Integrated metabolome, full-length sequencing, and transcriptome analyses unveil the molecular mechanisms of color formation of the canary yellow and red bracts of Bougainvillea × buttiana 'Chitra'.

Bougainvillea is a typical tropical flower of great ornamental value due to its colorful bracts. The molecular mechanism behind color formation is not well-understood. Therefore, this research conducted metabolome analysis, transcriptome analysis, and multi-flux full-length sequencing in two color bracts of Bougainvillea × buttiana 'Chitra' to investigate the significantly different metabolites (SDMs) and differentially expressed genes (DEGs). Overall, 261 SDMs, including 62 flavonoids and 26 alkaloids, were detected, and flavonols and betalains were significantly differentially accumulated among the two bracts. Furthermore, the complete-length transcriptome of Bougainvillea × buttiana was also developed, which contained 512 493 non-redundant isoforms. Among them, 341 210 (66.58%) displayed multiple annotations in the KOG, GO, NR, KEGG, Pfam, Swissprot, and NT databases. RNA-seq findings revealed that 3610 DEGs were identified between two bracts. Co-expression analysis demonstrated that the DEGs and SDMs involved in flavonol metabolism (such as CHS, CHI, F3H, FLS, CYP75B1, kaempferol, and quercetin) and betacyanin metabolism (DODA, betanidin, and betacyanins) were the main contributors for the canary yellow and red bract formation, respectively. Further investigation revealed that several putative transcription factors (TFs) might interact with the promoters of the genes mentioned above. The expression profiles of the putative TFs displayed that they may positively and negatively regulate the structural genes' expression profiles. The data revealed a potential regulatory network between important genes, putative TFs, and metabolites in the flavonol and betacyanin biosynthesis of Bougainvillea × buttiana 'Chitra' bracts. These findings will serve as a rich genetic resource for future studies that could create new color bracts.

Flower color modification in Torenia fournieri by genetic engineering of betacyanin pigments.

BACKGROUND: Betalains are reddish and yellow pigments that accumulate in a few plant species of the order Caryophyllales. These pigments have antioxidant and medicinal properties and can be used as functional foods. They also enhance resistance to stress or disease in crops. Several plant species belonging to other orders have been genetically engineered to express betalain pigments. Betalains can also be used for flower color modification in ornamental plants, as they confer vivid colors, like red and yellow. To date, betalain engineering to modify the color of Torenia fournieri-or wishbone flower-a popular ornamental plant, has not been attempted. RESULTS: We report the production of purple-reddish-flowered torenia plants from the purple torenia cultivar "Crown Violet."  Three betalain-biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were constitutively ectopically expressed under the cauliflower mosaic virus (CaMV) 35S promoter, and their expression was confirmed by quantitative real-time PCR (qRT-PCR) analysis. The color traits, measured by spectrophotometric colorimeter and spectral absorbance of fresh petal extracts, revealed a successful flower color modification from purple to reddish. Red pigmentation was also observed in whole plants. LC-DAD-MS and HPLC analyses confirmed that the additional accumulated pigments were betacyanins-mainly betanin (betanidin 5-O-glucoside) and, to a lesser extent, isobetanin (isobetanidin 5-O-glucoside). The five endogenous anthocyanins in torenia flower petals were also detected. CONCLUSIONS: This study demonstrates the possibility of foreign betacyanin accumulation in addition to native pigments in torenia, a popular garden bedding plant. To our knowledge, this is the first report presenting engineered expression of betalain pigments in the family Linderniaceae. Genetic engineering of betalains would be valuable in increasing the flower color variation in future breeding programs for torenia.

Exploring antiviral activity of Betanin and Glycine Betaine against dengue virus type-2 in transfected Hela cells.

