Exploring the mechanism underlying the therapeutic effects of butein in colorectal cancer using network pharmacology and single-cell RNA sequencing data.

BACKGROUND: Butein has shown substantial potential as a cancer treatment, but its precise mechanism of action in colorectal cancer (CRC) remains unclear. This study aimed to uncover the underlying mechanisms through which butein operates in CRC and to identify potential biomarkers through a comprehensive investigation. METHODS: Target genes associated with butein were sourced from SwissTargetPrediction, CTD, BindingDB and TargetNet. Gene expression data from the GSE38026 dataset and the single-cell dataset (GSE222300) were retrieved from the Gene Expression Omnibus database. The activation of disease-related pathways was assessed using Kyoto Encyclopedia of Genes and Genomes, Gene Ontology and differential gene analysis. Disease-associated genes were identified through differential analysis and weighted gene co-expression network analysis (WGCNA). The protein-protein interaction network was utilized to pinpoint potential drug targets. Molecular complex detection (MCODE) analysis was employed to uncover relevant genes influenced by butein within key subgroup networks. Machine learning techniques were applied for the screening of potential biomarkers, with receiver operating characteristic curves used to evaluate their clinical significance. Single-cell analysis was conducted to assess the pharmacological targets of butein in CRC, with validation performed using the external dataset GSE40967. RESULTS: A total of 232 target genes for butein were identified. Functional enrichment analysis revealed significant enrichment of signaling pathways, including mitogen-activated protein kinase, JAK-STAT and NF-κB, among these genes. Differential analysis, in conjunction with WGCNA, yielded 520 disease-related genes. Subsequently, a disease-drug-gene network consisting of 727 targets was established, and a subnetwork containing 56 crucial genes was extracted. Important pathways such as the FoxO signaling pathway exhibited significant enrichment within these key genes. Machine learning applied to the 56 important genes led to the identification of a potential biomarker, UBE2C. Receiver operating characteristic analysis demonstrated the excellent clinical predictive utility of UBE2C. Single-cell analysis suggested that butein's therapeutic effects might be linked to its influence on epithelial and T cells, with UBE2C expression associated with these cell types. Validation using the external dataset GSE40967 further confirmed the exceptional clinical predictive capability of UBE2C. CONCLUSION: This study combines network pharmacology with single-cell analysis to unravel the mechanisms underlying butein's effects in CRC. Notably, UBE2C emerged as a promising biomarker with superior clinical efficacy. These research findings contribute significantly to our understanding of specific molecular mechanisms, potentially shaping future clinical practices.

Identification of two 6'-deoxychalcone 4'-glucosyltransferase genes in dahlia (Dahlia variabilis).

MAIN CONCLUSION: Two glycosyltransferase genes belonging to UGT88 family were identified to have 6'-deoxychalcone 4'-glucosyltransferase activity in dahlia. 6'-Deoxychalcones (isoliquiritigenin and butein) are important pigments for yellow and orange to red flower color. 6'-Deoxychalcones are glucosylated at the 4'-position in vivo, but the genes encoding 6'-deoxychalcone 4'-glucosyltransferase have not yet been identified. In our previous study, it was indicated that snapdragon (Antirrhinum majus) chalcone 4'-O-glucosyltransferase (Am4'CGT) has isoliquiritigenin 4'-glucosylation activity. Therefore, to identify genes encoding 6'-deoxychalcone 4'-glucosyltransferase in dahlia (Dahlia variabilis), genes expressed in ray florets that shared high homology with Am4'CGT were explored. As a result, c34671_g1_i1 and c35662_g1_i1 were selected as candidate genes for 6'-deoxychalcone 4'-glucosyltransferases in dahlia. We conducted transient co-overexpression of three genes (c34671_g1_i1 or c35662_g1_i1, dahlia aldo-keto reductase1 (DvAKR1) or soybean (Glycine max) chalcone reductase5 (GmCHR5), and chili pepper (Capsicum annuum) MYB transcription factor (CaMYBA)) in Nicotiana benthamiana by agroinfiltration. Transient overexpression of c34671_g1_i1, DvAKR1, and CaMYBA resulted in increase in the accumulation of isoliquiritigenin 4'-glucosides, isoliquiritigenin 4'-O-glucoside, and isoliquiritigenin 4'-O-[6-O-(malonyl)-glucoside]. However, transient overexpression of c35662_g1_i1, DvAKR1, and CaMYBA did not increase accumulation of isoliquiritigenin 4'-glucosides. Using GmCHR5 instead of DvAKR1 showed similar results suggesting that c34671_g1_i1 has isoliquiritigenin 4'-glucosyltransferase activity. In addition, we conducted co-overexpression of four genes (c34671_g1_i1, c35662_g1_i1 or Am4'CGT, DvAKR1 or GmCHR5, CaMYBA, and chalcone 3-hydroxylase from dahlia). Accumulation of butein 4'-O-glucoside and butein 4'-O-[6-O-(malonyl)-glucoside] was detected for c35662_g1_i1, suggesting that c35662_g1_i1 has butein 4'-glucosyltransferase activity. Recombinant enzyme analysis also supported butein 4'-glucosyltransferases activity of c35662_g1_i1. Therefore, our results suggested that both c34671_g1_i1 and c35662_g1_i1 are 6'-deoxychalcone 4'-glucosyltransferases but with different substrate preference.

