key: cord-1047126-gqzk37aa
authors: refaat, Hesham; Mady, Fatma M.; Sarhan, Hatem A.; Rateb, Heba S.; Alaaeldin, Eman
title: Optimization and Evaluation of Propolis liposomes as a promising therapeutic approach for COVID-19
date: 2020-11-07
journal: Int J Pharm
DOI: 10.1016/j.ijpharm.2020.120028
sha: 8e70a1eb54194c3cd13c11bfecaa3405991edf76
doc_id: 1047126
cord_uid: gqzk37aa
The present work aimed to develop an optimized liposomal formulation for enhancing the anti-viral activity of propolis against COVID-19. Docking studies were performed for certain components of Egyptian Propolis using Avigan, Hydroxychloroquine and Remdesivir as standard antivirals against both COVID-19 3CL-protease and S1 spike protein. Response surface methodology and modified injection method were implemented to maximize the entrapment efficiency and release of the liposomal formulation. The optimized formulation parameters were as follow: LMC of 60 mM, CH% of 20% and DL of 5 mg/ml. At those values the E.E% and released % were 70.112 % and 81.801%, respectively with nanosized particles (117±11nm). Docking studies revealed that Rutin and Caffeic acid phenethyl ester showed the highest affinity to both targets. Results showed a significant inhibitory effect of the optimized liposomal formula of Propolis against COVID-3CL protease (IC50=1.183±0.06) compared with the Egyptian propolis extract (IC50=2.452±0.11), P< 0.001. Interestingly, the inhibition of viral replication of COVID-19 determined by RT_PCR has been significantly enhanced via encapsulation of propolis extract within the liposomal formulation (P<0.0001) and was comparable to the viral inhibitory effect of the potent antiviral (remdesivir). These findings identified the potential of propolis liposomes as a promising treatment approach against COVID-19.
Caffeic acid phenethyl ester (CAPE), Propolis-liposomes (PP-Lip), entrapment efficiency (%EE), Lipid molar concentration (LMC), cholesterol percentage (CH)% , drug loading (DL), deoxyribonucleotide triphosphates (dNTP), hydroxyquinone (HQ), receptor binding domain (RBD), RSM (response surface methodology).
Coronavirus 19 is the latest member of the coronavirus family that causes severe acute respiratory syndrome (SARS). However, it possess higher potential of infectivity and transmission than other SARS family members Zhu et al., 2020) .
Although ATP antagonists such as Remdesivir has been theoretically effective against the viral replication via RNA-dependent RNA polymerase (RdRP) inhibition (Gordon et al., 2020) , the concomitant general inhibition of other ATP-dependent enzymes like protein kinases and ATPases may result in numerous side effects. Therefore, the effective treatment of corona virus requires selective inhibition of certain host enzyme that is important for the viral replication with minimal effect on other enzymes which may affect the normal physiology of the host.
Fortunately, kinase PAK1 is a selective enzyme which is important for malarial and viral infection (Maruta, 2014) . Activation of PAK1 is responsible for viral infection, malarial infection, aging and even cancer (Maruta, 2014) .
Propolis or bee glue, a resinous material produced by bees to protect their hives, is rich in wide range of compounds such as flavonoids, polyphenolics, amino acids, resins and oils (Simone-Finstrom and Spivak, 2010) . Propolis is well-known for its antibacterial (Kujumgiev et al., 1999) , antiviral (Kujumgiev et al., 1999; Kumazawa et al., 2004) , anti-inflammatory (Banskota et al., 2001) and immunomodulatory effect (Marcucci, 1995) . Rutin, caffeic acid phenethyl ester, Quercetin, p-coumaric acid, benzoic acid, galangin, pinocembrin, chrysin, and Pinobankasin are among the active components responsible for the pharmacological effects of propolis (El Hady and Hegazi, 2002; Tolba et al., 2013; Lan et al., 2016; Badria et al., 2018) .
Caffeic acid phenethyl ester (CAPE), the major constituent of the Egyptian propolis, is one of PAK1 inhibitors which acts via the down regulation of RAC (a signaling protein in human cells) (Maruta and He, 2020) . In other words, CAPE is capable of blocking viral infection including corona virus and preventing coronavirus-induced lung fibrosis (Maruta, 2014; Maruta and He, 2020) .
