Lichens are a source of secondary metabolites with significant pharmacological potential. Data regarding their possible application in glioblastoma (GBM) treatment are, however, scarce. The study aimed at analyzing the mechanism of action of six lichen secondary metabolites: atranorin, caperatic acid, physodic acid, squamatic acid, salazinic acid, and lecanoric acid using two- and three-dimensional GBM cell line models. The parallel artificial membrane permeation assay was used to predict the blood-brain barrier penetration ability of the tested compounds. Their cytotoxicity was analyzed using the MTT test on A-172, T98G, and U-138 MG cells. Flow cytometry was applied to the analysis of oxidative stress, cell cycle distribution, and apoptosis, whereas qPCR and microarrays detected the induced transcriptomic changes. Our data confirm the ability of lichen secondary metabolites to cross the blood-brain barrier and exert cytotoxicity against GBM cells. Moreover, the compounds generated oxidative stress, interfered with the cell cycle, and induced apoptosis in T98G cells. They also inhibited the Wnt/β-catenin pathway, and this effect was even stronger in case of a co-treatment with temozolomide. Transcriptomic changes in cancer related genes induced by caperatic acid and temozolomide were the most pronounced. Lichen secondary metabolites, caperatic acid in particular, should be further analyzed as potential anti-GBM agents. Lichens are a source of ~1000 unique secondary metabolites of various chemical structures, including aliphatic, cycloaliphatic, aromatic, and terpenic compounds . These sGlioblastoma (GBM) is the most common and devastating primary malignant brain tumor in adults. It has the astrocytic lineage and is classified as a WHO grade IV tumor . With a Wnt/β-catenin pathway hyperactivation is one of the most important contributors to the malignant phenotype of GBM cells ,9,10. PhHypogymnia physodes), evernic acid (isolated from Evernia prunastri), salazinic acid (derived from Parmelia sulcata), and (−)-usinc acid  possess the anti-cancer properties and decrease the viability of GBM cells [C. uncialis is also the source of squamatic acid, which has not been analyzed in the context of GBM so far. The data about atranorin and caperatic acid, both derived from Platismatia glauca, and lecanoric acid from Hypocenomyce scalaris, in regard to GBM treatment, are also scarce.In our previous studies, we discovered that lichen-derived extracts as well as lichen secondary metabolites, such as physodic acid , Dulbecco’s modified Eagle medium (DMEM), and all other media components, including antibiotics solution , glutamine, nonessential amino acids (NEAA), and sodium pyruvate (NaP), were obtained from Merck . Fetal bovine serum (FBS) was obtained from Biowest . Other compounds used in the study, e.g., dimethylsulfoxide (DMSO), ribonuclease A (RNase A), 3--2,5-diphenyltetrazolium bromide (MTT), trypsin, Tris, PKF118–310, temozolomide (TMZ), and others were purchased from Merck .Cladonia uncialis, Hypocenomyce scalaris, and Hypogymnia physodes were collected from Jastrzębsko Stare ((52°17′56″ N 16°03′37″ E), Greater Poland, Poland (June 2018); Parmelia sulcata was found in Podlesice (50°33′49″ N 19°32′02″ E), Silesian region, Poland (July 2018); whereas Platismatia glauca originated from the forest belonging to the Piła commune , Greater Poland, Poland (October 2018). The lichens were authenticated by Dr. Daria Zarabska-Bożejewicz from the Institute for Agricultural and Forest Environment of Polish Academy of Sciences in Poznan and Dr. Wojciech Gruszka from the Department of Biological Sciences, Poznan University of Physical Education. A voucher specimen  was deposited in the herbarium of the Department of Pharmacognosy at Poznan University of Medical Sciences.Manual collection of the examined lichens was performed: P. glauca, 12.0 g for H. physodes, 33.5 g for C. uncialis, 6.0 g for P. sulcata, and 1.5 g for H. scalaris). For P. glauca, the extraction with shaking at room temperature was effectuated. Hexane (eight times with 200 mL) , followed by diethyl ether (five times with 200 mL) , was used. H. physodes was extracted under the same experimental conditions but using hexane (five times with 200 mL) and then acetone (three times with 150 mL)  as extractants. The thallus of C. uncialis, P. sulcata, and H. scalaris were extracted using an ultrasound bath at 35–40 °C. For C. uncialis, hexane (five times with 150 mL), followed by acetone (six times with 150 mL), was used. For P. sulcata, the hexane (three times with 80 mL) and acetone (fourteen times with 50 mL) were applied, and for H. scalaris, acetone extraction only was carried out (six times with 20 mL). Next, the extracts obtained with one kind of solvent were poured together and filtered using Whatman filter paper No. 1. A concentration under a vacuum at 35–40 °C (until dried) was effectuated next. The extracts were obtained with different yields: 0.8% for hexane and 4.9% for acetone extract from P. glauca, 0.3% for hexane and 6.5% for acetone extract from H. physodes, 0.9% for hexane and 0.8% for acetone extract from C. uncialis, 1.8% for hexane and 4.7% for acetone extract from P. sulcata, and 18.9% for acetone extract from H. scalaris.Next, the lichen-derived extracts were prepared by gradually extracting the dried, cleaned, and fragmented thallus . The method was performed to obtain caperatic acid with a yield of 35%. Acetone extract from H. physodes was used to obtain the physodic acid. Thus, physodic acid was isolated by applying silica column chromatography  using the increasing gradient mixtures of solvents toluene-ethyl acetate 110:0 to 100:10 as the mobile phase. The compound was obtained with a yield of 4%. Squamatic acid was obtained from the C. uncialis acetone extract by the spontaneous crystallization during evaporation of acetone with a yield of 33%. Salazinic acid was obtained by recrystallizing the P. sulcata acetone extract from the acetone: water (8:2) mixture with a yield of 42%. Lecanoric acid was isolated from the H. scalaris acetone extract, using preparative thin layer chromatography . The identity of the compounds was confirmed using UV, 1H NMR, 13C NMR, and MS analysis, and the obtained data were compared to the published values [Atranorin was purchased from ChromaDex , but all other lichen secondary metabolites were isolated from the extracts as previously described . Brieflyd values . In addid values  or thin eP) of the compounds was calculated by using the following equation:eP is the effective permeability coefficient (cm/s), DV—donor volume, AV—acceptor volume, eqC—equilibrium concentration,S—membrane area, and t—incubation time (in seconds) [The parallel artificial membrane permeability assay (PAMPA) for the blood–brain barrier (BBB)  was used to analyze whether the lichen secondary metabolites tested in this study were able to cross the BBB. The stock solutions (5 mg/mL) of tested compounds were prepared using DMSO. Next, the donor solution (24.75 µg/mL) using Prisma buffer  were made. Firstly, 180 µL of the donor solution were added to the donor wells. Then the filter membranes were coated with 5 μL BBB-1 lipid solution  and the acceptor wells were filled with 200 µL BSB . The plates were sandwiched together and incubated for 4 h at 37 °C . The results were determined by spectroscopic analysis , with the appropriate wavelength for a given compound . The amount of permeated active compounds was calculated using the standard curves prepared for each tested compound. The theophylline and lidocaine were used as the negative and positive controls, respectively. Effective permeability (seconds) .eP (×10−6 cm/s) > 1.5 were classified as high permeation predicted, while eP (×10−6 cm/s) < 1.5 were classified as low permeation predicted [Compounds with redicted ,18. Samp6-methylguanine-DNA methyltransferase (MGMT), the key factor determining the response to alkylating agents, such as TMZ, is expressed in T98G and U-138 MG cells, whereas in A-172 the expression of MGMT is silenced by promoter methylation [Three human GBM cell lines were used in this study, i.e., A-172 (ATCC-CRL-1620), T98G (92090213), and U-138 MG (ATCC-HTB-16). The A-172 and U-138 MG cell lines were purchased from the American Type Culture Collection (ATCC), whereas the T98G cell line was obtained from the European Collection of Authenticated Cell Cultures (ECACC). It has been confirmed by us that Ohylation . TherefoThe cells were grown as monolayers (2D cell culture) in the recommended media: A-172 were grown in the ATCC-formulated DMEM , whereas T98G and U-138 MG cell lines were grown in the ATCC-formulated EMEM . These media were supplemented with FBS  to a final concentration of 10%, as well as antibiotics (penicillin and streptomycin)  to the final concentrations of 1%. For the experiments, the amount of FBS was reduced to 5%. The medium for the T98G cell line was supplemented with 2 mM glutamine, 1% nonessential amino acids, and 1% sodium pyruvate .®, Corning, NY, USA) (1000 cell per well) to form spheroids. The standard medium was supplemented with Corning® Matrigel® Matrix for Organoid Culture (3.5%)  and epidermal growth factor—EGF (10 ng/mL) . Both 2D and 3D cell cultures were grown in a standard humidified incubator  at 37 °C in 5% CO2 and 95% air atmosphere.Moreover, the 3D culture of the T98G cell line was also established. The cells were cultured in ultra-low attachment microplates , we performed the MTT test. The procedure of the test was described previously . BrieflyMGMT expression and methylation status, i.e., TMZ 30 µM was added to the A-172 cell line, and TMZ 100 µM was added to both the T98G and U-138 MG cell lines. In the case of the 2D culture, the cells were treated with the compounds 24 h after seeding, whereas, in regard to 3D culture, 5 days were needed to form spheroids. All subsequent analyses were performed 48 h after treatment.Based on the MTT test, the lichen secondary metabolite concentrations allowing the survival of more than 70% of cells were chosen for the subsequent experiments. The compounds were added as follows: A-172 cells—atranorin 25 µM, caperatic acid 50 µM, physodic acid 25 µM, squamatic acid 25 µM, salazinic acid 50 µM, and lecanoric acid 100 µM; T98G cells—atranorin 50 µM, caperatic acid 100 µM, physodic acid 25 µM, squamatic acid 50 µM, salazinic acid 100 µM, and lecanoric acid 100 µM; U-138 MG cells—atranorin 25 µM, caperatic acid 50 µM, physodic acid 25 µM, squamatic acid 50 µM, salazinic acid 50 µM, and lecanoric acid 100 µM. As far as the TMZ treatment is concerned, it was added in the concentrations depending on the TBP), a component of the DNA-binding protein complex TFIID, served as the endogenous RNA control. The results were expressed as N-fold differences in the gene expression relative to the TBP gene. The ΔΔCt method was used for fold-change calculation. All samples were analyzed in triplicate, and the reaction was repeated at least twice.The Universal RNA Purification Kit  was used for total RNA isolation from the A-172, T98G, and U-138 MG cells, and the Revert-Aid First Strand cDNA Synthesis kit  was chosen for cDNA synthesis. Quantitative real-time PCR (qPCR) was performed using Hot FIREPol EvaGreen qPCR Mix Plus  and LightCycler 96 . Primer sequences were previously published ,20 and t® Oxidative Stress Kit . Briefly, 100,000 cells per well were seeded on 6-well plates and after a 24 h incubation they were treated with the analyzed lichen secondary metabolites in the concentrations allowing >70% of cell survival; cells treated with DMSO were used as the negative control. After 48 h incubation, the cells were trypsinized, washed with PBS buffer, and resuspended in assay buffer containing Working Solution of Muse® Oxidative Stress Reagent. The cells were incubated at 37 °C for 30 min, and then run on Muse® Cell Analyzer . The obtained data were evaluated using Muse® Analysis Software ver. 1.5 .The intracellular detection of superoxide radicals was detected by flow cytometric analysis after the staining with dihydroethidium, using The MuseThe Muse Cell Cycle Kit and the Muse Cell Cycle Software  were used to quantitatively measure the percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle. The assay utilizes the nuclear DNA intercalating stain propidium iodide and RNAse A in a formulation. Propiodium iodide discriminates cells at different stages of the cell cycle, based on differential DNA content, while the presence of RNAse increases the specificity of DNA staining. Briefly, the cells were seeded on 6-well plates  and incubated for 24 h. Afterward, the lichen secondary metabolites were added to the concentrations chosen based on the MTT test, allowing >70% of cell survival. 