key: cord-0980855-8syza774 authors: Mahmoud, Dina B.; Ismail, Walaa M.; Moatasim, Yassmin; Kutkat, Omnia; ElMeshad, Aliaa N.; Ezzat, Shahira M.; El Deeb, Kadriya S.; El-Fishawy, Ahlam M.; Gomaa, Mokhtar R.; Kandeil, Ahmed; Al-karmalawy, Ahmed A.; Ali, Mohamed A.; Mostafa, Ahmed title: Delineating a potent antiviral activity of Cuphea ignea extract loaded nano-formulation against SARS-CoV-2: In silico and in vitro studies date: 2021-09-15 journal: J Drug Deliv Sci Technol DOI: 10.1016/j.jddst.2021.102845 sha: 4fbf38e918c877a517d883d326509de6459f09cd doc_id: 980855 cord_uid: 8syza774 The outbreak of coronavirus disease-2019, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a worldwide emerging crisis. Polyphenols are a class of herbal metabolites with a broad-spectrum antiviral activity. However, most polyphenols encounter limited efficacy due to their poor solubility and degradation in neutral and basic environments. Thus, the effectiveness of their pharmaceutical application is critically dependent on the delivery systems to overcome the aforementioned drawbacks. Herein, Polyphenols-rich Cuphea ignea extract was prepared and its constituents were identified and quantified. Molecular docking was conducted for 15 compounds in the extract against SARS-CoV-2 main protease, among which rutin, myricetin-3-O-rhamnoside and rosmarinic acid depicted the most promising antiviral activity. Further, a self-nanoemulsifying formulation, composed of 10% oleic acid, 40% tween 20 and propylene glycol 50%, were prepared to improve the solubility of the extract components and enable its concurrent delivery permitting combined potency. Upon dilution with aqueous phases, the formulation rapidly form nanoemulsion of good stability and excellent dissolution profile in acidic pH when compared to the crude extract. It inhibited SARS-CoV-2 completely in vitro at a concentration as low as 5.87 μg/mL presenting a promising antiviral remedy for SARS-CoV-2, which may be attributed to the possible synergism between the extract components. The outbreak of coronavirus disease-2019, caused by severe acute respiratory syndrome 27 coronavirus-2 (SARS-CoV-2) is a worldwide emerging crisis. Polyphenols are a class of herbal 28 metabolites with a broad-spectrum antiviral activity. However, most polyphenols encounter 29 limited efficacy due to their poor solubility and degradation in neutral and basic environments. 30 Thus, the effectiveness of their pharmaceutical application is critically dependent on the delivery 31 systems to overcome the aforementioned drawbacks. Herein, Polyphenols-rich Cuphea ignea 32 extract was prepared and its constituents were identified and quantified. Molecular docking was The latest outbreak of coronavirus disease 2019 was primarily recognized in Wuhan, 64 China [1] by the end of 2019. This disease is caused by a novel Betacoronavirus that has been 65 given the name of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the 66 International Committee on Taxonomy of Viruses [2] . SARS-CoV-2 belongs to the enveloped 67 positive-sense single-stranded RNA viruses and it can be transmitted from human to human [3] . In 68 October 10, 2020, the World Health Organization (WHO) recorded 37.1 million confirmed cases 69 of COVID-19 and 1.07 million confirmed deaths worldwide [4] , signifying that COVID-19 is a 70 life-threatening disease that affects public health. Thus, substantial efforts that are currently being 71 exerted to discover effective vaccines or drugs. In numerous countries, positively tested cases are 72 subjected to off-label and repurposed therapies such as chloroquine, hydroxychloroquine, 73 lopinavir-ritonavir, azithromycin, remdesivir, favipiravir, nitazoxanide, convalescent plasma and 74 interleukin-6 inhibitors [5, 6] ; yet there is a critical and urgent necessity to discover other effective 75 compounds as potential therapies for . 76 Computational studies are very promising tools in drug discovery processes. Several 77 computational methods like molecular docking studies help scientists in the discovery of new drug 78 candidates [8] . The SARS-CoV-2 main protease (M pro ) is responsible for its replication and 79 transcription as well, through the conversion of polypeptides into functional proteins [9, 10] . 