Another mechanism that may be implicated in the anti-viral effect of propolis against COVID-19 is the improved inhibition potential of propolis components, rutin, myricetin and caffeic acid phenethyl ester , on ACE II receptors (Güler et al., 2020) . ACE II has been proven to be strongly recognized by SARS-CoV-2 than SARS CoV (Wan et al., 2020) , hence increasing the opportunity to be transmitted from person to person. Therefore, blocking ACE receptors has an essential role in treatment of SARS-CoV-2.
Collecting all those together, essentiates the need for a good delivery system for this promising natural product for the treatment of that pandemic disease. The efficient delivery of propolis may be hindered by the sticky and the resinous nature of the extract. Moreover, a special dosage form is required to deliver both the hydrophilic and the lipophilic contents of propolis extract. Therefore, this study aimed at optimizing a liposomal formulation for the efficient delivery of propolis components. To the best of our knowledge, it is the first study to formulate a nanocarrier dosage form to make the best use of propolis in treatment of COVID-19.
Lipoid S75 (70 % phosphatidylcholine-containing fat free soybean phospholipids) was kindly given by lipoid company (Germany). Alcoholic extract of Propolis (PE) was purchased from VACSERA-EGYPT (Cell Culture Department). Cholesterol was obtained from Fluka chemical co. (India). Ethanol (absolute) was obtained from El-Nasr Pharmaceuticals, (Egypt).
All chemicals and reagents were of analytical grade and purchased from (Sigma Aldrich).
In this study, some compounds detected in the ethanolic propolis extracts were used as ligands for COVID-19 3CL-protease (main protease) (PDB ID: 6LU7) (Jin et al., 2020) ; COVID-19 S1 spike protein subunit (PDB ID: 7BZ5) (Wu et al., 2020) ; as viral targets in order to evaluate their binding affinities and identify their inhibition activities and binding modes at the active site of each target. The crystal structure of them was downloaded from protein data bank web site. All bound water molecules, ligands and cofactors were removed from the protein. All components were constructed on ChemDraw 3D structures using ChemDraw 3D ultra 9.0 software then they were energetically minimized by using MM2, Jop
Type with 100 iterations and minimum RMS gradient of 0.01 and saved as MDL MolFile.
Docking studies were performed using Molsoft Internal Coordinate Mechanics (ICM) 3.4-8C
program as reported (https://www.rcsb.org/).
PP-Lip were prepared by spraying technique reported by Refaat et al (Refaat et al., 2019) , nevertheless we aimed to modify the formulation parameters to increase the entrapment efficiency and release of the prepared liposomes with maintaining small particle size in the nanoscale. Briefly, propolis, cholesterol and lipoid S75 were dissolved in the minimal volume of absolute ethanol then sprayed (40
L per second stirred at 1500 rpm at 80ºC) on the surface of sucrose-containing distilled water (9% w/v). After evaporation of ethanol by stirring, the spontaneously formed liposomes were kept overnight at 4°C for optimum annealing of the formed lipid bilayer (Fueldner, 1981) . Manual removal of the cooled aggregated free unencapsulated propolis at the surface of the liposomal suspension was carried out.
The percentage of entrapped content of flavonoids was measured exactly as reported by
Refaat et al (Refaat et al., 2019) . First, free drug was removed by centrifugation of liposomal suspension at 15000 rpm at 4º C for 2h. Separated liposomes were washed twice in distilled water to confirm the complete removal of free propolis. Liposomes were decomposed by sonication in absolute alcohol then vortexed to form homogenous suspension which was centrifuged at 15000 rpm for 30 minutes. The entrapped amount of flavonoids was calculated in 100µl of the separated supernatant by addition of 100µl of 10% alcoholic aluminum chloride.
The volume was completed to 2 ml using absolute alcohol; then, the absorbance was dignified
In vitro release of propolis from the prepared PP-Lip was studied using the semipermeable membrane which was immersed in 0.9 % sodium chloride solution one hour before use. Then the membrane was fitted on modified Franz cell with a reservoir compartment containing 20 ml phosphate citrate buffer containing 1 % tween 80 (pH 7.4) as a receptor media. Aliquots of PP-Lip formulations with equivalent drug content (1 mg) were added to the sample compartment.