0.5% DMSO and 100 nM topotecan were used as negative and positive controls, respectively. The analysis was performed after a 48 h incubation with lichen secondary metabolites and started with washing the cells with PBS, followed by cell fixation with ice-cold ethanol and overnight incubation at −20 °C. Following steps included cell re-washing with PBS, then a 30 min incubation in the dark with propiodium iodide-RNAse A formulation, and a final analysis on Muse™ Cell Analyzer .® Caspase-3/7 Kit was performed in order to analyze the percentage of live, early, and late apoptotic; total apoptotic; and dead cells in A-172, T98G, and U-138 MG GBM cells treated with lichen secondary metabolites. The principle of the method relies on the measurement of the activity of executioner caspases, i.e., caspase-3 and caspase-7, as well as analyzing the integrity of the cell membrane with the use of a dead cell marker 7-AAD. Cell seeding and treatment followed the same protocol as for cell cycle or ROS generation analysis, while the subsequent procedures were performed according to the manufacturer’s recommendations.The fluorescent-based analysis of apoptosis using MuseAfter 5 days, the spheroids were fixed in 4% phosphate-buffered formalin, embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin (H&E), according to routinely used Department of Histology and Embryology protocols, to visualize cellular content. They were subsequently analyzed under a light microscope.The total RNA from lichen secondary metabolites and TMZ supplemented spheroids culture was isolated with the RNeasy MinElute, according to the manufacturer’s instructions . The RNA for transcriptome study was received from three independent replicates for each experimental variant: control (1), TMZ 100 µM (2), atranorin 25 µM + TMZ 100 µM (3), caperatic acid 50 µM + TMZ 100 µM (4), physodic acid 25 µM + TMZ 100 µM (5), squamatic acid 25 µM + TMZ 100 µM (6), salazinic acid 50 µM + TMZ 100 µM (7), and lecanoric acid 100 µM + TMZ 100 µM (8), and then pooled together into a single sample per group.The complete procedure for preparing RNA for hybridization was performed using the GeneChip WT PLUS Reagent Kit . The detailed procedure was described earlier . First, p-values of selected GO terms were corrected using Benjamini-Hochberg correction. In similar way, the “RDAVIDWebService” library was carried out to analyze enrichment of the KEGG (Kyoto Encyclopedia of Genes and Genomes) signaling pathway database. The associations of individual DEGs with relevant KEGG signaling pathways were presented using Cytoscape (v. 3.7.2) software [The obtained CEL files were further analyzed using the R statistical language and Bioconductor package, including the relevant Bioconductor libraries. For the normalization, background correction, and calculation of the expression values of the examined genes, the robust multiarray average (RMA) normalization algorithm implemented in the “Affy” library was applied . A complsoftware .p < 0.05 considered as significant was applied for the PAMPA-BBB assay.The statistical significance between the experimental groups and their respective controls  was assessed using Student’s t test, while ANOVA with Duncan’s multiple range post hoc test  variance analysis with e coefficient, followed by atranorin and lecanoric acid.The PAMPA-BBB assay revealed that all analyzed lichen secondary metabolites, except squamatic acid and salazinic acid, can cross the BBB . In thisMGMT methylation status nor the sensitivity to TMZ. Among the four compounds analyzed in this study, the most cytotoxic were squamatic acid and atranorin, which reduced the number of living cells in the whole analyzed concentration range, i.e., 1–100 µM . Importantly, caperatic acid in the highest tested concentration also had a significant impact on GBM cell viability. In the A-172 cell line, it reduced the viability of cells to only 11.67 ± 5.51%. Lecanoric acid was, on the other hand, not cytotoxic; only the A-172 cell line was sensitive to its maximal tested concentration (100 µM), which killed ~30% of cells. Referring to the previously published results of physodic acid, its cytotoxicity was remarkable, and the U-138 MG cell line was the most prone to its cytotoxic influence [50 values of the analyzed compounds together with their chemical structures are presented in Here, as shown in nfluence . SalazinOne of the most important features of anticancer agents is the ability to generate oxidative stress. The results of our study show that physodic and squamatic acids induce oxidative stress in the T98G cell line . HoweverAxin2, c-MYC, CCND1, BIRC5, and NEDD9. The results shown in Axin2, its expression was mostly affected in the A-172 and U-138 MG cell lines. In this context, atranorin, caperatic, and squamatic acids downregulated its expression in both cell lines; in U-138 MG physodic acid also decreased Axin2 expression. As far as c-MYC expression is concerned, we observed either no impact of lichen secondary metabolites on its mRNA level, or rather the upregulation; in this context lecanoric acid upregulated its expression in A-172 cell line, while salazinic acid did so in T98G cell line. In the latter cell line, only physodic acid downregulated c-MYC expression, while for atranorin and squamatic acid we observed a trend towards downregulation, but with no statistical significance. However, it has to be noted that in both A-172 and T98G cell lines even PKF118-310 did not diminish the level of c-MYC oncogene. Furthermore, we also analyzed CCND1 expression, as this master cell cycle regulator is also one of the Wnt pathway target genes. Here the most profound effects were observed in the U-138 MG cell line, where physodic and salazinic acids downregulated CCND1 expression by around half. Additionally, atranorin and squamatic acid exhibited similar downregulatory effects in the T98G cell line. Importantly, salazinic acid significantly upregulated CCND1 expression in the latter cell line. The next gene analyzed was BIRC5, encoding survivin, the key apoptosis inhibitor. Here the most profound effects were observed in the T98G cell line, where physodic and salazinic acids downregulated BIRC5 expression, similarly to what was observed for PKF118-310. Moreover, squamatic and salazinic acids diminished its expression in the A-172 and U-138 MG cell lines, respectively. Finally, NEDD9 was also analyzed, as it regulates signaling complexes important in cell attachment, migration, and invasion as well as apoptosis and the cell cycle. Our study shows, that caperatic acid downregulated NEDD9 expression in all analyzed cell lines. On the other hand, the effects of physodic acid treatment in the context of NEDD9 expression were cell line dependent; in the A-172 cell line it downregulated, while in U-138 MG it upregulated its transcript level. Moreover, we also observed the downregulation of NEDD9 after the treatment with squamatic acid in T98G cells.Since Wnt/β-catenin signaling is regarded as a therapeutic target for GBM , we deciReduced cell viability can also be mediated by the cell cycle arrest and apoptosis induction. Therefore, we investigated these two phenomena using flow cytometry analysis after 48 h of treatment  was also observed. Detailed results are presented in The analysis performed on the A-172 cell line revealed the downregulation, or a trend towards downregulation, of β-catenin and the genes controlled by the β-catenin/TCF/LEF transcriptional complex as a response to the treatment with lichen secondary metabolites and TMZ. Mostly, the mRNA level of Axin2, was also observed). Also, squamatic acid and lecanoric acid treatment with TMZ led to the decrease of Wnt pathway target genes expression. In the case of squamatic acid, the downregulated genes were as follows: CTNNB1, c-MYC, CCND1, and NEDD9; while concerning lecanoric acid, these were CTNNB1, Axin2, c-MYC, and CCND1. Atranorin and TMZ decreased the expression of only β-catenin, caperatic acid and TMZ reduced the expression of CCND1, while physodic acid and TMZ did not downregulate any genes.As far as the T98G cell line is concerned, the best results were obtained for salazinic acid, which downregulated five out of six analyzed genes , 161 (atranorin 25 µM + TMZ 100 µM), 530 (caperatic acid 50 µM + TMZ 100 µM), 58 (physodic acid 25 µM + TMZ 100 µM), 76 (squamatic acid 25 µM + TMZ 100 µM), 28 , and 79 (lecanoric acid 100 µM + TMZ 100 µM) and upregulated: 15 (TMZ 100 µM), 34 (atranorin 25 µM + TMZ 100 µM), 54 caperatic acid 50 µM + TMZ 100 µM, 28 (physodic acid 25µM + TMZ 100 µM), 33 (squamatic acid 25 µM + TMZ 100 µM), 34 , and 28 (lecanoric acid 100 µM + TMZ 100 µM).The T98G spheroids were exposed to the influence of lichen secondary metabolites for 48 h and subjected to microarray analysis. A pairwise scatter plot analysis was performed to determine the general profiles of the whole gene expression in the control (1), TMZ 100 µM (2), atranorin 25 µM + TMZ 100 µM (3), caperatic acid 50 µM + TMZ 100 µM (4), physodic acid 25µM + TMZ 100 µM (5), squamatic acid 25 µM + TMZ 100 µM (6), salazinic acid 50 µM + TMZ 100 µM (7), and lecanoric acid 100 µM + TMZ 100 µM (8) experimental groups; this pairwise analysis revealed the patterns of upregulated and downregulated genes compared to the control A. Each cDifferentially expressed genes from each group  were then assigned to Gene Ontology (GO). The GO analysis showed that the addition of lichen secondary metabolites and TMZ alters the expression of certain genes that play an essential role in the regulation of signaling pathways, inter alia: “positive regulation of cell proliferation”, “double−strand break repair via nonhomologous end joining”, extracellular matrix organization”, “cell migration”, “cell adhesion”, “protein deubiquitination”, “ubiquitin−dependent protein catabolic process”, “transforming growth factor beta receptor signaling pathway”, “nucleosome assembly”, and “regulation of actin cytoskeleton organization”. Note that the variant, caperatic acid 50 µM + TMZ 100 µM vs. control, resulted in the greatest number of altered biological processes B.Due to the structure of the GO database, single genes can often be assigned to many ontological terms. For this reason, the relationship between genes and GO terms were mapped with circos plots, with visualization of logFC values and gene symbols. All of those genes were either upregulated (green color) or downregulated (red color) C in the p < 0.05). Differentially expressed genes belonging to these pathways were assigned to a predetermined color scale, which was subsequently imposed on the gene/protein symbol field  database. This analysis allowed us to determine the biological pathways “Cell cycle”, “TGF-beta signaling pathway”, “Wnt signaling pathway”, “MAPK signaling pathway”, “Focal adhesion”, and “Focal adhesion”, which involve a significant enrichment of differentially expressed genes in the