Dengue virus (DENV) infection is a worldwide public health concern infecting approximately 400 million individuals and about 40,000 mortalities yearly. Despite this, no licensed or readily available antiviral medication is currently available specifically for DENV infection, and therapy is typically symptomatic. Therefore, the objective of the study was to investigate the antiviral activity of Beta vulgaris L. phytoconstituents against DENV-2 targeting NS3 protein. The antiviral activity of phytochemicals was examined through virtual ligand-based screening, antiviral inhibition and dosage response assays, western blotting analysis and MD simulations. We conducted toxicological, and pharmacokinetic analysis to assess plant-based natural compound's efficacy, safety, and non-toxic doses. Molecular docking and MD simulation results revealed that the nonstructural protein-3 (NS3) might prove as a funamental target for Betanin and Glycine Betaine against Dengue virus. Betanin and Glycine betaine were initially studied for their non-toxic doses in HeLa, CHO, and Vero cells via MTT assay. HeLa cells were transiently transfected with cloned vector pcDNA3.1/Zeo(+)/DENV-2 NS3 along with non-toxic doses (80 µM-10 µM) of selected phytochemicals. The dose-response assay illustrated downregulated expression of DENV-2 NS3 gene after administration of Betanin (IC50 = 4.35 µM) and Glycine Betaine (IC50 = 4.49 µM). Dose response analysis of Betanin (80 µM-10 µM) depicted the significant inhibition of NS3 protein expression as well. These results suggested downregulated expression of DENV-2 NS3 at mRNA and protein level portraying the DENV replication inhibition. Based on our study findings, NS3 protease is depicted as distinctive DENV-2 inhibitor target. We will channel our study further into in vitro characterization employing the mechanistic study to understand the role of host factors in anti-flavi therapeutic.

Protective effects of betanin, a novel acetylcholinesterase inhibitor, against H2O2-induced apoptosis in PC12 cells.

BACKGROUND: Dysfunction of the cholinergic system and increased oxidative stress have a crucial role in cognitive disorders including Alzheimer's disease (AD). Here, we have investigated the protective effects of betanin, a novel acetylcholinesterase (AChE) inhibitor, on hydrogen peroxide (H2O2)-induced cell death in PC12 cells. METHODS AND RESULTS: The protective effects were assessed by measuring cell viability, the amount of reactive oxygen species (ROS) production, AChE activity, cell damage, and apoptosis using resazurin, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), Ellman method, lactate dehydrogenase (LDH) release, propidium iodide (PI) staining and flow cytometry, and Western blot analysis. H2O2 (150 µM) resulted in cell viability reduction and apoptosis induction while, pretreatment with the betanin (10, 20, and 50 µM) and N-Acetyl-L-cysteine (NAC) (2.5 and 5 mM) significantly increased the viability (P < 0.05, P < 0.01 and P < 0.001) and at 5-50 µM betanin decreased ROS amount (P < 0.05, P < 0.01 and P < 0.001). Whereas, pretreatment with the betanin (10, 20, and 50 µM) decreased AChE activity (P < 0.001), also at 20 and 50 µM betanin reduced the release of LDH (P < 0.001), and at 10-50 µM decreased the percentage of apoptotic cells (P < 0.001). Apoptosis biomarkers such as cleaved poly (ADP-ribose) polymerase (PARP) (P < 0.01 and P < 0.001) and cytochrome c (P < 0.05 and P < 0.001) were attenuated after pretreatment of PC12 cells with betanin at 10-20 µM and 10-50 µM respectively. Indeed, survivin (P < 0.001) increased after pretreatment of cells with betanin at 10-20 µM. CONCLUSIONS: Overall, betanin may use the potential to delay or prevent cell death caused by AD through decreasing the activity of AChE as well as attenuating the expression of proteins involved in the apoptosis pathway.

Elucidating the therapeutic mechanism of betanin in Alzheimer's Disease treatment through network pharmacology and bioinformatics analysis.