The system was shaken in a thermostatic shaker at 37 ± 0.5 °C at 50 ± 10 rpm. Samples of 1 ml of the release medium were withdrawn at predetermined time intervals over a period of 6 hours and replaced with the same volume of fresh media maintained at the same temperature.
flavonoidal concentration was determined spectrophotometrically at 420 nm according to woisky and salatino (Woisky and Salatino, 1998) . All these experiments were accomplished in a triplicate manner, the average values were reported, and cumulative percentage of released flavonoids was calculated.
Lipid molar concentration, cholesterol % and flavonoidal loading were the three chosen factors to study their effect on the entrapment efficiency and release of the prepared PP-Lip.
The factors were analyzed at three levels, Lipid molar concentration (LMC) (80, 60, 40 mM), cholesterol percentage (CH)% (66, 43, 20%), and drug loading (DL) (5, 3.25, 1.5 mg) ( Table 1 ). The 17 formulations were prepared according to box Behnken design and the response surface diagram was assembled using Design Expert software, version 11 (StatEase®, Minneapolis, MN, USA) and were used to optimize a liposomal formulation of propolis extract.
To gain more insight into the effect of both propolis extract and the propolis liposomes on the inhibition of viral RNA 3CL-protease and consequently blocking viral replication, 3CL-Protease (SARS-CoV-2) Assay Kit was used . 3CL-protease inhibition was tested for propolis extract, propolis liposomes, solvent of the extract (alcohol) and Remdesivir as a positive control. 3CL-Protease in Assay buffer was diluted with 1 mM DTT at 3-5 ng/µl (90-150 ng per reaction). Thirty μl diluted 3CL-Protease enzyme solution was added to wells.
Ten µl Remdesivir (500 µM) was added to the wells of the positive control. The inhibitor solution was prepared by dilution in 1% DMSO. Ten µl inhibitor solution was added to each well of "Test Sample". Five percentage was added to "Blank" and "Positive Control" wells.
3CL-Protease enzyme was Pre-incubated with the inhibitor solution for 30 min at room temperature with slow shaking. Five mM 3CL-Protease substrate was diluted (1:20) in assay buffer with DTT, to make a 250 µM solution. Reaction was started by adding 10 µl of the substrate solution to each well. Plate was sealed and incubated overnight. fluorescence intensity was measured in a microtiter plate-reading fluorimeter (TECAN spark reader).
To evaluate the antiviral potential of propolis extract and liposomal propolis against corona virus, real time PCR using classical cell culture was adopted (Günther et al., 2004) .
Vero cells were seeded in a 24-well plate (4×104 /well). After twenty-four hours cells were infected with COVID-19 at a multiplicity of infection (MOI) of 0.01. One hour later, the inoculum was replaced by fresh medium containing predetermined concentrations of test compound (extract solvent (alcohol), lipids, propolis extract, propolis liposomes, Remdesivir as positive control). Percentage of inhibition of COVID-19 replication was determined via estimation of viral RNA concentration using RT-PCR (Drosten et al., 2003) . Briefly, A 25-μl reaction was maintained using 5 μl of RNA, 12.5 μl of 2 X reaction buffer solution introduced with the Superscript III one step RT-PCR system accompanied with Platinum Taq
To predict the antiviral activity of the components of the Egyptian propolis on a structural basis, automated docking studies were carried out using Molsoft ICM 3.4-8C program (https://www.rcsb.org/) the scoring functions and hydrogen bonds formed with the surrounding amino acids found in COVID-19 main protease and spike protein sequences are used to predict their binding modes, their binding affinities and orientation of these compounds at the active site of the single-crystal structures are available through the RCSB Protein Data Bank (PDB entries 6LU7, 7BZ5 respectively). The scoring functions of the compounds were calculated from minimized ligand-enzyme complexes.
The 3CL-protease (main protease or M pro ) in COVID is essential for the proteolytic maturation of the virus and has been examined as a potential target protein to prevent the spread of infection by inhibiting the cleavage of the viral polyprotein (Abagyan and Totrov, 1994) .
The discovery of the 3CL-protease structure in COVID-19 provides a great opportunity to identify potential drug candidates for treatment. Proteases represent potential targets for the inhibition of COVID replication. The 3CL-protease amino acids Thr24, Thr26, and Asn119 (Verma et al., 2020) . N3, the native ligand binds to amino acid residues PHE140, ASN142, GLU166, HIS163, HIS172, HIS41, MET49, TYR54, MET165, ASP187. MET165, LEU167, PHE185, GLN192, GLN189, PRO168, THR190, ALA191, THR24 and THR25 at the active site of main protease (Liu and Wang, 2020; Wan et al., 2020) . Other amino acid residues as LYS102, GLN110, THR111, ASP295, ASN151, ILE152, ASP153, SER158, PHE294, THR292, are participating in the interaction at the binding pocket of 6LU7 to FDA approved antiviral compounds and active phytochemicals (Chandel et al., 2020; Dayer, 2020; Liu and Wang, 2020 (Figure 1, Table 2 ).