examined groups , C. uncialis (squamatic acid), H. physodes (physodic acid), P. sulcata , and P. glauca (caperatic acid) were analyzed. We also analyzed atranorin, which is present in many lichen species [Therefore, in this study we examined the anti-GBM effects as well as the underlying molecular mechanisms, including the impact on Wnt/β-catenin and other cancer-related signaling pathways of lichen secondary metabolites used as a single compound or combined with TMZ. To the best of our knowledge, such a therapeutic approach has never been studied before. The metabolites derived from  species . AtranorThe initial obstacle that therapies against GBM must overcome is the BBB. Our results show that the permeability coefficient of all the analyzed compounds, except salazinic acid, was high enough to treat these compounds as BBB permeable. This opens up the pathway for their simple delivery to the CNS. In the case of salazinic acid, novel drug delivery systems, such as ligand-anchored dendrimers that utilize receptor-mediated transcytosis should be taken into consideration .50 concentration of 5.36  ±  0.85 μM and 7.55  ±  1.2 μM, respectively [Our previous studies confirmed the cytotoxic potential of physodic acid and salazinic acid ,14. Hereectively . The cytectively  or melanectively . Importaectively ,35,36.CD44, CCND1, and c-MYC in lung cancer cells [Our previous studies revealed that lichen secondary metabolites, especially caperatic acid and physodic acid, exert anticancer properties, inhibiting Wnt/β-catenin pathway in colorectal cancer cells ,37. Moreer cells . To the er cells . Taking er cells , lichen Pseudevernia furfuracea and its major constituent, physodic acid, resulted in intrinsic caspase-dependent cell death induction, which was associated with increased oxidative stress, DNA damage, and cell cycle arrest [P. furfuracea and physodic acid treatment [Flavocetraria cucullata exhibited the most potent cytotoxicity and pro-apoptotic effect in several human cancer cell lines and usnic acid, but also salazinic acid, squamatic acid, baeomycesic acid, d-protolichesterinic acid, and lichesterinic acid were found as major subcomponents of its acetone extracts [Since Wnt signaling and apoptosis are interconnected, we assumed that lichen secondary metabolites might also induce apoptosis in GBM cells. This was found to be true in the case of the T98G cell line, but not in two other cell lines. Among all the compounds analyzed, physodic acid was found to induce the strongest pro-apoptotic effects, leading to almost 80% of apoptotic cells detected following 48 h treatment. Similar results were also obtained by Cardile et al., who found that physodic acid activated an apoptotic process, probably involving the reduction of Hsp70 expression, in A375 melanoma cancer cells . In anote arrest . The autreatment . In our extracts . Moreoveextracts . Harikriextracts . These rFOSL1, another of its target genes. Additionally, TGF-β and MAPK signaling pathways were identified as additional targets of lichen secondary metabolites combined with TMZ. This is an important finding, as TGF-β and MAPK pathways play critical roles in cell cycle regulation, as well as in tumor formation and metastasis [CCND1 expression in GBM cells [Additional lichenochemical target genes and signaling pathways were also revealed by our microarray analysis. Since spheroids mimic the in vivo behavior of cells better than monolayer culture, we decided to establish a 3D culture of the T98G cell line. This cell line was chosen for the spheroid formation for three reasons. First, because it was able to grow in a 3D culture; next, because it seemed the most prone to lichenochemical treatment, based on our ROS generation, cell cycle and apoptosis analysis; and finally, because it is TMZ resistant and exemplifies the most challenging tumor. Interestingly, our transcriptome profiling showed the most promising anti-GBM properties of the combination of caperatic acid and TMZ. Treatment with these two compounds resulted in the most significant downregulatory transcriptomic changes in cancer-related genes involved in e.g., vasculogenesis, TGF-β signaling, or positive regulation of proliferation, among others. Moreover, genes involved in cell adhesion, cell migration, and cell-matrix adhesion were found to be downregulated after the treatment with caperatic acid and TMZ. KEGG analysis also confirmed that all lichen secondary metabolites, but in particular caperatic acid and TMZ, interfere with the Wnt pathway, downregulating tastasis . It has BM cells . Indeed,BM cells . The intBM cells . FurtherWe conclude that lichen secondary metabolites, in particular caperatic acid, but also atranorin, physodic acid, squamatic acid, salazinic acid, and lecanoric acid, ameliorate the response of GBM cells to TMZ treatment, in particular due to the Wnt pathway inhibition mechanism. We also showed that lichen secondary metabolites were able to generate oxidative stress, interfered with the cell cycle distribution, and induced apoptosis in the TMZ-resistant T98G cell line. The co-treatment of GBM cells with caperatic acid and TMZ resulted in the most significant and potentially therapeutic transcriptomic changes. Further studies are required to determine the potential clinical application of lichen secondary metabolites in GBM therapy. The safety of a co-treatment with TMZ for normal astrocytes and neurons should be evaluated and the best drug delivery system selected. Recognition of the subpopulation of GBM patients who would benefit from adjuvant lichen secondary metabolites therapy seems crucial. However, the good anti-GBM profile and BBB permeability of those compounds give hope for their successful implementation into treatment.


Secondly, the problems of these TMOs in practical application are presented and the corresponding feasible solutions are clarified. Then, we summarize the latest developments of the six TMOs for supercapacitor electrodes. Finally, we discuss the developing trend of supercapacitors and give some recommendations for the future of supercapacitors.In the past decades, the energy consumption of nonrenewable fossil fuels has been increasing, which severely threatens human life. Thus, it is very urgent to develop renewable and reliable energy storage devices with features of environmental harmlessness and low cost. High power density, excellent cycle stability, and a fast charge/discharge process make supercapacitors a promising energy device. However, the energy density of supercapacitors is still less than that of ordinary batteries. As is known to all, the electrochemical performance of supercapacitors is largely dependent on electrode materials. In this review, we firstly introduced six typical transition metal oxides (TMOs) for supercapacitor electrodes, including RuO E represents the energy density, C represents the specific capacitance, and V represents the potential window, we can know that the energy density of a supercapacitor depends on both capacitance and operating voltage, and it can be enhanced by increasing the potential window and using an electrode material with high capacitance [Owing to the rapid development of the global economy, accelerating the consumption of fossil fuels like coal, fuel, and natural gas, the problems of global climate change and environmental pollution are increasing to significant levels. Developing solar, wind, tidal, and other renewable clean energy is a way to mitigate current energy and environmental pollution problems ,3,4,5,6.acitance ,26. It iacitance ,28,29,30−1), higher energy density than carbon materials, and better chemical stability than conductive polymers [2 is thought to be an optimal pseudocapacitive electrode material [3O4, MnO2, and ZnO have the advantages of being natural abundant and high specific capacitance, which makes them substitutes for RuO2 [2O4 , have more active reaction sites and high electrical conductivity than binary metal oxides [2O4, X = Ni, Cu, Zn, Mn, and so on) and metal molybdate  have received tremendous research interest because of their low cost, enhanced electrochemical activity, and being a natural abundant resource [Electrode materials can be divided into three categories: carbon materials, conductive polymers, and transition metal oxides (TMOs). Carbon materials are usually used as electrode materials for EDLCs. They have the characteristics of high specific surface area, adjustable pore size distribution, and excellent electrical conductivity ,32. Owinpolymers ,42,43. Tmaterial ,45,46. Hmaterial ,48,49. Cfor RuO2 ,51. The for RuO2 . Becausel oxides . In addiresource ,55.2, Co3O4, MnO2, ZnO, XCo2O4 , and AMoO4  are firstly introduced. Secondly, existing problems of those TMOs in practical application and the corresponding methods are presented. The methods include synthesizing composites, preparing material in nano scale, introducing battery-type material or oxygen vacancies, and modification of quantum dots. Particularly, we introduce the latest developments of six TMOs for supercapacitor electrode according to the strategies. Finally, the future development of supercapacitors is discussed and recommendations on supercapacitors are exhibited.In this review, six transition metal oxide materials including RuO2 has high theoretical capacitance (2000 F g−1) and it can carry out rapid faraday redox reaction. It has good electrical conductivity, high chemical and thermal stability, as well as a big voltage window. Thus, RuO2 is called the most ideal pseudocapacitive electrode material [RuOmaterial ,56. Howe2 and reduce the cost of RuO2 electrode [2-based electrodes have been prepared such as metal sulfide-RuO2 electrode, metal oxide-RuO2 electrode, carbon material-RuO2 electrode, and multicomponent RuO2-based electrode [2 nanorods (RuO2-NRs) via chemical vapor deposition (CVD) and the annealing process. The prepared electrode has the characteristics of both supercapacitors and lithium batteries. The results show that the designed material has high specific capacitance (176 F g−1) and superior cycling stability   .−2 at the current density of 100 mA cm−2 as a Li-ion batteries electrode. The excellent electrochemical performance lies in the synergistic effect of metal oxide NRs and the unique structure of CNTS-CC. Enlarged surface area and exposed active sites allow accessibility of the ions/electrolytes to the electrodes. Ternary single-walled carbon nanotubes/RuO2/polyindole (SWCNT/RuO2/PIn) nanocomposite fabricated by Zhu’s group [−1 at 1.0 A g−1. At 500 W kg−1 and 5000 W kg−1, the energy densities of the SWCNT/RuO2/PIn electrode-based symmetric supercapacitor are 42 W h kg−1 and 33 W h kg−1, respectively. It also shows good cycling stability and capacitive performance of 1203 F g−1 at 1.0 A g−1. Via the hydrothermal and annealing process, CuCo2O4/CuO nanoneedles were synthesized directly on the conductive Ni foam (NF), on which RuO2 nanoparticles were deposited [2O4/CuO@RuO2 can work as water oxidation catalysis and has low overpotential (279 mV at 10 mA cm−2), low Tafel slope, and stable long-term performance. Besides, it achieves a high areal capacity up to 862.5 mAh cm−2 and a capacity retention of about 90.1% after 8000 cycles. The hybrid supercapacitor fabricated by CuCo2O4/CuO@RuO2 and activated carbon achieves an energy density of 0.84 mW h cm−2 at 8 mW cm−2.In addition, they demonstrate a reversible capacity of ~3.85 mAh cm’s group  presentseposited . The CuC2 particles, which results in reduced surface area, limited electrochemical properties, and decreased double layer capacitance [2 is a kind of nanocrystalline material with RuO2 as the core and a small amount of water hydrogen bond on the surface. The form of porous structure provides conduction paths for protons to enter the inner part of RuO2 [x•nH2O composites  obviously improves the discontinuity of the electron path in particles. Further, the obstacle of electron hopping between particles is reduced because a large amount of active carbon (AC) powder is introduced. The specific capacitance of the RuOx•nH2O is 1340 F g−1 (at 25 mV s−1) [2@SWCNTs/graphene (S/G) composites by the sol–gel method. The hybrid electrode material, RuO2@S/G-60, has a specific capacitance up to 988 F g−1 and excellent rate capability at 1 A g−1. Hexagon WO3 (h-WO3) and hydrous RuO2 were respectively prepared on three-dimensional conducting carbon cloth (CC) via the hydrothermal method [2 at different magnifications. 