Homotherapy for heteropathy: therapeutic effect of Butein in NLRP3-driven diseases.

BACKGROUND: Aberrant inflammatory responses drive the initiation and progression of various diseases, and hyperactivation of NLRP3 inflammasome is a key pathogenetic mechanism. Pharmacological inhibitors of NLRP3 represent a potential therapy for treating these diseases but are not yet clinically available. The natural product butein has excellent anti-inflammatory activity, but its potential mechanisms remain to be investigated. In this study, we aimed to evaluate the ability of butein to block NLRP3 inflammasome activation and the ameliorative effects of butein on NLRP3-driven diseases. METHODS: Lipopolysaccharide (LPS)-primed bone-marrow-derived macrophages were pretreated with butein and various inflammasome stimuli. Intracellular potassium levels, ASC oligomerization and reactive oxygen species production were also detected to evaluate the regulatory mechanisms of butein. Moreover, mouse models of LPS-induced peritonitis, dextran sodium sulfate-induced colitis, and high-fat diet-induced non-alcoholic steatohepatitis were used to test whether butein has protective effects on these NLRP3-driven diseases. RESULTS: Butein blocks NLRP3 inflammasome activation in mouse macrophages by inhibiting ASC oligomerization, suppressing reactive oxygen species production, and upregulating the expression of the antioxidant pathway nuclear factor erythroid 2-related factor 2 (Nrf2). Importantly, in vivo experiments demonstrated that butein administration has a significant protective effect on the mouse models of LPS-induced peritonitis, dextran sodium sulfate-induced colitis, and high-fat diet-induced non-alcoholic steatohepatitis. CONCLUSION: Our study illustrates the connotation of homotherapy for heteropathy, i.e., the application of butein to broaden therapeutic approaches and treat multiple inflammatory diseases driven by NLRP3.

Spectral and HPLC Analyses of Synthesized Butin and Butein.

Butin and butein are significant bioactive flavanones derived from plants, existing as tautomers of each other. However, their physicochemical attributes, such as their spectral profiles under varying experimental conditions in aqueous solutions and established chromatographic methods for distinguishing between them, remain undetermined. In this study, we determined the basic properties of butin and butein using conventional spectroscopic, reversed-phase, and chiral HPLC analyses. The spectra of the synthesized butin and butein were analyzed using a UV-Vis spectrophotometer in several solvents with different polarities as well as in aqueous solutions at various pH values. Furthermore, the behavior of the measured spectra was reproduced by calculations to reveal the effects of the solvent and pH on the spectra of butin and butein in organic and aqueous solutions. Subsequently, we assessed the structural stability of butin and butein using reversed-phase HPLC, which revealed that butein is unstable compared with butin in a general culture medium. The synthesized butin was effectively separated into R- and S-isomers with positive and negative Cotton effects, respectively, via HPLC using a chiral column. These findings will aid in uncovering the individual properties of both butin and butein that may have been concealed by their tautomerism and enable the synthesis of S-butin, which is typically challenging and time-consuming to isolate.