The descending role of affinity to COVID-19 main protease is: Rutin > Caffeic acid phenethyl ester > Quercetin > Kaempferol > Pinobanksin > Galangin > Chrysin > p-cumaric acid > Benzoic acid. On comparison of these components of Egyptian propolis with Avigan, it is obviously observed that all propolis components have higher binding affinity than Avigan.
Interestingly, Rutin has showed higher affinity than HQ. Moreover, Rutin showed a perspective binding affinity comparable to the potent antiviral drug, Remdesivir.
The spike protein, S protein, is a class I fusion protein. Each S protomer consists of S1 and S2 domains with the receptor binding domain (RBD) located on the S1 domain (Narkhede et al., 2020) . S1 domain amino acid extended from 1 -745 where (RBD) located in it extended from 375 -604 (Han et al., 2017) . Amino acid residues of the SARS-CoV-2 RBD LYS417, GLY446, TYR449, TYR453, LEU455, PHE456, ALA475, PHE486, ASN487, LYS489, GLN493, GLY496, GLN498, THR500, ASN501, GLY502 and LYS505 Are the residues in contact to ACE2 (Lan et al., 2020) . On the other hand, amino acid residues in Contact between COVID-19 virus RBD and both heavy and light chains of B38 are reported (Jin et al., 2020) . In this study, we docked certain components of Egyptian propolis, Avigan, hydroxychloroquine and Remdesivir into the active site of S1 spike protein of COVID-19 (Figure 2, Table 3 ).
The spraying technique has successfully produced a nanosized uniform PP-Lip, with the least aggregation of free unencapsulated propolis during preparation and with high propolis entrapment and release percentage mostly above 50% for both entrapped and release percentage ( Table 4 ).
The effect of the three nominated factors was studied to optimize the formulation parameters for high entrapped and released flavonoidal content. The E.E% and released % were estimated for the 17 runs as the average of three replicates for each run and the results were provided in table 4.
Design expert software was used to analyze the measured data and the equations of regression were produced as follows:
Y F = X o + X 1 A + X 2 B + X 1 X 1 A 1 A 1 +X 2 X 2 B 2 B 2
Where Y F represents the independent variable, X o is the response of the arithmetic mean of the seventeen runs, and X 1 is the assessed coefficient factor for A. The average produced by changing a dependent variable once is represented by A and B. Non-linearity is estimated using Table 5 shows that ANOVA of model of regression for the E.E% of PP-Lip was estimated by F-test and p-value. The highly significant p-value (P=0.0003) and the p-value of lack-of-fit which is greater than 0.05 (0.5248) indicate the capability of the fitted regression equation in justifying and expecting the results.
Data obtained from the ANOVA was confirmed by studying the effect of each factor on the E.E % (Figure 4) . It is obvious that the results of individual effect of each factor confirmed the positive effect of LMC and DL on E.E%.