80 Accordingly, targeting M pro of this virus can be a very promising target to examine the anti-viral 81 efficacy of any compound [11, 12] . 82 Numerous herbal metabolites possess many health benefits. However, the pharmaceutical use of 83 the herbal extracts and ingredients or phytopharmaceuticals is restricted because of their low 84 solubility and bioavailability which is considered the biggest obstacle in the application of herbal the hydrophobic nature of most polyphenolic compounds limits their water solubility and hence 104 they exhibit poor bioavailability which greatly affect their efficacy [20] . Furthermore, the 105 stability of polyphenolic compounds in gastrointestinal tract is an important issue that needs to be 106 considered while designing a successful drug delivery system, as they are stable at acidic pH of 107 stomach while they degrade in neutral or basic pH environments resulting in limited 108 bioavailability [21] . Consequently, a formulation with rapid dissolution which is able to rapidly 109 dissolve the polyphenolic compounds allowing their rapid absorption in the stomach before 110 reaching the intestine is highly demanded. Therefore, our aims were to explore the phenolic metabolites of Cuphea ignea A. DC. 132 ethanolic extract, study their ability to target SARS-CoV-2 M pro using molecular docking and 133 propose a rapidly dissolving self-nanoemulsifying formulation to overcome the poor solubility 134 and pH dependent instability of polyphenols, hence their bioavailability can be improved 135 accordingly. Moreover, our aim was to assess the viability of utilizing the developed formulation 136 as an inhibition therapy for SARS-CoV-2. We envisioned that this formulation will be a 137 promising tool to improve the delivery of Cuphea ignea polyphenols and offers a combined was involved in our studies as a reference standard. 189 The examined compounds were obtained from the PubChem and prepared for docking using the The SARS-CoV-2 main protease X-ray structure (code: 6LU7) [26] was obtained from the Protein 196 Data Bank and it was subjected for the detailed preparation steps described previously [27] . The aforementioned database was docked according to the general docking methodology applied 206 Six formulations were prepared with changing the concentrations of oleic acid, Tween 20 and 207 propylene glycol as an oily phase, a surfactant and a co-solvent, respectively; as shown in Table 1 . The excipients were weighed into glass vials of 10 mL capacity and stirred at 300 rpm and 50° C, 209 to ensure the complete mixing of the components. The mixtures were allowed to cool to 37 °C 210 then 1 g of each mixture was transferred to a glass beaker containing 500 mL of distilled water 226 In order to assess the ability of SNEDDS to solubilize Cuphea ignea extract with the acceptable 227 stability. SNEDDSs were subjected to centrifugation at 3500 rpm for 30 min. The SNEDDS 228 formulations which exhibited stability without any precipitation of the extract or phase separation 229 of the SNEDDSs components were exposed to 6 heating cooling cycles (45°C and 4°C). Additionally, passed formulations were further exposed to 6 freeze thaw cycles (between Table 3 and supplementary data. 339 The results of docking simulation showed that Rutin (4), Myricetin-3-O-rhamnoside (2), and 340 Rosmarinic acid (10) have the best binding scores towards SARS-CoV-2 main protease equal to -341 9.28, -7.86, and -7.05 kcal/mol, respectively (Table 3) . These values were close to the binding 342 energy of the docked N3 inhibitor (S = -10.11 kcal/mol). The detailed binding mode of N3 was; the docked N3 inhibitor fitted the asymmetric pocket of (Table 4) . In general, the docking results of the tested compounds of Cuphea ignea extract, compared to N3, 353 presented a good idea about their binding affinities towards SARS-CoV-2 main protease. Some 354 compounds of the extract showed ideal and promising binding, which corresponds to high