3 at different magnifications.In the reports above, many electrode materials have the addition of carbon materials. Theoretically, adding carbon materials can improve the surface area and conductivity of electrodes . Howeveracitance ,67. Hydr of RuO2 ,69. The  mV s−1) . Similar mV s−1)  synthesil method , which m2SO4 aqueous electrolyte with h-WO3/CC as negative electrode and hydrated RuO2/CC as positive electrode. At an operating voltage of 1.6 V, the asymmetric supercapacitor device is optimized with volumetric capacitance of 3.52 F cm−3 at 5 mA cm−2. Moreover, at a power density of 40 W cm−3, the asymmetric supercapacitor device shows brilliant energy density of 1.25 W h cm−3 and it also shows promising electrochemical stability. By comparison, an all-pseudocapacitive asymmetric device [−2 at 40 mW cm−2, but a remarkable 96% retention after 20,000 charge/discharge cycles.The asymmetric supercapacitor was fabricated in Hc device  has an e3O4 is a kind of transition metal oxide belonging to the spinel family, and the faradic redox reaction of Co3O4 can be described as follows [Co follows :(1)Co3O43O4 has the advantages of high theoretical specific capacitance (3560 F g−1), low price, environmental friendliness, and good chemical durability, so it is a promising active material [3O4 are limited [Comaterial ,76,77. H limited ,79.3O4 nanomaterials have been synthesized, such as nanowires [3O4/reduced graphene oxide (rGO) composites. Tiny Co3O4 nanoparticles are dispersed on rGO flakes and a three-dimensional structure is formed. The prepared electrode has more active sites than Co3O4 nanowires [3O4/rGO-120-12 shows high specific capacitance of 1152 F g−1 at 1 A g−1. Indira Priyadharsini [3O4 nanoparticles by the sol–gel method. The obtained Co3O4 nanoparticles, carbon, and polyvinylidene fluoride (PVDF) were then mixed with a mass ratio of 8:1:1. The electrode was prepared by coating the synthesized slurry uniformly on the Ni foam substrate. The specific capacitance of the electrode at 11 mA cm−2 is 761.25 F g−1. The reason for the low specific capacitance is that the addition of PVDF increases the resistance between the collector and the redox active material, which results in a decrease in the number of active sites and the electron/charge transport rate [3O4 active materials on conductive substrates such as carbon fiber, carbon cloth, stainless steel sheet, and metal foam can avoid the addition of binder and conductive additive [3O4 nanosheets synthesized on Ni foam [2 g−1) larger than the original Co3O4 (108 m2 g−1). There is still a superior specific capacitance of 1323 F g−1 at the current density of 10 A g−1 , indicating outstanding rate capability of the sample. Furthermore, it exhibits 104.7% of the initial capacitance at the first 2000 cycles. The electrode materials prepared by Yang’s group [−1 and 1606.6 F g−1 at the current density of 1 A g−1, respectively). Electrode prepared by Wei [3O4/NF with different heating times are shown in 3O4 nanoflakes.Nanomaterials have a large surface with numerous active sites. The ultra-small size of nanomaterials shortens the electron/ion diffusion path, providing favorable transfer pathways . Becauseanowires , nanofibanowires , nanoparanowires ,84, and anowires . Yue andanowires  used a sanowires  to achiedharsini  firstly ort rate . Howeveradditive . Ag dope Ni foam  possess ’s group  and Wangd by Wei  has uniq3O4 nanowires were anchored on the surface of the Co3O4 nanosheet, effectively expanding the surface area, providing rich active states for the faradaic redox reaction and promoting the diffusion rate of electrolyte ions. The sample prepared at hydrothermal heating for 8 h (Co3O4/NF-8 h) shows a high specific capacitance of 2053.1 F g−1 at 1 A g−1. The working voltage of the hybrid supercapacitor device assembled by the hetero-structured Co3O4 array and graphene can reach 1.6 V. The maximum energy density of 22.2 W h kg−1 is delivered and the excellent cycle is proved by the 93.3% capacitance retention after 10,000 cycles.Co3O4 nanomaterials [3O4 nanomaterials. With ZIF-67 crystals as precursor, porous Co3O4 nanoparticles [3O4 nanoparticles exhibit high specific capacitance (190 F g−1 at 5 A g−1) and capacitance retention (71.42% after 5000 cycles). Bao and co-workers [3O4 embedded α-Co/Ni(OH)2 hollow nanocages. The prepared composite (α-Co/Ni(OH)2@Co3O4-70) has abundant electrolyte diffusion channels and reactive active sites. At the same time, it possesses excellent conductivity and stability. Therefore, the high capacitance of 1000 F g−1 is obtained at 1 A g−1 and the capacitance retention is 74% at 10 A g−1. Meanwhile, the hybrid nanocages still maintain 72.34% of original capacitance after 8000 charge/discharge cycles. In addition, α-Co/Ni(OH)2@Co3O4-70//active carbon (AC) device has an energy density of 23.88 W h kg−1 at a power density of 0.075 kW kg−1. Wei et al. [3O4 ultrathin nanoparticles by thermal treatment. The energy density at 790.7 W kg−1 of asymmetric supercapacitors prepared with Co3O4//AC is 46.5 W h kg−1. Moreover, the overpotential of 2D Co3O4 ultrathin nanoparticles at the onset potential is 230 mV and Tafel slope is 74.0 mV dec−1. This shows 2D Co3O4 ultrathin nanoparticles’ great performance in oxygen evolution reaction. Besides, Co-based MOFs (Co-MOFs) have become fascinating templates for thermal transformation to generate porous Co3O4 structures [3O4 composites by pyrolysis of 3D graphene/Co-MOF precursor. The electrochemical performance of the composites is not good (specific capacitance of 423 F g−1 at 1 A g−1 and capacitance attenuation of about 17% after 2000 cycles). This is because MOFs-based electrodes are usually composed of insulation binder, conductivity additives, and MOFs, which hinders the electrolyte penetration and reduces the electron transport between electroactive sites of active material and electrolytes [In recent years, metal organic frameworks (MOFs)-based materials have attracted attention in various fields of gas adsorption, energy device, pollutant degradation, electrocatalysis, and drug release . Besidesaterials ,95. Commarticles  were tra-workers  used ZIFi et al.  developeructures . Li et atrolytes .3O4@CoNi2S4 nano-wall arrays on carbon cloth. The synergistic effect of Co-MOF-derived Co3O4 skeleton is utilized to improve the electrochemical activity of cobalt nickel sulfide. The specific capacitance of the Co3O4@CoNi2S4 electrode is 244.4 mAh g−1 at 1A g−1 and a specific capacitance attenuation of 18.7% at 16 A g−1. At a power density of 884.4 W kg−1, the energy density of the Co3O4@CoNi2S4//AC device reaches 55.6 W h kg−1. After 10,000 cycles, the capacitance retention of Co3O4@CoNi2S4 reaches 86%. Liao and co-workers [3O4 nanoparticles on graphene nanosheets supported by carbon fabric. The electrode shows an unbelievable specific capacitance of 3480 F g−1, which is close to the theoretical value (3560 F g−1). Besides, they fabricated a flexible symmetric supercapacitor device with two pieces of prepared electrode. The flexible supercapacitor has no sacrifices of electrochemical performance when it is bent under bending angles from 0° to 150°. Furthermore, the supercapacitor delivers capacitance of 580 F g−1 and 13.8% capacitance decline after 20,000 cycles. The maximum energy density of the device is 80 W h kg−1 , MnO2 has been widely studied as the most competitive transition metal oxide [Owing to its rich natural content, no environmental pollution, and high theoretical specific capacitance . When the power density of the asymmetric supercapacitor is 1198.4 W kg−1, the energy density can reach 49.8 W h kg−1. After 5000 cycles, the capacitance retention of the asymmetric supercapacitor can reach 90.6%. Lei and co-workers [2 nanosheets@graphenated CNTs network on 316 L stainless steel by the CVD method followed by annealing treatment . 3D-graphene/MnO2 foam composite [2 composite electrodes in the absence of carbon black have a high specific capacitance of 333.4 F g−1 at 0.2 A g−1 and excellent cycling stability.Providing an effective electronic pathway, graphenated CNTs are interconnected to form porous structures, with MnOomposite  was prep2/biomass-based porous carbon (BPC) composites by the hydrothermal method. The MnO2/BPC composites electrode has a great specific capacitance of 139.6 F g−1 at the current density of 300 mA g−1 and 70 F g−1 at 10 A g−1. In addition, it loses 7.7% of the specific capacitance after 1000 cycles. In Shen’s research [2/soybean pod carbon (SPC) composites by a one-step in situ hydrothermal method. The prepared material retains the original tubular structure of the pods, with a large number of δ-MnO2 nanorods covered on the surface. According to the research, the MnO2/SPC electrode has a better specific capacitance than the MnO2 electrode . Besides, the symmetric MnO2/SPC-based supercapacitor displays high capacitance retention of 91% after 6000 cycles and maintains the energy density of 35.1 W h kg−1 at a high power density of 9000 W kg−1. The hollow structure of MnO2/carbon spheres (CSs) [4 aqueous solution by sonication. Then, this aqueous solution was heated at 160 °C for 3 h in a Teflon-lined stainless steel autoclave. After the reaction and cooling down, the product was vacuum filtered, washed, thoroughly dried at 60 °C, and then labeled as MnO2/hollow CS-20. An asymmetrical supercapacitor is assembled with the MnO2/hollow CS-30 material as the positive electrode and the hollow CS as the negative electrode. At the 2.0 V voltage window, the maximum energy density (41.4 W h kg−1) is obtained at a power density of 500 W kg−1. Moreover, the asymmetric supercapacitor shows a good cycling stability of 93.9% capacitance retention after 5000 cycles.However, the high cost and complex preparation process of commercial carbon materials are not conducive to large-scale preparation. Therefore, it is very important to develop low price and renewable materials to meet the increasing demand. Biomass resource is a renewable resource with high utilization value. Biomass resource utilization has become a hot area and pillar of economic and social development in the future . Biomassresearch , soybeanes (CSs)  was prep2-based electrodes has been thought to be an effective way to increase the capacity and energy density of supercapacitors. Rapid redox reactions and synergistic effects of MnO2 and battery metal oxides improve the overall performance of the electrode. The electrode [2 on NiO nanosheets for 30 min has an ultra-high specific capacitance of 1227.2 F g−1 (at 10 A g−1) and a capacitance retention rate of 76.7% after 10,000 cycles. Via a low temperature facile chemical bath deposition, Liu et al. [3O4@MnO2 nanosheets on the carbon cloth fibers (Co3O4@MnO2CC) with a hierarchical core–shell structure. The leaf-like Co3O4@MnO2 nanosheets exhibit brilliant electrochemical performance, including super-high specific capacitance (616.7 F g−1 at 2 A g−1) and superior cyclic stability  as well as high output potential of 1.2 V. The asymmetric supercapacitor (Co3O4@MnO2CC-90 as the cathode and AC as the anode) has energy density of 54.71 W h kg−1 at the power density of 1.06 kW kg−1 and 86.3% retention of specific capacitance after 10,000 cycles at 10 A g−1.According to Nie’s study , introdulectrode  obtainedu et al.  fabricat−1 (at 1 A g−1) and capacitance retention of 94% . ZnO nanomembranes (NMs) [Electrode materials largely determine the performance of supercapacitors . Therefoes (NMs)  were pre−1 (6 M KOH at 1 A g−1), 465 F g−1 (1 M KCl at 1 A g−1), and 65 F g−1 (6 M Na2SO4 at 1 A g−1), respectively. However, those materials mentioned all have the problem of capacity attenuation during long-term cycling [In different electrolytes, the ZnO NMs have different specific capacitance, 846 F g cycling ,136.