The positive significant interaction between LMC and DL shown by ANOVA was confirmed by the interaction plot (Figure 5) . The interaction plot showed slight increase in E.E% on increasing the LMC at lower level of DL (1.5 mg/ml). On the other hand, there was a significant increase in the E.E % on increasing LMC at higher level of DL (5mg/ml). This observation was also evident in the 3D plot of E.E % and its relationship with LMC and DL ( Figure 6 ) where E.E % reached to higher values at maximum levels of LMC and DL. this is in consistence with Mostafa et al (Mostafa et al., 2018) who stated the increased E.E% of thymoquinone due to increased LMC. This could be attributed to increased width of lipid bilayer membrane. Interestingly, Arafa et al (Arafa et al., 2018) stated the increased E.E% of propolis with increased DL which could be attributed to the enhanced capacity of the prepared nanostructure. Unlikely, Results showed that CH% had no effect on E.E % of the prepared PP-Lip. This was in contrast with Mclntosh et al (McIntosh, 1978) who stated that increasing CH% leads to increased E.E % due to increased particle size and width of the prepared liposomes to entrap more drug. This discrepancy may be due the small particle size of the prepared PP-Lip prepared by modified spraying technique. Table 4 shows the results of the release study for the 17 formulations. The release % of (Figure 7) . This emphasizes the significance of the model and efficiency of the equation to predict the release % within the factors' levels used in the experiments. ANOVA of regression model for the release % of PP-Lip revealed a highly significant p-value, P= 0.0003 ( Table 5 ). The p-value of lack-of-fit was greater than 0.05 (0.6627) indicating that the equation of the fitted regression was good and capable of elucidating and expecting the results. Results show that CH% (B) has a dramatic significant negative effect on release unlike LMC (A) which has a slight positive effect on release % while DL has no effect on the release % (Table 4, Figure 8 ). The 3D plot (Figure 9 ) approves the effect of the LMC and CH% on release %. At LMC 40 mM, increasing the CH% from 20% to 66% decreases the release % significantly from 77.5±2.73 to 27.5±2.3, respectively. At CH% equals 20%, the release % shows the highest values with varying LMC. The slight positive effect of LMC a on release % may be attributed to the enhanced engagement of the lipid bilayer to the structurally similar cell membrane. Increasing Cholesterol % has resulted in lower release % which could be attributed increasing the rigidity and decreasing the fluidity of the lipid bilayer (Gier et al., 1969; Niven and Schreier, 1990) . Reduction of cholesterol concentration makes the liposomes more fluid enhancing the drug release and so drug pharmacological effects (A Ghaffar et al., 2017) .
The preparation conditions were set in the box-behnken design to formulate PP-Lip with the highest E.E% and release %. The optimized formulation parameters were as follow: LMC of 60 m.mole, CH% of 20% and DL of 5 mg/ml. At those values the E.E% and released % were 70.112 % and 81.801%, respectively. Five verification experiments were carried out at these optimium conditions to ensure the model capablility to optimize the conditons as directed. The E.E% and released % were 68+2.4% and 76+3.2 %, respectively (Figure 10) . Release pattern follows the 1 st order kinetics with R 2 0.992. Furthermore, the Particle size of the optimized PP-Lip was 117+11nm (Figure 11) . These results indicate that the optimization process is successful in practical lab work .
3CL protease has been an important target for the prevention of the replication of corona virus especially that it has not been found in hostcells (Akaji et al., 2011) . Results show that propolis extract possess a good inhibitory effect against covid-3CL-protease (IC50=2.452±0.11) (Figure 12) . That could be justified by binding of the studied propolisflavonoids to the active site of 3CL-protease. Competitive inhibition of the protease resulted from strong binding to such flavonoids could result in blocking the enzyme activity (Pillaiyar et al., 2016) . Fortunately, that inhibitory effect was significantly enhanced via the encapsulation of the extract within the optimized liposomal formulation (IC50=1.183±0.06), P<0.001. We believe that the prepared liposomal system had the potential to flawlessly introduce both the hydrophilic and the lipophilic components of the extract via an enhanced surfaced surface area with avoiding the sticky nature of the extract.
To gain more insight into the effect of the formulation of the propolis extract on the enhancement of the anti-viral effect on COVID-19 virus, viral replication was determined using RT-PCR. Results show the great impact of the optimized propolis liposomal formulation in enhancing the inihbitory effect of the encasulated propolis against covid viral replication compared to the unformulated propolis extract (87.9±1.2, 72.4±0.5, respectively) (P<0.0001).
We suppose that encapsulation of propolis within an optimized liposomal system would enhance cell permeability of propolis components due to increased surface area available for endocytosis and the silmilarity of lipid structure of the liposomal membrane to cell membrane.
Moreover, the liposomal system is capable of introducing both the hydrophilic and the lipophilic components of propolis for cellular uptake avoiding retaining of lipophilic components within the liposomal mebrane and/or poor cellular uptake of the hydrophilic components of propolis extract.
Interestnigly, the optimized propolis liposomes has comparatively inhibited the replication of human corona virus such as remedsivir, antiviral drug with reported promising in vitro inhibitory effect on covoid-19 (Hashemian et al., 2020) (87.9±1.2, 91.2 ± 2.5, respectively), Figure 13 .