Betanin, a natural compound with anti-inflammatory and antioxidant properties, has shown promise in mitigating Alzheimer's disease (AD) by reducing amyloid plaque production. Employing network pharmacology, this study aimed to elucidate betanin's therapeutic mechanism in AD treatment. Through integrated analyses utilizing SwissTargetPrediction, STITCH, BindingDB, Therapeutic Target Database (TTD), and OMIM databases, potential protein targets of betanin in AD were predicted. Gene ontology analysis facilitated the identification of 49 putative AD targets. Subsequent gene enrichment and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed associations between these targets and AD. Network pharmacology techniques and molecular docking aided in prioritizing essential genes, with APP, CASP7, ITPR1, CASP8, CASP3, ITPR3, and NF-KB1 emerging as top candidates. The results provide novel insights into betanin's therapeutic efficacy, shedding light on its potential clinical application in AD treatment. By targeting key genes implicated in AD pathology, betanin demonstrates promise as a valuable addition to existing therapeutic strategies. This holistic approach emphasizes the relevance of network pharmacology and bioinformatics analysis in understanding natural chemical disease therapy processes.

Semisynthesis of Betaxanthins from Purified Betacyanin of Opuntia dillenii sp.: Color Stability and Antiradical Capacity.

The availability of pure individual betalains in sufficient quantities which permit deeper understanding is still a challenge. This study investigates the high-yielding semisynthesis of betaxanthins using betalamic acid from a natural source (Opuntia dillenii), followed by condensation with ʟ-amino acids and further purification. Moreover, the color stability of the four synthesized individual betaxanthins, namely proline (ʟ-ProBX), alanine (ʟ-AlaBX), leucine (ʟ-LeuBX), and phenylalanine (ʟ-PheBX) betaxanthins, was investigated at different pHs. Their relative contribution to free radical scavenging was also scrutinized by TEAC and DPPH. ʟ-AlaBX and ʟ-LeuBx showed a significantly (p < 0.05) higher antioxidant activity, whereas ʟ-ProBX was the most resistant to the hydrolysis of betaxanthin and hence the least susceptible to color change. The color stability was strongly influenced by pH, with the color of ʟ-ProBX, ʟ-LeuBX, and ʟ-AlaBX at pH 6 being more stable, probably due to the easier hydrolysis under acid conditions. The semisynthesis and purification allowed us to have available remarkable quantities of pure individual betaxanthins of Opuntia dillenii for the first time, and to establish their color properties and antioxidant capacity. This study could be a step forward in the development of the best natural food colorant formulation, based on the betalain structure, which is of special interest in food technology.

Effect of betanin synthesis on photosynthesis and tyrosine metabolism in transgenic carrot.

BACKGROUND: Betalain is a natural pigment with important nutritional value and broad application prospects. Previously, we produced betanin biosynthesis transgenic carrots via expressing optimized genes CYP76AD1S, cDOPA5GTS and DODA1S. Betanin can accumulate throughout the whole transgenic carrots. But the effects of betanin accumulation on the metabolism of transgenic plants and whether it produces unexpected effects are still unclear. RESULTS: The accumulation of betanin in leaves can significantly improve its antioxidant capacity and induce a decrease of chlorophyll content. Transcriptome and metabolomics analysis showed that 14.0% of genes and 33.1% of metabolites were significantly different, and metabolic pathways related to photosynthesis and tyrosine metabolism were markedly altered. Combined analysis showed that phenylpropane biosynthesis pathway significantly enriched the differentially expressed genes and significantly altered metabolites. CONCLUSIONS: Results showed that the metabolic status was significantly altered between transgenic and non-transgenic carrots, especially the photosynthesis and tyrosine metabolism. The extra consumption of tyrosine and accumulation of betanin might be the leading causes.

Metabolic engineering of betacyanin in vegetables for anti-inflammatory therapy.