Butein, a potential drug for the treatment of bone cancer pain through bioinformatic and network pharmacology.

Bone cancer pain is a difficult-to-treat pathologic condition that impairs the patient's quality of life. The effective therapy options for BCP are restricted due to the unknown pathophysiology. Transcriptome data were obtained from the Gene Expression Omnibus database and differentially expressed gene extraction was performed. DEGs integrated with pathological targets found 68 genes in the study. Butein was discovered as a possible medication for BCP after the 68 genes were submitted to the Connectivity Map 2.0 database for drug prediction. Moreover, butein has good drug-likeness properties. To collect the butein targets, we used the CTD, SEA, TargetNet, and Super-PRED databases. Furthermore, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses revealed butein's pharmacological effects, indicating that butein may aid in treating BCP by altering the hypoxia-inducible factor, NF-kappa B, angiogenesis, and sphingolipid signaling pathways. Moreover, the pathological targets integrated with drug targets were obtained as the shared gene set A, which was analyzed by ClueGO and MCODE. Biological process analysis and MCODE algorithm further analyzed that BCP related targets were mainly involved in signal transduction process and ion channel-related pathways. Next, we integrated targets related to network topology parameters and targets of core pathways, identified PTGS2, EGFR, JUN, ESR1, TRPV1, AKT1 and VEGFA as butein regulated hub genes by molecular docking, which play a critical role in its analgesic effect. This study lays the scientific groundwork for elucidating the mechanism underlying butein's success in the treatment of BCP.

Butein Inhibits the Glycation of α-Crystallin: An Approach in Prevention of Retinopathy.

The aggregation of lens proteins induced by glycation is one of the key drivers of diabetic retinopathy and development of diabetic cataracts. Moreover, glycation also causes numerous alterations not only to the tertiary structure of lens proteins but also to serum proteins. There are also evidences of covalent crosslinking among lens crystallins resulting in development of cataract. In this article, the inhibitory potential of butein was tested against the glucose induced glycation and the aggregation α-crystallin (α-cry). The results showed that there was inhibition of advanced glycation products (78.28%) and early glycation products (86.30%) following the treatment of butein. Additionally, the presence of butein caused a significant improvement in the tested biochemical markers of glycation. The treatment with butein reduced the free lysine modification to 23.67%. The secondary and tertiary structural distortions of α-cry were also protected. The mechanism of inhibition further investigated at the molecular level using biophysical and computational techniques. The interaction data showed the butein exhibited strong affinity towards the α-cry. The binding event was entropically driven and energetically favourable. The Gibb's free energy of the interaction was found to be -5.99 to -7.17 kcal mol-1. The binding site of butein in α-cry was deciphered by molecular docking and the dynamics was studied using molecular dynamics (MD) simulations. The simulation data showed that butein formed stable complex with α-cry under physiological conditions. Most of the tested parameters from molecular simulations, such as secondary structure, was found to be stable. The data clearly show the potential of butein in inhibiting the glycation induced aggregation of α-cry and hence can be developed as useful inhibitor in the management of diabetic cataract and retinopathy.

A novel aldo-keto reductase gene is involved in 6'-deoxychalcone biosynthesis in dahlia (Dahlia variabilis).