The present study has revealed the anti-viral potential of flavonoidal components of Egyptian propolis. Molecular docking has shown that all propolis components have high binding affinity to COVID 3-CL protease and spike protein compared to Avigan, hydroxychloroquine (HQ) and Remdesivir. An optimized liposomal formulation could guarantee both the enhanced delivery to the target cells and the improved cellular uptake of encapsulated propolis. To the best of our knowledge, that has been the first study that estimates the effect of a nanocarrier dosage form on the enhancement of the anti-viral effect of Egyptian propolis extract against Covid-19. Further clinical studies are being carried out to estimate the effeciency of the optimized formulation against COVID-19.
The authors declare no conflict of interest. 6. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, ornot-for-profit sectors.
Authors declare no conflict of interest
The role of size in development of mucosal liposome-lipopeptide vaccine candidates against group A Streptococcus
Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins
Structure-based design, synthesis, and evaluation of peptide-mimetic SARS 3CL protease inhibitors
Propolisbased niosomes as oromuco-adhesive films: A randomized clinical trial of a therapeutic drug delivery platform for the treatment of oral recurrent aphthous ulcers
Chemical and biological diversity of propolis samples from Bulgaria, Libya, and Egypt
Recent progress in pharmacological research of propolis
Silico Identification of Potent COVID-19 Main Protease Inhibitors from FDA Approved Antiviral Compounds and Active Phytochemicals through Molecular Docking: A Drug Repurposing Approach
Old drugs for newly emerging viral disease, COVID-19: Bioinformatic Prospective
Identification of a novel coronavirus in patients with severe acute respiratory syndrome
Egyptian propolis: 2. Chemical composition, antiviral and antimicrobial activities of East Nile Delta propolis
Characterization of a third phase transition in multilamellar dipalmitoyllecithin liposomes
The role of cholesterol in lipid membranes
The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus
Investigation of potential inhibitor properties of ethanolic propolis extracts against ACE-II receptors for COVID-19 treatment by Molecular Docking Study
Application of real-time PCR for testing antiviral compounds against Lassa virus, SARS coronavirus and Ebola virus in vitro
Structure of the S1 subunit C-terminal domain from batderived coronavirus HKU5 spike protein
A Review on Remdesivir: A Possible Promising Agent for the Treatment of COVID-19
Potential of coronavirus 3C-like protease inhibitors for the development of new anti-SARS-CoV-2 drugs: Insights from structures of protease and inhibitors
Structure of M pro from SARS-CoV-2 and discovery of its inhibitors
Antibacterial, antifungal and antiviral activity of propolis of different geographic origin
Antioxidant activity of propolis of various geographic origins
Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor
The natural flavonoid pinocembrin: molecular targets and potential therapeutic applications
Potential inhibitors against 2019-nCoV coronavirus M protease from clinically approved medicines
The reproductive number of COVID-19 is higher compared to SARS coronavirus
Effect of cholesterol and ethanol on dermal delivery from DPPC liposomes
Propolis: chemical composition, biological properties and therapeutic activity
Herbal therapeutics that block the oncogenic kinase PAK1: a practical approach towards PAK1-dependent diseases and longevity
PAK1-blockers: Potential Therapeutics against COVID-19
The effect of cholesterol on the structure of phosphatidylcholine bilayers
Optimization and characterization of thymoquinone-loaded liposomes with enhanced topical antiinflammatory Activity
The molecular docking study of potential drug candidates showing anti-COVID-19 activity by exploring of therapeutic targets of SARS-CoV-2
Nebulization of liposomes. I. Effects of lipid composition
An overview of severe acute respiratory syndrome-coronavirus
3CL protease inhibitors: peptidomimetics and small molecule chemotherapy
Modified spraying technique and response surface methodology for the preparation and optimization of propolis liposomes of enhanced anti-proliferative activity against human melanoma cell line A375
Propolis and bee health: the natural history and significance of resin use by honey bees
Caffeic acid phenethyl ester, a promising component of propolis with a plethora of biological activities: A review on its anti-inflammatory, neuroprotective, hepatoprotective, and cardioprotective effects
Potential inhibitors of SARS-CoV-2 Main protease (Mpro) identified from the library of FDA approved drugs using molecular docking studies
Receptor recognition by the novel coronavirus from Wuhan: an analysis based on decadelong structural studies of SARS coronavirus
Analysis of propolis: some parameters and procedures for chemical quality control
A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2
A novel coronavirus from patients with pneumonia in China
LMC: lipid molar concentration, CH%: cholesterol percentage, DL: drug loading