affinities and 355 predicted intrinsic activities as well. 358 Results revealed that SN1 and SN2 formed clear, isotropic, transparent grade A nanoemulsions of 388 It was noticed that emulsification rate of SN1 was significantly faster than that of SN2, as SN1 389 took only 5 ± 1 min to disperse and emulsify in water while SN2 took 12.5 ± 1.5 min (p < 0.05; 390 student t-test). This may be justified by the presence of higher surfactant (tween 20) concentration 391 in SN2 than SN1 (80% and 40%, respectively) which impart SN2 higher viscosity than SN1. In vitro dissolution study 419 The in vivo bioavailability data for herbal medicines is not readily attainable as a result of the SNEDDSs (SN1 and SN2) were found to be greater as compared with that of the suspension of 428 the crude extract powder. Results are shown in Fig. 1 . It was observed that after 5 min, the highest 429 J o u r n a l P r e -p r o o f % extract dissolved was achieved in case of SN1 as it could dissolve 77.25 ± 4.1% of the extract, 430 followed by SN2 that dissolved 52.76 ± 3.3%, while only 12.17 ± 2.9% dissolved in case of the 431 crude extract. Moreover, complete dissolution of the extract was observed after 60 min from SN1 432 (100 ± 0.1 %) while the maximum % dissolved from the crude extract was as low as 21.56 ± 2.2% 433 at the end of the experiment (60 min). By applying ANOVA statistical analysis, results proved that there is a statistically significant 543 This work has been submitted for patent application to patent office Academy of Scientific J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f Table 4 : 3 D pictures of the binding interactions and the M pro positioning between the most promising tested compounds of Cuphea ignea A. DC leaves extract (Myricetin-3-Orhamnoside (2), Rutin (4), and Rosmarinic acid (10)) and N3-binding site compared to the N3 (docked, 16). Red dash represents H-bonds and black dash represent H-pi interactions. Remdesivir and chloroquine effectively inhibit the recently emerged novel 548 coronavirus (2019-nCoV) in vitro Clinical features of patients infected with 2019 novel coronavirus in Wuhan The lancet A familial cluster of pneumonia associated with the 2019 novel coronavirus 552 indicating person-to-person transmission: a study of a family cluster. The Lancet COVID-19): Global epidemiological situation 555 report Polyphenols Have a Role Against Coronavirus Infection? An Overview 558 of in vitro Evidence Drug repurposing of nitazoxanide: can it be an effective 560 therapy for COVID-19? Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling 562 of its spike protein for risk of human transmission Computational approaches for drug discovery Coronaviridae in Fields virology Publishing in face of the COVID-19 pandemic. International journal of antimicrobial 569 agents Molecular docking, molecular dynamics, and in vitro studies reveal the potential 571 of angiotensin II receptor blockers to inhibit the COVID-19 main protease. Heliyon Molecular docking reveals the potential of Cleome amblyocarpa isolated 574 compounds to inhibit COVID-19 virus main protease Antitumor activity of Cuphea ignea extract against benzo (a) pyrene-induced 577 lung tumorigenesis in Swiss Albino mice Angiotensin-converting enzyme and renin inhibition activities, antioxidant 579 properties, phenolic and flavonoid contents of Cuphea ignea A. DC Antiulcerogenic effect of Cuphea ignea extract against ethanol-induced 582 gastric ulcer in rats Polygonum cuspidatum and its active components inhibit replication of the 584 influenza virus through toll-like receptor 9-induced interferon beta expression Resveratrol inhibits the TRIF-dependent pathway by upregulating sterile alpha and 587 armadillo motif protein, contributing to anti-inflammatory effects after respiratory syncytial virus 588 infection Resveratrol inhibits rhinovirus replication and expression of inflammatory 590 mediators in nasal epithelia Resveratrol-mediated gamma interferon reduction prevents airway inflammation 592 and airway hyperresponsiveness in respiratory syncytial virus-infected immunocompromised mice Encapsulation of