MAIN CONCLUSION: A novel gene belonging to the aldo-keto reductase 13 family is involved in isoliquiritigenin biosynthesis in dahlia. The yellow pigments of dahlia flowers are derived from 6'-deoxychalcones, which are synthesized via a two-step process, involving the conversion of 3-malonyl-CoA and 4-coumaloyl-CoA into isoliquiritigenin in the first step, and the subsequent generation of butein from isoliquiritigenin. The first step reaction is catalyzed by chalcone synthase (CHS) and aldo-keto reductase (AKR). AKR has been implicated in the isoflavone biosynthesis in legumes, however, isolation of butein biosynthesis related AKR members are yet to be reported. A comparative RNA-seq analysis between two dahlia cultivars, 'Shukuhai' and its butein-deficient lateral mutant 'Rinka', was used in this study to identify a novel AKR gene involved in 6'-deoxychalcone biosynthesis. DvAKR1 encoded a AKR 13 sub-family protein with significant differential expression levels, and was phylogenetically distinct from the chalcone reductases, which belongs to the AKR 4A sub-family in legumes. DNA sequence variation and expression profiles of DvAKR1 gene were correlated with 6'-deoxychalcone accumulation in the tested dahlia cultivars. A single over-expression analysis of DvAKR1 was not sufficient to initiate the accumulation of isoliquiritigenin in tobacco, in contrast, its co-overexpression with a chalcone 4'-O-glucosyltransferase (Am4'CGT) from Antirrhinum majus and a MYB transcription factor, CaMYBA from Capsicum annuum successfully induced isoliquiritigenin accumulation. In addition, DvAKR1 homologous gene expression was detected in Coreopsideae species accumulating 6'-deoxychalcone, but not in Asteraceae species lacking 6'-deoxychalcone production. These results not only demonstrate the involvement of DvAKR1 in the biosynthesis of 6'-deoxychalcone in dahlia, but also show that 6'-deoxychalcone occurrence in Coreopsideae species developed evolutionarily independent from legume species.

Butein combined with radiotherapy enhances radioresponse of gastric cancer cell by impairing DNA damage repair.

Radiation therapy is common in the current procedures of cancer treatment, but in many cases, radiation resistance of cancerous tissue limits efficacy in clinical applications. Therefore, the use of radiosensitizers has been introduced as an effective strategy to increase the efficiency of radiotherapy. Butein (2', 3, 4, 4'-Tetrahydroxychalcone), a polyphenolic compound of flavonoids family, presents anti-cancer properties and inhibits the signaling pathways associated with radiation resistance. Therefore, we hypothesized that butein in combination with radiation may increase radiosensitivity. To evaluate the radiosensitizing effect of butein, we used MKN-45 cell line and performed several assays such as MTT, soft-agar colony formation, apoptosis, cell cycle, and comet assays. Based on obtained results, butein significantly enhanced radiosensitivity of MKN-45 cells. Butein treatment abrogated the radiation-induced G2/M cell cycle arrest, increased DNA damage, enhanced apoptosis, and reduced colony-forming ability of irradiated cells. This study on MKN-45 cells demonstrates that combination of butein with radiotherapy increases its radiosensitivity by abrogating the radiation-induced G2/M blockage, impairing DNA repair, and enhancing apoptotic and reproductive cell death. Therefore, we suggest butein as a candidate for combination with radiation therapy to decrease dose of radiation delivered to the patients and its corresponding side effects.

Butein induces cellular senescence through reactive oxygen species-mediated p53 activation in osteosarcoma U-2 OS cells.

Butein is a flavonoid isolated from various medicinal plants. It is known to have different biological activities including anti-inflammation, anti-adipogenesis, and anti-angiogenesis. In the study, we demonstrated the anti-proliferative effect of butein in human osteosarcoma U-2 OS cells. Our data showed that butein significantly suppressed the viability and colony formation ability of U-2 OS cells. Further experiments revealed butein exposure resulted in a cell cycle arrest at S and G2/M phase in U-2 OS cells. Importantly, we found that butein activated the tumor suppressor p53, and trigged a p53-dependent senescence in U-2 OS cells. Knockdown of p53 suppressed the senescence and rescued the viability in butein-treated U-2 OS cells. Furthermore, we observed that butein exposure significantly enhanced reactive oxygen species (ROS) levels in U-2 OS cells. Co-administration of the ROS inhibitor NAC largely abolished the up-regulated p53 protein level, and rescued the suppressed viability and colony formation ability in butein-exposed U-2 OS cells. Taken together, our data proposed the increased ROS by butein exposure activated p53, and the activated p53 was involved in the anti-proliferative effect of butein via inducing senescence in U-2 OS cells. This report suggests that butein is a promising candidate for cancer therapy against osteosarcoma.

Butein and Frondoside-A Combination Exhibits Additive Anti-Cancer Effects on Tumor Cell Viability, Colony Growth, and Invasion and Synergism on Endothelial Cell Migration.