−1 at 20 A g−1 (277 F g−1 at 0.05 A g−1) and capacitance loss of 2.6% after 15,000 cycles. Chebrolu et al. [−2 with a specific capacitance of 1248 F g−1 and long-term cycling stability). Because of ZnO/NiO nanosheets’ unique surface morphology, increasing the electron transfer rate and electrical conductivity, the energy storage properties are improved. Di’s group [2O3 could prevent the ZnO nano-framework from collapsing. Meanwhile, the specific surface area of the material could be increased to enlarge the contact area between the composite and the electrolyte solution. The Al2O3-ZnO nanorod displays a high specific capacity (463.7 F g−1) and favorable cyclic stability (96.9%), which is higher than Al2O3 and ZnO. In addition, the ion transferred number and the ion diffusion coefficient of the Al2O3-ZnO nanorod are 6.3 and 7.6 × 10−13 cm2 s−1. Studies have shown that the electrochemical properties can be further improved by synthesizing the ZnO-based multicomponent electrodes [3O4-CuO-ZnO@GO nanocomposite films by the hydrothermal method and then examined the effect of carbon ion irradiation on the properties of the prepared nanocomposite. The results of X-ray diffraction (XRD) suggest that low doses of carbon ion irradiation enhance the crystallinity of the materials, and high doses irradiation lead to deficiencies and disorder to the Co3O4-CuO-ZnO@GO, resulting in distortion and defects in the structure of the material. According to the study of electrochemical performance, the highest specific capacitance (1950 F g−1 at 10 mV s−1) is obtained at a radiation dose of 5.0 × 1015 ions cm−2, while the specific capacitance of 208 F g−1 and 1356 F g−1 are obtained at 1.0 × 1015 ions cm−2 and 7.0 × 1015 ions cm−2, respectively. Self-assembled ZnO-CoO@nitrogen-doped carbon (NC) electrode [−1  without morphology change. Besides, the graphene//ZnO-CoO@NC device also has 94% capacitance retention after 10,000 cycles at 2 A g−1. At a potential window of 1.6 V, the device possesses an energy density of 16.5 W h kg−1 at a power density of 396.51 W kg−1 and remains at 5.5 W h kg−1 at 5634.5 W kg−1.Numerous ZnO-based electrode materials are fabricated, such as carbon material-ZnO electrode ,138,139,u et al.  fabricat’s group  reportedectrodes ,149,150.ectrodes  synthesilectrode  was prep42− modified carbon layer (Mo-C) [2 g−1) than pure zinc oxide (0.835 m2 g−1). In addition, compared with the electrode material prepared by Mohamed’s group [−1 at 1 A g−1 and 650 F g−1 at 10 A g−1). He and co-workers [2. The unique structure increases the charge and ion transfer rates and improves the contribution of pseudoactive substances in faradaic reactions. An asymmetric supercapacitor is assembled in 3 M NaOH with NF@ZnO@Co(OH)2 as positive electrode and NF@cellulose paper as the negative electrode. In cell voltage of 1.5 V, the asymmetric supercapacitor exhibits maximum energy density of 62.57 W h kg−1 at a power density of 71 1.28 W kg−1 and, at 16,985.55 W kg−1 (maximum power density), it delivers an energy density of 16.04 W h kg−1. Ding et al. [−2, ZnO@CoS shows a high specific capacity of 898.9 C g−1, while specific capacity of pristine ZnO and ZnO precursor are 614.4 C g−1 and 530.6 C g−1, respectively. The assembled button-type asymmetric supercapacitor displays an energy density of 45.2 W h kg−1 at 1039.1 W kg−1 with an areal capacity of 2438 mC cm−2. In addition, the capacity retention of 107% after 10,000 cycles shows its remarkable cycling stability. A study  pointed r (Mo-C) . The car’s group , ZnO@Mo--workers  construcg et al.  anchored2O4 has a spinel structure. Generally, the two metal ions exhibit high capacity and conductivity, so ternary transition metal oxide has more abundant redox reactions and higher electrochemical activity than single metal oxides [2O4 is used as electrode material, the corresponding equations can be described as follows [Ternary transition metal oxide XCol oxides . Thus, i follows ,159:(3)M2O4 electrode by a simple solution combustion method. At the scanning rate of 10 mV s−1, the MnCo2O4 electrode shows the maximum specific capacitance of 270 F g−1, and the capacitance attenuation after 1000 cycles is 7.6%. MnCo2O4 nanosheets [−1 at 0.2 mA cm−2. Furthermore, after 1500 cycles, the MnCo2O4 electrode possesses a capacity retention rate of 51.8%. A battery-type flexible electrode [2O4 on an ultrafine nickel wire by electrodeposition, dealloying, and oxidation  was obtained. An asymmetric supercapacitor was assembled with the prepared sample as the positive electrode and AC as the negative electrode. The measurement results show that, with the potential window of 1.5 V, the asymmetric supercapacitor delivers a specific capacitance of 112 F g−1 at 1 mA cm−2 and a maximum energy density of 35 W h kg−1. In Zhu’s study [2O4@Ni(OH)2 provides large specific surface area, which enables rapid response and enhances electrochemical properties (2160 F g−1 at 1 A g−1 and a good capacitance retention of 82.7% at 20 A g−1).Wang’s group  synthesi-workers  designed’s study , the uni9S8@NiCo2O4 nanobrushes by NiCo2O4 nanosheets grown on Co9S8 hollow nanoneedles. Owing to the good electrical conductivity and specific capacitance of Co9S8 and large specific surface area provided by the hollow nanoneedles structure, remarkable electrochemical properties are displayed (a high specific capacitance of 1966 F g−1 at 1 A g−1 and excellent capacitance retention of 92.9% after 5000 cycles). Owing to its unique structure, CuCo2S4/CuCo2O4 has the advantages of electrochemical surface area, excellent conductivity, short ion diffusion path, and rapid electron and ion transport rate [2S4/CuCo2O4, the asymmetric supercapacitor is synthesized, which delivers energy density of 33.2 W h kg−1, power density of 13.3 kW kg−1, and outstanding long-term stability of 73% capacitance retention after 10,000 cycles.Transition metal sulfides have the characteristics of high faradic redox activities, unique physical or chemical properties, and long cycle performance. The conductivity of metal sulfides is higher than that of corresponding oxides, which is more convenient for electron transfer. In addition, low electroconductivity restricts TMOs further application in supercapacitors. At the same time, transition metal sulfides can make up for the shortcoming of TMOs. Moreover, because of the synergistic effect between the two individual materials, the mixed structure electrode can also improve the whole active region and promote the transport of ions from the electrolyte ,166,167.ort rate . Based o2O4 electrode material, Feng et al. [2O4 nanoflowers in hypoxic atmosphere via a hydrothermal method followed by thermal treatment. The presence of oxygen defect sites and impurity bands provides massive reaction sites and fast ion intercalation, which improves electrical conductivity and hydrophilic properties. The electrode material shows a brilliant specific capacitance of 1.2 F cm−2 at 1.2 mA cm−2 and rate capability of 69.4% capacitance retention at 20 A g−1. Yan et al. [2O4 nanostructures in the different concentration of NaBH4 solution  to change the content of oxygen vacancies  Introduction of oxygen vacancies can adjust the surface chemical structure and enlarge the electroactive sur-face area exposed to the electrolyte. (ii) Oxygen vacancies can help the diffusion for charge carriers. Therefore, the conductivity of TMOs is improved. (iii) Electrochemical active sites are created when oxygen vacancies are introduced because the oxygen vacancies can serve as active sites for redox reactions. The kinetics of surface reactions is accelerated and the capacitance is increased ,170,171.acancies .2O4 nanosheets electrode treated with 0.5 M NaBH4 has the best electrochemical performance .The results indicate that an appropriate oxygen vacancy content can increase the diffusion rate of carriers. In addition, oxygen vacancies as active sites for redox reactions can accelerate the surface kinetics reaction. The NiCo2 [Compared with carbon materials, pseudocapacitive materials like metal oxides and organic conductive materials have higher specific capacitance owing to redox reaction on the surface of the material. In addition, ternary metal oxides have more active sites and faster redox reactions than single metal oxides, which become one of the materials to replace RuO2 ,173,174.2 . The per2 .4@CoS core–shell heterostructures on rGO/Ni foam. 4, CoS, and CoMoO4@CoS with different magnification.According to Yi’s study , the des4@CoS composite shows 81.1% retention of the initial capacitance (3380.3 F g−1 at 1 A g−1) after 6000 cycles at 10 A g−1. In order to further improve the performance of MCo2O4 materials, Hussain and co-workers [4 with ternary metal sulfide NiCo2S4 to synthesize three-dimensional walking palm-like core–shell CoMoO4@NiCo2S4@NF nanostructure by a two-step hydrothermal method. At a current density of 5 mA cm−2, the synthesized composite exhibits a high capacitance of 17.0 F cm−2. Besides, 114% capacitance retention after 10,000 charge/discharge cycles reveals its remarkable cycling stability. The asymmetric supercapacitor fabricated with CoMoO4@NiCo2S4@NF and AC@NF as electrodes shows a high energy density of 60.2 W h kg−1 at a power density of 188 W kg−1. Moreover, it lights 22 parallel-connected red light emitting diodes for over 60 s.The unique material structure, the excellent conductivity of rGO, and the synergistic effect between the two materials help to increase the electrochemical active sites and enhance the capacitance. According to the electrochemical performance tests, the core–shell CoMoO-workers  combined4-based electrode. To the best of our knowledge, there are still relatively few reports in this regard. Zhu et al. [2O4 sphere composites. The CQDs/NiCo2O4 composite electrode possesses a specific capacitance (856 F g−1 at 1 A g−1) and excellent rate capability of 83.9%, 72.5%, and 60.8% capacity retention rate at 20, 50, and 100 A g−1, respectively. It also has 1.25% capacitance loss over 10,000 cycles at 5 A g−1, which indicates its exceptional cycling stability. The maximum capacitance of graphene quantum dot modified nano-needle electrode [−1 is four times higher than that of pure MnCo2O4.5 nano-spherical electrode . In Zhang’s research [4 microspheres were modified by silver quantum dots to fabricate the Ag QDs/NiMoO4 microspheres (Quantum dots (QDs) are zero-dimensional nanoparticles of metal or semiconductor crystals with a diameter between 2 and 8 nanometers . Quantumu et al.  synthesilectrode  at 1 A gresearch , mesoporospheres .4 microspheres exhibit specific capacitance of 3342.7 F g−1 at 1 mV s−1 and 2074 F g−1 at 1 A g−1  Preparing nano scale materials. The small size enlarges the contact surface and shortens the ion diffusion distance between materials and electrolyte. The size of material largely decides the substantial variation of fundamental electrical and optical properties. In addition, optical, electronic, and mechanical properties of QDs are different from corresponding nanoparticles. That is why QDs are very attractive in various fields. (ii) Utilizing the synergistic effect. It is very important that, through different combinations of carbon materials, transition metal oxides, transition metal