nutraceutical ingredients in 595 liposomes and their potential for cancer treatment Bioavailability of anthocyanins and derivatives Self-emulsifying drug delivery systems (SEDDS) with polyglycolyzed glycerides for 600 improving in vitro dissolution and oral absorption of lipophilic drugs. International journal of 601 pharmaceutics Self-emulsifying drug delivery systems: formulation and biopharmaceutic 603 evaluation of an investigational lipophilic compound Molecular Operating Environment (MOE), 2013.08. Montreal, QC, Canada: Chemical 606 Computing Group Inc Molecular modelling of mebendazole polymorphs as a 608 potential colchicine binding site inhibitor Structure of M pro from SARS-CoV-2 and discovery of its inhibitors Tanshinone IIA synergistically enhances the antitumor activity of doxorubicin 613 by interfering with the PI3K/AKT/mTOR pathway and inhibition of topoisomerase II: in vitro and 614 molecular docking studies Empagliflozin and Doxorubicin Synergistically Inhibit the Survival of Triple-616 Negative Breast Cancer Cells via Interfering with the mTOR Pathway and Inhibition of Calmodulin The performance of current methods in ligand-620 protein docking In vitro and in vivo evaluation of self-622 nanoemulsifying drug delivery systems of cilostazol for oral and parenteral administration. 623 International journal of pharmaceutics Design of a liquid nano-sized 625 drug delivery system with enhanced solubility of rivaroxaban for venous thromboembolism 626 management in paediatric patients and emergency cases The use of liquid self-microemulsifying drug delivery 629 systems based on peanut oil/tween 80 in the delivery of griseofulvin. Drug development and 630 industrial pharmacy Design and evaluation of microemulsions for improved 632 parenteral delivery of propofol Cold maceration application in red wine production and its 634 effects on phenolic compounds: A review. LWT Characterization of phenolic compounds in strawberry 636 (Fragaria× ananassa) fruits by different HPLC detectors and contribution of individual compounds 637 to total antioxidant capacity Crystal Violet Assay for Determining Viability of 640 Cultured Cells. Cold Spring Harbor protocols Flavonoids and phenolic acids: Role and biochemical 643 activity in plants and human Structure properties and mechanisms of action of naturally originated phenolic 645 acids and their derivatives against human viral infections Self-nanoemulsifying drug delivery systems of 648 tamoxifen citrate: design and optimization Self-dispersing lipid formulations for improving oral absorption of 651 lipophilic drugs Enhancement of oral absorption of curcumin by self-microemulsifying drug delivery 654 systems Repositioned Phenytoin for Healing of Corneal Ulcers Selection of bioavailability markers for herbal extracts based on in 658 silico descriptors and their correlation to in vitro permeability Formulation of simvastatin chitosan nanoparticles for controlled delivery in 661 bone regeneration: Optimization using Box-Behnken design, stability and in vivo study Size, stability, and porosity of mesoporous nanoparticles characterized 664 with light scattering. Nanoscale research letters Unexpected low-dose toxicity of the universal solvent DMSO Dimethyl sulfoxide (DMSO): a review. The Cornell Veterinarian Adverse reactions of dimethyl sulfoxide in humans: a systematic review Structure-based drug designing for potential antiviral activity of selected 672 natural products from Ayurveda against SARS-CoV-2 spike glycoprotein and its cellular receptor In-silico identification of potent inhibitors of COVID-19 main protease (Mpro) 675 and angiotensin converting enzyme 2 (ACE2) from natural products: Quercetin, hispidulin, and 676 cirsimaritin exhibited better potential inhibition than hydroxy-chloroquine against COVID-19 main 677 protease active site and ACE2 Naringenin, a flavanone with antiviral and anti-inflammatory effects: A 679 promising treatment strategy against COVID-19 a S: the score of a compound inside the protein binding pocket (Kcal/mol), b RMSD: The Root Mean Squared Deviation between the predicted pose and the crystal structure