Despite the significant advances in targeted- and immuno-therapies, lung and breast cancer are at the top list of cancer incidence and mortality worldwide as of 2020. Combination therapy consisting of a mixture of different drugs taken at once is currently the main approach in cancer management. Natural compounds are extensively investigated for their promising anti-cancer potential. This study explored the anti-cancer potential of butein, a biologically active flavonoid, on two major solid tumors, namely, A549 lung and MDA-MB-231 breast cancer cells alone and in combination with another natural anti-cancer compound, frondoside-A. We demonstrated that butein decreases A549 and MDA-MB-231 cancer cell viability and colony growth in vitro in addition to tumor growth on chick embryo chorioallantoic membrane (CAM) in vivo without inducing any noticeable toxicity. Additionally, non-toxic concentrations of butein significantly reduced the migration and invasion of both cell lines, suggesting its potential anti-metastatic effect. We showed that butein anti-cancer effects are due, at least in part, to a potent inhibition of STAT3 phosphorylation, leading to PARP cleavage and consequently cell death. Moreover, we demonstrated that combining butein with frondoside-A leads to additive effects on inhibiting A549 and MDA-MB-231 cellular viability, induction of caspase 3/7 activity, inhibition of colony growth, and inhibition of cellular migration and invasion. This combination reached a synergistic effect on the inhibition of HUVECs migration in vitro. Collectively, this study provides sufficient rationale to further carry out animal studies to confirm the relevance of these compounds' combination in cancer therapy.

Insights into Intra-Tumoral Heterogeneity: Transcriptional Profiling of Chemoresistant MPM Cell Subpopulations Reveals Involvement of NFkB and DNA Repair Pathways and Contributes a Prognostic Signature.

Chemoresistance is a hallmark of malignant pleural mesothelioma (MPM) management and the expression of ALDH1A3 is responsible for the survival and activity of MPM chemoresistant cell subpopulations (ALDHbright cells). We enriched mesothelioma ALDHbright cells to near homogeneity by FACS sorting and an Aldefluor assay and performed unbiased Affymetrix gene expression profiling. Viability and ELISA assays were used to rule out significant apoptosis in the sorted cell subpopulations and to assess target engagement by butein. Statistical analysis of the results, pathway enrichment and promoter enrichment were employed for the generation of the data. Q-RTPCR was used to validate a subset of the identified, modulated mRNAs In this work, we started from the observation that the mRNA levels of the ALDH1A3 isoform could prognostically stratify MPM patients. Thus, we purified MPM ALDHbright cells from NCI-H2595 cells and interrogated their gene expression (GES) profile. We analyzed the GES of the purified cells at both a steady state and upon treatment with butein (a multifunctional tetrahydroxy-chalcone), which abates the ALDHbright cell number, thereby exerting chemo-sensitizing effects in vitro and in vivo. We identified 924 genes modulated in a statistically significant manner as a function of ALDH status and of the response to the inhibitor. Pathway and promoter enrichment identified the molecular determinant of high ALDH status and how butein treatment altered the molecular portrait of those chemoresistant cell subpopulations. Further, we unraveled an eighteen-gene signature with high prognostic significance for MPM patients, and showed that most of the identified prognostic contributors escaped the analysis of unfractionated samples. This work proves that digging into the unexplored field of intra-tumor heterogeneity (ITH) by working at the cell subpopulation level may provide findings of prognostic relevance, in addition to mechanistic insights into tumor resistance to therapy.

X-ray Structure Determination, Antioxidant Voltammetry Studies of Butein and 2',4'-Dihydroxy-3,4-dimethoxychalcone. Computational Studies of 4 Structurally Related 2',4'-diOH Chalcones to Examine Their Antimalarial Activity by Binding to Falcipain-2.

Malaria is a huge global health burden with resistance to currently available medicines resulting in the search for newer antimalarial compounds from traditional medicinal plants in malaria-endemic regions. Previous studies on two chalcones, homobutein and 5-prenylbutein, present in E. abyssinica, have shown moderate antiplasmodial activity. Here, we describe results from experimental and computational investigations of four structurally related chalcones, butein, 2',4'-dihydroxy-3,4-dimethoxychalcone (DHDM), homobutein and 5-prenylbutein to elucidate possible molecular mechanisms by which these compounds clear malaria parasites. The crystal structures of butein and DHDM show that butein engages in more hydrogen bonding and consequently, more intermolecular interactions than DHDM. Rotating ring-disk electrode (RRDE) voltammetry results show that butein has a higher antioxidant activity towards the superoxide radical anion compared to DHDM. Computational docking experiments were conducted to examine the inhibitory potential of all four compounds on falcipain-2, a cysteine protease that is involved in the degradation of hemoglobin in plasmodium-infected red blood cells of the host. Overall, this work suggests butein as a better antimalarial compound due to its structural features which allow it to have greater intermolecular interactions, higher antioxidant activity and to create a covalent complex at the active site of falcipain-2.