sulfides, and other materials, the synergistic effect between different components can be used to improve the electrochemical performance effectively. In addition, some electrode materials containing metal sulfides exhibit good electrochemical performance, which indicates that other binary metal materials such as metal phosphides and metal nitrides also have potential for electrode materials. (iii) Synthesizing composites with a special structure. Porous structure, core–shell structure, and hollow structure can enlarge the surface area with generous electrochemical active sites and reduce the resistance of ion and electron transfer, thus increasing the conductivity and the rate of redox reaction. Further, the introduction of oxygen vacancies can not only increase active sites, but also adjust the surface chemical structure, improving the conductivity. (iv) Ternary transition oxides have higher conductivity, faster faradic reaction, and more active sites than single metal oxides. At the same time, In this review, we have summarized recent developments of TMOs-based electrode materials for supercapacitors, including design, fabrication, and electrochemical performances. RuOAlthough much promising progress has been obtained in TMOs-based electrode materials, low energy density is still an obstacle for the development of supercapacitors. We have three recommendations on the development of supercapacitors. (i) Researchers should aim efforts towards how to obtain raw material of electrode with low cost and no pollution, and the use of biomass carbon materials is a good example. (ii) The development of portable and wearable electronic devices drives the study of flexible supercapacitors. That means, apart from increasing the energy density, it is necessary to achieve the stability of the capacitance performance under different bending and deformation conditions. (iii) At present, there are many reports on transparent and flexible electrodes for solar batteries. Moreover, supercapacitors based on battery-type electrodes are also widely reported. At the same time, hybrid supercapacitors can shorten the gap between conventional metal ion batteries and supercapacitors . We wond


Cladosporium michoacanense, Cladosporium sp. (cladosporioides complex), and Didymella sp., this last being reported for the first time in this area. The secretion of seven enzymes, namely, endoglucanase, exoglucanase, β-D-glucosidase, endo-1,4-β-xylanase, β-D-xylosidase, acid, and alkaline phosphatases, were analyzed under agitation and static conditions optimized for the growth period and incubation temperature. Cladosporium strains under agitation as well as incubation for 72 h mostly showed the substantial activation for endoglucanase reaching up to 4563 mU/mL and xylanase up to 3036 mU/mL. Meanwhile, other enzymatic levels varied enormously depending on growth and temperature. Didymella sp. showed the most robust activation at 8 °C for endoglucanase, β-D-glucosidase, and xylanase, indicating an interesting profile for applications such as bioremediation and wastewater treatment processes under cold climatic conditions.The isolation of filamentous fungal strains from remote habitats with extreme climatic conditions has led to the discovery of a series of enzymes with attractive properties that can be useful in various industrial applications. Among these, cold-adapted enzymes from fungi with psychrotrophic lifestyles are valuable agents in industrial processes aiming towards energy reduction. Out of eight strains isolated from soil of the paramo highlands of Ecuador, three were selected for further experimentation and identified as  The High Andean mountain region is marked by the paramo ecosystem, neotropical grassland covering mountainsides from 3500 up to 5000 m of altitude from Southern Venezuela, Colombia, Ecuador, and Northern Peru . It is cThe Paramo Region of South America is a vastly unexplored area in terms of soil-derived microorganisms. Geospatial separation by Andean mountain chains creates a series of unique habitats, forcing microbes to adapt to a variety of given conditions, increasing the biological diversity with unique species within different paramo regions . Most suEnzymes can break down plant cell wall components, such as those of the cellulolytic system , for efficient cellulose cleavage and of the xylanolytic system (mainly endoxylanase and β-xylosidase) for xylan breakdown to the level of reducing sugars. They are widely applied to biotechnological processes in areas such as the food industry, production of fuels, detergents, and biopulping ,22,23,24Fungi further gain increasing importance as biofertilizers to improve crop yield. Given the broad range of climatic zones where crops are planted, biofertilizers must be equally adapted to these conditions including cold regions with generally lower productivity . Cold adThe present study, therefore, aimed to investigate filamentous fungi from paramo soil and optimize incubation conditions for optimal enzyme activity yield to analyze whether their extreme living conditions led to adapted enzymes of the carbon and phosphate cycle with possible interesting applications in the industry comparable to reported enzyme activities for mesophilic fungi. Unfortunately, soil-associated fungi of this region are poorly described and have never been analyzed for their enzymatic hydrolyzation capacities to the best of our knowledge.Two sampling sites were established, each at two different altitudes within the grassland paramo (4000 m asl) and at the frontier between grassland paramo and superparamo (4150 m asl), lying on a lineal transect towards the peak of the volcano Northern Iliniza to guarantee similar climatic conditions and soil characteristics . Each saSoil samples were diluted with sterile water at dilutions of 0.1 g in 10, 100, and 1000 mL with three replicates each, and 50 µL of each dilution were distributed with a sterilized spreading rod on 10 cm Petri dish containing 12 mL of potato dextrose agar (PDA)  with streptomycin at 1 mg/mL . Then, Petri dishes were incubated for 48 h at 20 °C. After incubation, eight samples of clearly isolated and visibly round-shaped fungal colonies were picked with sterile toothpicks and transferred to a fresh PDA petri dish for cultivation.For the growth assay, each fungus was picked with a sterile toothpick and inoculated at the center of a PDA Petri dish. Then dishes were incubated at 4, 30, and 40 °C, respectively, for a total of 35 days. Growth was measured weekly in cm growth-diameter of the halo through the inoculation point.g for 10 min at 4 °C. The supernatant was isolated and mixed with 50 µL sodium acetate 3 M (pH 5.2) and 300 µL isopropanol. After the second centrifugation under the same conditions, the supernatant was discarded, and the mixture was washed twice with 600 µL of 70% ethanol following centrifugation steps. After discarding the final supernatant, the resulting pellet was diluted in 50 µL TE buffer  and 5 µL RNAse (10 mg/mL).To obtain genomic DNA of three fungal strains, the mycelia were macerated with a mortar and pestle in TES lysis buffer . First, lysed tissue was incubated at 65 °C for 15 min. Then, 140 µL of 5 M NaCl were added, and the mixture was incubated on ice for 30 min. Afterward, 600 µL of chloroform/isoamyl alcohol (24:1) were added and centrifuged at 10,000× ® SV Gel kit and PCR Clean-Up System (Promega) following the kit’s instructions. Finally, the PCR product was quantified on a NanoDrop® .Genomic DNA was used to amplify the fungal ITS region (ITS1-5.8S-ITS2) applying the primer pairs ITS4 and ITS5 . For amp® Terminator Cycle Sequencing Kit  following the manufacturer’s instructions and analyzed with ABI 3500 XL sequencer system (Life Technologies). Resulting forward and reverse sequences were quality checked and merged into a consensus sequence with BioEdit v.7.0.5.3 [www.ncbi.nlm.nih.gov) and the Trichokey database (http://isth.info/) were used to compare contigs with homologous sequences (both accessed on 12 April 2019). After a second, quality control sequences were aligned with homologous sequences from culture collections applying the ClustalW tool [Sequencing reactions were performed with the BigDye.7.0.5.3 . The BLAalW tool . Then, talW tool .6–107 spores per mL were established in sterile water using a Neubauer chamber for counting. These spores were then used to inoculate 50 mL of sterile Adam’s medium (pH = 6) with 1% wheat bran in 125 mL Erlenmeyer flasks . Triplicates were incubated at 20 °C under static and shaking conditions (120 rpm), respectively, for 120 h harvesting each 24 h. Each crude extract sample from flasks was filtered over Whatman paper using a vacuum pump and applied on the same day for enzyme assays.Isolated fungi were pre-cultured at 20 °C on inclined agar PDA medium in test tubes until sporulation. Concentrations of 10®) and 0.5% xylan beechwood (endoxylanase measurement) in the following relation: 25 µL substrate, 10 µL buffer sodium acetate 50 mM pH 5.0, and 15 µL crude extract. Mixes were incubated in a thermocycler  for 20 min at 30 °C. In the assay to determine optimal incubation temperatures, a range was established from 4 to 32 °C. Then, 50 µL DNS was added to interrupt enzyme activity, and sample-absorbance was measured at 540 nm on a spectrophotometer  compared to glucose and xylose standard curves (0–1 mg/mL). Blancs were established adding enzyme extract after incubation, directly interrupting the reaction with DNS. The results were expressed as milliunits per mL (mU/mL), defined as the enzyme quantity that releases one µmol of reducing sugars per minute per mL.To measure enzyme activity, the crude enzyme extract was mixed with respective water-diluted substrates to obtain activity over time according to the Miller method . Reducinp-nitrophenol as a result of enzyme cleavage was measured for the following substrates: p-nitrophenol-β-D-glycopyranoside (β-D-glucosidase measurement), p-nitrophenol-β-D-xylanopyranoside (β-D-xylosidase measurement), and p-nitrophenol-β-D-cellobiose (exoglucanase measurement)  used in the following relation: 25 µL substrate, 10 µL sodium acetate buffer 50 mM pH 5.0, and 15 µL crude extract [To measure enzyme activity, the crude enzyme extract was mixed with respective water-diluted substrates to obtain activity over time. The amount of released  extract .p-nitrophenyl phosphate , the assay mix was prepared accordingly using 10 µL crude extract, 40 µL substrate, and 100 µL buffer, where acid phosphatase was measured with acetate buffer 100 mM, pH 4.5 and alkaline phosphatase with Tris-HCl 100 mM, pH 8.0 (modified according to [®) for 20 min at 30 °C. In the assay to determine optimal incubation temperatures, a range was established from 4 to 32 °C in steps of 4 °C. Then, 50 µL (100 µL for phosphatases) of 0.2 M Na2CO3 solution was added to interrupt enzyme activity and sample-absorbance was measured at 410 nm compared to a p-nitrophenol–standard curve (0–1 mg/mL) [2CO3 solution. The results are expressed as milliunits per mL (mU/mL), defined as the enzyme quantity that releases one µmol of p-nitrophenol per minute per mL.For the measurement of phosphatases with rding to ). Mixes 1 mg/mL) . Blancs t-test for independent variables or Mann–Whitney U test, respectively. To analyze maximum yields among the three studied fungi, one-way ANOVA or Kruskal–Wallis’s test were applied following Sidak–Bonferroni or Dunn post-hoc tests. All statistical analyses were performed using licensed STATA 16.0.Measured enzyme activity is expressed as the mean ± standard deviation using Microsoft EXCEL. The Shapiro–Wilk test was used to prove the normal distribution of data and the Levene’s test for homoscedasticity. To test for statistical difference between incubation types , averages of the maximum enzyme activities were