advances in Pharmacological and Pharmaceutical Sciences 11 1,2,3-Benzenetriol_4YOI 1,2,3-Benzenetriol_6M17 1,2,3-Benzenetriol_6YB7 (a) 1,2,3-Benzenetriol_1M17 1,2,3-Benzenetriol_5DXT (b) Figure 9: (a) Docking interactions of lead compound 1, 2, 3-Benzenetriol with the SARS-CoV-2 target proteins; (b) docking interactions of lead compound 1, 2, 3-Benzenetriol with the breast cancer target proteins. Figure 10: Pharmacophore hypothesis of 1, 2, 3-Benzenetriol. 12 Advances in Pharmacological and Pharmaceutical Sciences RMSD Backbone after 1sq fit to Backbone RMS fluctuation 0.5 0.6 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 10000 20000 30000 40000 50000 0 50 100 150 200 250 300 Time (ps) Residue Hydrogen Bonds 0 10000 20000 30000 40000 50000 Time (ps) Hydrogen bonds Figure 11: Molecular dynamics simulation on the lead complex 1, 2, 3-Benzenetriol_4YOI. 5. Conclusion Data Availability Overall, based on the fndings of the tests mentioned above, All data used to support the fndings of this study are it was evident that the compounds identifed in the GC MS available from the corresponding author upon reasonable profle may aid in the function of this drug as a potent request. antioxidant and anticancer agent. Molecular docking and dynamics, ADME prediction, and pharmacophore hy- Conflicts of Interest pothesis are some of the computational methods used in the current work to inhibit expression. According to a molecular Te authors declare that they have no conficts of interest. dynamics simulation study, reveals the lead docked complex showed improved stability and reduced fuctuation with Authors’ Contributions a signifcant number of hydrogen bond interactions. Te phytocompounds’ potential efectiveness against the SARS- LSR, PK, EP, and RKRM conceptualized the study. NKT, KL, CoV 2 and breast cancer target receptors was discovered and GS wrote the original draft. AS, SC, KC, and BRS using in silico approaches. Tus, these results indicate the supported in revision and editing the manuscript. SSK su- genius of the complementary and alternative medicine pervised the study. All authors have read and agreed to the practitioners. published version of the manuscript. Langeswaran RMSD (nm) Number (nm) Advances in Pharmacological and Pharmaceutical Sciences 13 cancer cell death via triggering apoptotic pathway,” Bio- Kulanthaivel, Anitha Shanmuganathan, Kirubhanand medicine and Pharmacotherapy, vol. 103, pp. 562–573, 2018. Chandrasekaran, Bharat Ramrao Sontakke and Gowtham [13] S. Kumari, P. B. Katare, R. 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Advances in Pharmacological Sciences – Hindawi Publishing Corporation

Published: Apr 28, 2023

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