Exploring the 2'-Hydroxy-Chalcone Framework for the Development of Dual Antioxidant and Soybean Lipoxygenase Inhibitory Agents.

2'-hydroxy-chalcones are naturally occurring compounds with a wide array of bioactivity. In an effort to delineate the structural features that favor antioxidant and lipoxygenase (LOX) inhibitory activity, the design, synthesis, and bioactivity profile of a series of 2'-hydroxy-chalcones bearing diverse substituents on rings A and B, are presented. Among all the synthesized derivatives, chalcone 4b, bearing two hydroxyl substituents on ring B, was found to possess the best combined activity (82.4% DPPH radical scavenging ability, 82.3% inhibition of lipid peroxidation, and satisfactory LOX inhibition value (IC50 = 70 µM). Chalcone 3c, possessing a methoxymethylene substituent on ring A, and three methoxy groups on ring B, exhibited the most promising LOX inhibitory activity (IC50 = 45 µM). A combination of in silico techniques were utilized in an effort to explore the crucial binding characteristics of the most active compound 3c and its analogue 3b, to LOX. A common H-bond interaction pattern, orienting the hydroxyl and carbonyl groups of the aromatic ring A towards Asp768 and Asn128, respectively, was observed. Regarding the analogue 3c, the bulky (-OMOM) group does not seem to participate in a direct binding, but it induces an orientation capable to form H-bonds between the methoxy groups of the aromatic ring B with Trp130 and Gly247.

PPAR-Gamma as putative gene target involved in Butein mediated anti-diabetic effect.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder caused due to varied genetic and lifestyle factors. The search for a potential natural compound to enhance the treatment of diabetes is the need of the hour. Butein, a flavonoid, found sufficiently in Faba bean, is said to possess an anti-diabetic property. In-silico analysis, Butein is predicted as a potential anti-diabetic compound, due to its regulatory action on PPAR-Gamma. Based on this evidence, the Butein's anti-diabetic action is studied in diabetic induced rat models. The drug property of Butein is studied through in-silico analysis to determine the metabolic properties. In animal models, the biochemical analysis, histopathological and gene expression against PPAR-Gamma were studied comparatively. Butein being a hydrophobic compound, the bioavailability is said to be minimum. Hence, Butein formulation was made using biopolymer Chitosan for the synergistic anti-diabetic action. The Butein Chitosan formulation was optimized and characterized using analytical techniques. Further, the anti-diabetic activity of Butein and Butein Chitosan formulation was studied in diabetic induced rats. The obtained in-silico analysis results showed that Butein is the most favorable drug. Apparently, in the rat model, Butein and Butein Chitosan formulation effectively controlled the blood glucose levels without any side effects. The histopathological observations of the tissue samples showed nontoxic activity. Additionally, the gene expression analysis predicted the possible mechanism of anti-diabetic action exhibited through the down regulation of PPAR-Gamma. Whereas, the Butein Chitosan formulation failed, to show synergetic anti-diabetic activity as expected. This study is vital in introducing Butein as a safe anti-diabetic compound, which can be used in the treatment of T2DM.

Impact of aminated carbon quantum dots as a novel co-reactant for Ru(bpy)32+: resolving specific electrochemiluminescence for butein detection.