compared using the This sampling sites in the Iliniza National Reserve were chosen due to the fact of their proximity to glacial areas of stratified volcanos in the High Andean region. At a 4000 m altitude, the vegetation zone of conserved paramo highlands is grassland, mainly characterized by herbaceous plants such as sphagnum mosses, tussock grass, and rosette plants b,c. At 4The first screening for cultivable fungal strains from soil samples showed relatively few growing colonies clearly separated from each other, allowing for easy isolation of distinct strains. Out of 12 soil samples, eight colonies were chosen for the study. These strains were further screened for their capacity to grow at different temperature conditions. Generally, all strains showed better growth at 4 °C than 30 °C, indicating their adaptation to cold environments . At 40 °Cladosporium: C. michoacanense and Cladosporium sp. 3.1 (C. Cladosporioides species complex), respectively, and 3.3 to the genus Didymella  [Trichoderma guizhouense, Ghazali et al. (2019) [Trichoderma asperellum and 5.6 U/mL for Aspergillus niger. Unfortunately, most studies for psychrotrophic fungi do not apply optimization assays, so comparability is limited. However, considering the advantage of growing under extreme conditions, the Cladosporium michoacanense strain is an interesting candidate for potential industrial application under harsh conditions. Moreover, the relatively early onset of enzyme secretion, indicating efficient substrate hydrolyzation at 72 h, represents an important advantage confirming previous results from studies with Cladosporium strains [The combination of optimal incubation time, agitation, and optimal substrate incubation temperature led to several enzymes’ considerably higher activity . Cladosp. (2019)  describe. (2019)  reported strains ,65.Cladosporium sp. 3.1 did not reach values as high as strain 1.1 but managed to perform well for all selected enzymes with values above 1 U/mL except alkaline phosphatase. However, the onset of enzyme production appeared relatively late. Interestingly, strain 3.1 showed the significantly highest activation of 2.04 U/mL for exoglucanase at low temperatures, although former studies indicated a relatively poor performance of paramo-derived fungi for these enzymes [Trichoderma asperellum and Aspergillus niger, respectively. It is also important to mention that this high activity was reached under conditions of only 4 °C. Furthermore, β-glucosidase, which works synergistically with exoglucanase, also performed well at low temperatures (8 °C). Given that Cladosporium cladosporioides had formerly been described to exhibit laccase activity [On the other hand,  enzymes . Compariactivity ,66,67, factivity , indicatDidymella 3.3 performed well only for endoglucanase, β-glucosidase, and xylanase, indicating poor performance for the rest of the enzymes, especially phosphatases, which could be related to the phytopathological behavior of many Didymella species allowing for the absorption of nutrients from hosts [Finally, om hosts . Neverthom hosts .Wide-ranging comparison of psychrotrophic fungi enzyme activity is difficult, given that most screening studies indicate qualitative and semi-quantitative data ,69,70,71Cladosporium sp. strain 1.1 showed the most competitive enzyme activity of all analyzed strains for endoglucanase and xylanase at 72 h, and agitation and assay temperatures between 24 and 28 °C, indicating an interesting potential for the industrial application processes where reducing sugars are used such as feed animal, food industry, and fuel production. Cladosporium strain 3.1 of the cladosporioides complex indicated average performance for a broad spectrum of different enzymes except for exoglucanase, which showed the highest activity. Its good performance of acid phosphatase at 12 °C could be applied in the industry of biofertilization to improve the crop yield of plants grown under cold conditions and in the eutrophication of phosphorus from animal feces due to the consumption of phytate in grains [Didymella strain 3.3 showed high endoglucanase, β-glucosidase, and xylanase activities at temperatures as low as 8 °C, where most enzymes are inactivated. This strain responded closest to a psychrotrophic profile, which could be specifically interesting in industrial processes such as wastewater management and bioremediation in cold conditions, an aspect that might become increasingly important in an industrialized world tending towards energy reduction.The present study directly isolated cold-adapted fungal strains from high Andean Paramo soil, of which three strains were chosen to identify the genus and characterize their enzymatic capacity. Selecting for optimal conditions concerning growth period, incubation conditions, and optimal temperature for enzyme performance, maximum enzyme activities were determined for each strain.


Cupriavidus species have been known as versatile microorganisms in the field of industrial biotechnology. Cupriavidus sp. strain EM10 was isolated from sewage sludges. Here, we report the complete genome sequence of this bacterium, which contains 6,658,510 bp  and 6,248 genes. Cupriavidus, belonging to the family Burkholderiaceae, have received much attention as bioplastic producers . To achieve this, 1 g sludge sample was added to lysogeny broth (LB)  and cultivated at 37°C with agitation (200 rpm) overnight. Serially diluted cultures were spread onto LB agar medium supplemented with 10 mM NiCl2 and incubated at 37°C for 7 days to obtain a pure single colony. Genomic DNA was extracted from 10 ml overnight culture using a Wizard genomic DNA isolation kit  according to the manufacturer’s instructions.Here, we report the complete genome sequence of N50 values of the reads were determined to be 971,301,097 bp, 15,324 bp, and 21,194 bp, respectively. The average quality of the reads was 0.84. De novo genome assembly was performed using the Hierarchical Genome Assembly Process (HGAP) v2 protocol (dnaA gene or predicted genes near its center. Gene prediction and annotation were processed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v5.2 with the GeneMarkS2+ annotation system , 12 rRNAs, 64 tRNAs, 4 noncoding RNAs (ncRNAs), and 895 pseudogenes. This complete genome sequence provides a genetic basis for elucidating the potential of the newly isolated Cupriavidus strain EM10.Genome sequencing was performed by Chunlab, Inc. . The DNA library preparation was performed using the PacBio DNA template prep kit 1.0 . Briefly, 10 μg genomic DNA was sheared and size-selected to 20 kb using g-TUBE devices . Blunt-end SMRTbell adapters were then ligated. Subsequently, the prepared library was sequenced on the PacBio RS II platform using PacBio P6-C4 chemistry in 8-well single-molecule real-time (SMRT) v3 cells  accession numbers are SRX11156676, SRX11156677, and SRX11156678 (PacBio). The BioSample and BioProject numbers are SAMN19357574 and PRJNA733020, respectively.The genome sequence reported here has been deposited at DDBJ/EMBL/GenBank under accession numbers


Peganum harmala L. (Zygophyllaceae family) have been reported to have antimicrobial, antifungal, and anticancer activities. In the present study, a 2-hydroxy propyl-β-cyclodextrin (HPβCD)/harmala alkaloid-rich fraction (HARF) host–guest complex was prepared using a thin-film hydration method to improve the water solubility and bioavailability of HARF. The designed complex was then co-encapsulated with ascorbic acid into PLGA nanoparticles coated with polyethylene glycol (HARF–HPßCD/AA@PLGA-PEG NPs) using the W/O/W multiple emulsion-solvent evaporation method. The average particle size, PDI, and zeta potential were 207.90 ± 2.60 nm, 0.17 ± 0.01, and 31.6 ± 0.20 mV, respectively. The entrapment efficiency for HARF was 81.60 ± 1.20% and for ascorbic acid was 88 ± 2.20%. HARF–HPßCD/AA@PLGA-PEG NPs had the highest antibacterial activity against Staphylococcus aureus and Escherichia coli (MIC of 0.025 mg/mL). They also exhibited high selective antiviral activity against the H1N1 influenza virus (IC50 2.7 μg/mL) without affecting the host (MDCK cells). In conclusion, the co-encapsulation of HPCD–HARF complex and ascorbic acid into PLGA-PEG nanoparticles significantly increased the selective H1N1 killing activity with minimum host toxic effects.Antimicrobial drugs face numerous challenges, including drug resistance, systemic toxic effects, and poor bioavailability. To date, treatment choices are limited, which warrants the search for novel potent antivirals, including those extracted from natural products. The seeds of  Microbial infections, including bacterial and viral infections, represent a significant health burden responsible for many deaths worldwide. Influenza A virus causes the hospitalization of about 4 million cases and the death of 600,000 patients each year. The pervasive use of antimicrobials is the leading cause of resistance to the existing medications . MoreovePeganum harmala L. (Zygophyllaceae family) is a member of the Zygophyllaceae family native to the area from the eastern Mediterranean region to India [P. harmala are rich in harmala alkaloids such as quinazoline and β-carboline alkaloids. The major alkaloid, harmine, is responsible for the various pharmacological activities of the harmala seeds [P. harmala extracts [Nature provides an enormous library of novel chemicals that can be explored to develop antimicrobials. to India ,4. The sla seeds ,6,7. Sevextracts ,9.Ascorbic acid is an essential dietary nutrient required for various biological functions . SeveralDespite the global trend to replace synthetic antimicrobials with natural ones, the use of natural products to treat microbial infections may face challenges similar to those of synthetic drugs, such as poor water solubility, low bioavailability, and non-selective targeting the infected organ. Thus, numerous nanoplatforms, including liposomes, supramolecular systems, and polymeric nanoparticles, were designed, being reported to remarkably ameliorate the therapeutic activities of different types of biologically active compounds ,16,17,18Supramolecular host molecules, such as cyclodextrins (CDs), are involved in overcoming the poor water solubility of the natural compounds via the formation of host–guest complexes. CDs are amphiphilic cyclic oligosaccharides obtained from the enzymatic hydrolysis of starch. They comprise three types, namely, α, β, and γ-CDs, consisting of six, seven, and eight glucopyranose units, respectively . In thisPolymeric nanoparticles (NPs) have been extensively involved in drug delivery, owing to their ability to encapsulate different therapeutically active compounds into their matrix and the ability of their surfaces to be decorated with different functional groups ,21,22. C1H NMR spectroscopy and phase solubility study. The prepared complex was then co-encapsulated with ascorbic acid into PEGylated PLGA (HARF–HPßCD/AA@PLGA-PEG NPs) utilizing the W/O/W multiple emulsion-solvent evaporation method. The designed NPs were physically characterized in terms of average size, surface charge, polydispersity index (PDI), entrapment efficiency, and morphology. Additionally, the release of HARF–HPβCD complex and ascorbic acid (AA) from HARF–HPßCD/AA@PLGA-PEG NPs was studied, and the release profile was acquired. Finally, the antibacterial and antiviral activities of HARF–HPßCD/AA@PLGA-PEG NPs were evaluated.In the present study, a 2-hydroxy propyl-β- cyclodextrin (HPβCD)/harmala alkaloid-rich fraction (HARF) host–guest complex was fabricated using the thin-film hydration method to alleviate the water solubility of HARF. The complex formation was investigated by 2-Hydroxy propyl ß cyclodextrin was purchased from BLD Pharmatech Co., Limited . Poly  (PLGA) and polyethylene glycol 6000 (PEG) were obtained from Sigma-Aldrich . Polyvinyl alcohol  was obtained from Acros Organics . Dimethylsulfoxide was purchased from Fisher Chemicals . Tween 80 was purchased from El-Nasr Pharmaceutical Chemicals Co. . Tryptic soy agar was purchased from Millipore . Phosphate-buffered saline, streptomycin, penicillin, fetal bovine serum, trichloroacetic acid, Dulbecco’s modified Eagle’s medium (DMEM), SRB, and tris (hydroxymethyl)aminomethane were purchased from Lonza .P.harmala L. were purchased from the local Egyptian market. The major alkaloids of P. harmala seeds were extracted, isolated, and characterized according to our previously reported method [Dried mature seeds of d method ,6.w/v) of HPßCD solution was prepared and subsequently added to the film, followed by 30 min sonication in a SONOREX DIGITAL 10 P sonicator . Afterward, the complex solution was stirred for another 30 min and purified through filtration using a 0.22 μm nylon filter. The filtrate was then dried in a lyophilizer .HARF–HPßCD inclusion complex was designed via the thin-film hydration method, as described elsewhere, with some modifications . Briefly1H NMR spectra of HPßCD and the inclusion complex were measured on a 400 MHz FT-NMR spectrometer  in D2O solutions. The scanning range of the hydrogen spectrum is 1–13 ppm.The phase solubility study was performed, as described elsewhere with some modifications , to confK1:1) was estimated utilizing the slope of the linear correlation and the intrinsic solubility (S0) using Equation (1) [The stability constant (tion (1) ,32.