Development of novel nanomaterial-based co-reactant is highly desired for enhancing ECL intensity and widespread analytical applications. Herein, we report the distinct role of amine-functionalized carbon quantum dots (f-CQDs) as a co-reactant, for the first time, augmenting the ECL property of Ru(bpy)32+ and demonstrating for biopharmaceutical (butein) detection. Unlike conventional co-reactants like tripropylamine (TPrA), 2-(dibutylamino)ethanol (DBAE), and pristine CQDs, the f-CQDs as a co-reactant yield superior ECL of Ru(bpy)32+. More importantly, the ECL intensity is independent of types of noble metals, metal oxide surfaces, and dissolved oxygen. Notably, the ECL intensity of Ru(bpy)32+-f-CQDs is linearly quenched with an increased concentration of butein, whereas no changes were observed with conventional co-reactants. ECL functionality of Ru(bpy)32+-f-CQDs has no interference with other similar phytochemicals and antioxidants. Enhanced selectivity is observed due to the formation of polyaminoquinone-like structures, which is confirmed by in situ spectroelectrochemical (UV-vis) and FT-IR studies. The present result envisaged that f-CQDs could be an alternative co-reactant for TPrA/DBAE, raising the ECL of Ru(bpy)32+ suitable for analytical studies. Graphical abstract.

Butein Promotes Lineage Commitment of Bone Marrow-Derived Stem Cells into Osteoblasts via Modulating ERK1/2 Signaling Pathways.

Butein is a phytochemical that belongs to the chalcone family of flavonoids and has antitumor, anti-inflammatory, and anti-osteoclastic bone resorption activities. This study aims to investigate the effects of butein on the differentiation potential of mouse primary bone marrow-derived mesenchymal stem cells (mBMSCs) into osteoblast and adipocyte lineages. Primary cultures of mBMSCs are treated with different doses of butein during its differentiation. Osteoblast differentiation is assessed by alkaline phosphatase (ALP) activity quantification and Alizarin red staining for matrix mineralization, while adipogenesis is assessed by quantification of lipid accumulation using Oil Red O staining. Osteoblastic and adipocytic gene expression markers are determined by quantitative real-time PCR (qPCR). Western blot analysis is used to study the activation of extracellular signal-regulated kinase (ERK1/2). Interestingly, butein promotes the lineage commitment of mBMSCs into osteoblasts, while suppressing their differentiation into adipocytes in a dose-dependent manner. A similar effect of butein is confirmed in human (h) primary BMSCs. Occurring at the molecular level, butein significantly upregulates the mRNA expression of osteoblast-related genes, while downregulating the expression of adipocyte-related genes. The mechanism of butein-induced osteogenesis is found to be mediated by activating the ERK1/2 signaling pathway. To conclude, we identify butein as a novel nutraceutical compound with an osteo-anabolic activity to promote the lineage commitment of BMSCs into osteoblast versus adipocyte. Thus, butein can be a plausible therapeutic drug for enhancing bone formation in osteoporotic patients.

Simultaneous Study of Anti-Ferroptosis and Antioxidant Mechanisms of Butein and (S)-Butin.

To elucidate the mechanism of anti-ferroptosis and examine structural optimization in natural phenolics, cellular and chemical assays were performed with 2'-hydroxy chalcone butein and dihydroflavone (S)-butin. C11-BODIPY staining and flow cytometric assays suggest that butein more effectively inhibits ferroptosis in erastin-treated bone marrow-derived mesenchymal stem cells than (S)-butin. Butein also exhibited higher antioxidant percentages than (S)-butin in five antioxidant assays: linoleic acid emulsion assay, Fe3+-reducing antioxidant power assay, Cu2+-reducing antioxidant power assay, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO•)-trapping assay, and α,α-diphenyl-ß-picrylhydrazyl radical (DPPH•)-trapping assay. Their reaction products with DPPH• were further analyzed using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS). Butein and (S)-butin produced a butein 5,5-dimer (m/z 542, 271, 253, 225, 135, and 91) and a (S)-butin 5',5'-dimer (m/z 542, 389, 269, 253, and 151), respectively. Interestingly, butein forms a cross dimer with (S)-butin (m/z 542, 523, 433, 419, 415, 406, and 375). Therefore, we conclude that butein and (S)-butin exert anti-ferroptotic action via an antioxidant pathway (especially the hydrogen atom transfer pathway). Following this pathway, butein and (S)-butin yield both self-dimers and cross dimers. Butein displays superior antioxidant or anti-ferroptosis action to (S)-butin. This can be attributed the decrease in π-π conjugation in butein due to saturation of its α,ß-double bond and loss of its 2'-hydroxy group upon biocatalytical isomerization.

SIRT1 activation by butein attenuates sepsis-induced brain injury in mice subjected to cecal ligation and puncture via alleviating inflammatory and oxidative stress.

Sepsis-induced brain injury is frequently encountered in critically ill patients with severe systemic infection. Butein (3,4,2',4'-tetrahydroxychalcone) has been demonstrated as the neuro-protective agent via reducing inflammation and oxidative stress on neurons. Moreover, activation of silent information regulator 1 (SIRT1) inhibits apoptosis, oxidation and inflammation thus alleviating sepsis-induced multiorgan injuries. In present study, we show that butein administrated intraperitoneally (10 mg/kg) saved mice from sepsis-induced lethality by increasing 7-day survival rate after cecal ligation and puncture (CLP) surgery. Additionally, butein treatment enhanced SIRT1 signaling thus decreasing the Ac-NF-κB, Ac-FOXO1 and Ac-p53 levels, thus attenuating the brain injury of mice after CLP surgery by decreasing cerebral edema, maintaining the blood-brain barrier integrity, inhibiting neuronal apoptosis, and decreasing pro-inflammatory cytokines production (IL-6, TNF-α and IL-1ß) and oxidative stress (downregulation of MDA, and upregulation of SOD and CAT) in both serum and cerebral cortex tissues. Moreover, butein treatment attenuated LPS induced neurological function loss. However, all above mentioned neuro-protective actions of butein were partially inhibited by EX527 co-treatment, one standard SIRT1 inhibitor. Collectively, butein attenuates sepsis-induced brain injury through alleviation of cerebral inflammation, oxidative stress and apoptosis by SIRT1 signaling activation.

Chalcone skeleton promotes transcription of gp91-phox gene but inhibits expression of gp91-phox protein, and hydroxyl groups in hydroxychalcones participate in the stable expression of gp91-phox protein.

The effects of chalcone and butein on the induction of the superoxide anion (O2 - )-generating system were studied in U937 cells by all-trans retinoic acid (RA). The chalcone skeleton, a common structural motif in them, significantly enhanced the transcription of gp91-phox in an epigenetic manner. In contrast, chalcone and butein showed opposite effects on the induction of the O2 - -generating activity by RA and the expression of gp91-phox protein. Chalcone inhibited, whereas butein promoted, the induction of O2 - -generating activity by RA and the expression of gp91-phox protein. These data raise the possibility that modification of the chalcone skeleton could produce more effective differentiation-promoting agents.

An Improved Method for the Synthesis of Butein Using SOCl2/EtOH as Catalyst and Deciphering Its Inhibition Mechanism on Xanthine Oxidase.

Butein (3,4,2',4'-tetrahydroxychalcone) belongs to the chalcone family of flavonoids and possesses various biological activities. In this study, butein was synthesized through aldol condensation catalyzed by thionyl chloride (SOCl2)/ethyl alcohol (EtOH) for the first time. The optimal reaction conditions including the molar ratio of reactants, the dosage of catalyst, and the reaction time on the yield of product were investigated, and the straightforward strategy assembles the yield of butein up to 88%. Butein has been found to inhibit xanthine oxidase (XO) activity. Herein, the inhibitory mechanism of butein against XO was discussed in aspects of inhibition kinetic, fluorescence titration, synchronous fluorescence spectroscopy, and molecular docking. The inhibition kinetic analysis showed that butein possessed a stronger inhibition on XO in an irreversible competitive manner with IC50 value of 2.93 × 10-6 mol L-1. The results of fluorescence titrations and synchronous fluorescence spectroscopy indicated that butein was able to interact with XO at one binding site, and the fluorophores of XO were placed in a more hydrophobic environment with the addition of butein. Subsequently, the result of molecular docking between butein and XO protein revealed that butein formed hydrogen bonding with the amino acid residues located in the hydrophobic cavity of XO. All the results suggested that the inhibitory mechanism of butein on XO may be the insertion of butein into the active site occupying the catalytic center of XO to avoid the entrance of xanthine and inducing conformational changes in XO.