(1)KCE) was computed employing the intrinsic solubility and the stability constant using Equation (2) [S0 is the intrinsic solubility, and K1:1 is the stability constant.Then, the complexation efficiency (tion (2) ,32.(2)CThe designed HARF–HPßCD complex was co-loaded with AA into PLGA NPs coated with PEG (HARF–HPßCD/AA@PLGA-PEG NPs) using the W/O/W multiple emulsion-solvent evaporation method, as previously reported with some modifications ,7,33,34.Briefly, 300 mg of PLGA and 100 mg PEG 6000 were dissolved in 8 mL dichloromethane (DCM), forming the emulsion’s organic phase (O). Then, HARF–HPßCD complex and AA were dissolved in water (the W/W ratio of each drug/PLGA was 0.2:1) to obtain the inner aqueous phase (W1). Into the previously prepared organic phase, we emulsified 800 µL of the internal aqueous phase using a homogenizer at 11,000 rpm for 8 min in an ice bath to form a W1/O emulsion (first emulsion). Then, 2.5% polyvinyl alcohol was dissolved in 100 mL deionized water to form the external aqueous phase (W2). The first emulsion was added to the external aqueous phase (W2) and emulsified by homogenization at 11,000 rpm for 8 min in an ice bath. The resulting W1/O/W2 emulsion (second emulsion) was stirred overnight using a magnetic stirrer to allow solvent evaporation and particle hardening. Afterward, the nanoparticle colloid was lyophilized .The average particle size and polydispersity index (PDI) were determined using a Zetasizer Nano ZS equipped with a 10 mW HeNe laser, allowing for measurements at 633 nm and a detection angle of 173° backscatter . The ζ-potential was measured using laser Doppler velocimetry ,36,37.The morphological features of the prepared nanoparticles were investigated using scanning electron microscopy (SEM) via high-resolution field emission scanning electron microscopy (FESEM)  with an acceleration voltage of 1.00 kV. Before conducting the SEM study, we coated freeze-dried PLGA NPs via gold sputtering (current: 10 mA) for 2 min under a nitrogen atmosphere. The measurements were performed using the image processing program ImageJ .EE) of HARF–HPßCD/AA@PLGA-PEG NPs was measured indirectly by centrifugation , followed by ultrafiltration of the supernatant. The quantities of the HARF and AA unencapsulated in the ultrafiltrate were determined by a FLUOstar Omega microplate reader  spectrophotometer at 373 and 287 nm, respectively, using Equation (3) [The entrapment efficiency (tion (3) ,39.(3)EThe cumulative release % of HARF and AA from HARF–HPßCD/AA@PLGA-PEG NPs was investigated utilizing the dialysis membrane method. Briefly, 1 mL of the NPs was loaded to a dialysis bag . The dialysis bag was inserted into 50 mL of PBS at pH 7.4 with 2% Tween in a proper jar. The whole system was left on a stirrer at 37 °C. A 1 mL aliquot of the sample was withdrawn for analysis and immediately replaced with another equal volume of warmed buffer at specific time intervals. The concentrations of HARF and AA were determined by a FLUOstar Omega microplate reader  spectrophotometer at 373 and 287 nm wavelengths, respectively.%) was calculated according to Equation (4) [c(n − 1) is the bulk concentration before sampling, Cn is the concentration of the sample, Vo is the bulk volume of the release medium, and mo is the original amount of the drug in the nanoparticles tested.The cumulative release (tion (4) .(4)CumuStaphylococcus aureus ATCC® 6538 (lot no. 4600502) and Escherichia coli ATCC® 8739 (lot no. 380063) were obtained from American Type Culture Collection . Tryptic soy broth (100 mL) was used as inoculation medium for either the Gram-positive or Gram-negative bacteria, then incubated at 37.0 ± 1.0 °C for 24 ± 2 h. A loopful of the broth was streaked onto tryptic soy agar medium and incubated at 37.0 °C for 21 ± 3 h. Three or four colonies (from each plate) were inoculated in broth, and the suspension was incubated till turbidity reached 0.5 McFarland standard. The inoculum density was standardized utilizing a 0.5 McFarland standard and DensiCHEK© optical device . The obtained suspensions contained about 1.0 × 108 CFU/mL of Staphylococcus aureus or Escherichia coli.5 CFU/mL. An additional 100 µL from each bacterial strain suspension was diluted and cultured to confirm the inoculum density. All plates were incubated at 37.0 °C for 24 h and then kept in the dark to monitor growth. All control wells yielded a turbid solution. The inoculum density was 4–6 × 105 CFU/mL for both evaluated bacterial strains, with reference to the Clinical and Laboratory Standards Institute (CLSI) procedures  [The antibacterial activities of plain PLGA-PEG NPs, HARF, and HARF–HPßCD/AA@PLGA-PEG NPs were evaluated by adding and mixing 5.0 mL from each sample to 5.0 mL broth (1:2 dilution) according to a previously reported method . Next, 507, A09) .The human influenza H1N1 and Madin–Darby Canine Kidney (MDCK) cells were obtained from American Type Culture Collection . Vero E6 cells were grown in DMEM medium supplemented with 10% fetal bovine serum and 0.1% antibiotic/antimycotic solution. Gibco BRL provided the antibiotic and antimycotic solution, trypsin-EDTA, fetal bovine serum, and DMEM medium .4 cells per well were infected with 0.1 mL (CCID50) influenza (H1N1) of the diluted viral suspension of Influenza H1N1 and then incubated at 37 °C for 60 min to facilitate the adsorption of the virus. The antiviral activity for each sample was determined using a twofold diluted concentration of 1–100 µg/mL. Culture plates were incubated at 37 °C in 5% CO2 for 72 h. The development of the cytopathic effect was monitored by light microscopy. The cell monolayers were stained . After washing and drying, the optical density was measured at 540/630 nm. The antiviral activities were calculated according to Pauwels et al. [Cytopathic effect (CPE) reduction assay was used to evaluate the antiviral activities of plain PLGA-PEG NPs, HARF, and HARF–HPßCD/AA@PLGA-PEG NPs in cell culture systems, as described elsewhere with some modifications ,43. Bries et al. , employi4 cells per well in a 96-well culture plate. After 24 h, the culture medium containing serially diluted samples was added to the cells and incubated for 72 h before being removed, and the cells were washed with PBS. The next steps were carried out in the same manner as described above for the antiviral activity assay. The 50% cytotoxic concentrations (CC50) and the 50% inhibitory concentration (IC50) were computed using GraphPad PRISM Software . Selectivity index (SI) was estimated as the ratio of CC50 to IC50.The cytotoxicity was evaluated against MDCK cells before conducting the aforementioned assay. Cells were seeded at a density of 2 × 101H NMR analyses performed in D2O could shed more light on the host–guest complexation between HPßCD and other guest molecules and could provide clues on the inclusion of guest molecules into the hydrophobic cavity of the host molecule. Upon the inclusion of the guest molecule into the HPßCD cavity, an upfield shift of the H-3 and H-5 (hydrogen atoms placed in the interior cavity of HPßCD) occurred due to their shielding by the guest molecule  of HPßCD protons in the presence and absence of HARF are presented in ith HARF . The 1H βCD concentration, as presented in R2 value of the extrapolated curve was 0.9807, suggesting that a remarkable linear correlation between HARF solubility and HPβCD concentration where the solubility of HARF increased with increasing the concentration of HPβCD. As previously described by Higuchi and Connors, this phase solubility diagram displays the AL-type pattern, indicating a complex stoichiometry of 1:1 (HARF/HPβCD) [The solubility of HARF was determined by plotting the change with HPF/HPβCD) ,31. More−1, and 0.66, respectively, similar to values reported previously [βCD had improved the water solubility of HARF via the formation of the host–guest complex.The stability constant and the complexation efficiency (CE) were 81.4 Meviously ,32,49. TThe average particle size and PDI of the designed HARF–HPßCD/AA@PLGA-PEG NPs were 207 ± 2.60 nm and 0.17 ± 0.01, respectively . These vThe entrapment efficiencies of the HARF and AA are presented in The high entrapment efficiencies of HARF and AA are attributed to the PEG coating the PLGA NPs founding a barrier that surrounds and protects the loaded drugs, preventing their loss during the homogenization step and retaining their stability . The capAs demonstrated in S. aureus and E. coli. HARF–HPßCD/AA@PLGA-PEG NPs exhibited high antibacterial activity against S. aureus and E. coli (MIC of 0.025 mg/mL), as compared to free HARF (MIC of 0.5 mg/mL), as shown in The bacterial activities of the HARF, plain PLGA-PEG NPs, and HARF–HPßCD/AA@PLGA-PEG NPs were assessed against Treatment choices for viral infections are limited and face many challenges, such as drug resistance, systemic toxic effects, and poor bioavailability. This warrants the search for effective antiviral agents that can overcome the shortcomings of their current counterparts. The antiviral activities of the HARF, PLGA-PEG NPs, and HARF–HPßCD/AA@PLGA-PEG NPs against influenza A (H1N) were investigated through employing the cytopathic effect (CPE) reduction assay. The PLGA-PEG showed no antiviral activity. The developed HARF–HPCD/PLGA-PEG NPs exhibited high selective antiviral activity against the H1N1 influenza virus  without Staphylococcus aureus and Escherichia coli, with a MIC value of 0.025 mg/mL, as compared to free HARF (MIC of 0.50 mg/mL). Moreover, the designed NPs exhibited high selective antiviral activity against the H1N1 influenza virus without affecting the host, MDCK cells. Biocompatible nanocarriers loaded with natural extracts with antimicrobial activity could be explored as future effective treatments of infections.In this work, HARF was first mixed with HPßCD as a host molecule to form an inclusion complex. This was done to improve the solubility of HARF, which is highly hydrophobic. Finally, AA and HARF–HPßCD inclusion complex were co-loaded into the PLGA-PEG nanoparticles to form the final biodegradable delivery system with enhanced release and bioavailability. The fabricated HARF–HPßCD/AA@PLGA-PEG NPs demonstrated acceptable cytotoxicity and enhanced antibacterial and antiviral activities compared to free HARF. They showed a higher bactericidal effect against