Betalains, which consist of the subgroups betaxanthins and betacyanins, are hydrophilic pigments that have classically been used for food colorants. Owing to their strong antioxidant property, their usefulness for application for therapeutic use is also expected. In addition, as betalains are mainly naturally available from plants of the order Caryophyllales, including beet (Beta vulgaris), metabolic engineering for betalain production in crops such as vegetables, fruits and cereals may provide new food resources useful for healthcare. Here we conducted metabolic engineering of betacyanins in tomato fruits and potato tubers. The transgenic tomato fruits and potato tubers with coexpression of betacyanin biosynthesis genes, CYP76AD1 from B. vulgaris, DOD (DOPA 4,5-dioxygenase) and 5GT (cyclo-DOPA 5-O-glucosyltransferase) from Mirabilis jalapa, under control of suitable specific promoters, possessed dark red tissues with enriched accumulation of betacyanins (betanin and isobetanin). The anti-inflammatory activity of transgenic tomato fruit extract was superior to that of wild-type fruit extract on macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS), as a result of decreased LPS-stimulated transcript levels of proinflammatory genes. These findings were in accord with the observation that administration of the transgenic tomato fruits ameliorated dextran sulfate sodium (DSS)-induced colitis as well as body weight loss and disease activity index in mice, via suppression of DSS-stimulated transcript levels of pro-inflammatory genes, including Tnf (encoding TNF-alpha), Il6, and Ptgs2 (encoding cyclooxygenae 2). Intriguingly, given the fact that the transgenic potato tuber extract failed to enrich the anti-inflammatory activity of macrophage cells, it is likely that metabolic engineering of betacyanins will be a powerful way of increasing the anti-inflammatory property of ordinary foods such as tomato.

The synergistic effects of betanin and radiotherapy in a prostate cancer cell line: an in vitro study.

BACKGROUND: Prostate cancer is among the most common cancers in men with an increasing incidence rate. Radiation therapy (RT) is a therapeutic strategy for the management of prostate cancer after surgery; nonetheless, it has different side effects on neighboring healthy cells/tissues. Moreover, radioresistance has been an increasing phenomenon in the recent years. Therefore, there is an urgent need for the introduction of a safe and effective radiosensitizing agent. Accordingly, the recent trend in the development of novel drugs is accompanied by a push toward natural compounds. Our study evaluated the effects of betanin combined with RT as a potential radiosensitizing agent in the PC-3 cell line. METHODS AND RESULTS: MTT assay was utilized to determine the growth inhibitory impact of betanin. The possible synergistic effect was evaluated with CompuSyn software upon Trypan blue exclusion test. Apoptosis-related gene expression was evaluated via Real-time PCR and the protein expression of P21 was determined using western blotting. A synergistic anticancer effect with an optimal combination index of 0.61 was achieved by treating PC-3 cells with betanin and RT. The results pointed out that betanin synergistically triggered RT-mediated apoptosis and cell cycle arrest through modulating gene and protein expression in comparison with each of the monotherapies. CONCLUSION: These findings shed light on the synergistic antitumor effect of betanin and RT in prostate cancer, indicating the potential use of betanin as a radiosensitizer agent.

Exploring the possible mitoprotective and neuroprotective potency of thymoquinone, betanin, and vitamin D against cytarabine-induced mitochondrial impairment and neurotoxicity in rats' brain.

It has been suggested that cytarabine (Ara-C) induces toxicity via mitochondrial dysfunction and oxidative stress. Therefore, we hypothesized that mitochondrial protective agents and antioxidants can reduce cytarabine-induced neurotoxicity. For this purpose, 48 male Wistar rats were assigned into eight equal groups include control group, Ara-C (70 mg/kg, i.p.) group, Ara-C plus betanin (25 mg/kg, i.p.) group, Ara-C plus vitamin D (500 U/kg, i.p.) group, Ara-C plus thymoquinone (0.5 mg/kg, i.p.) group, betanin group, vitamin group, and thymoquinone group. The activity of acetylcholinesterase (AChE), and butyrylcholinesterase (BChE), the concentrations of antioxidants (reduced glutathione and oxidized glutathione), oxidative stress (malondialdehyde) biomarkers, mitochondrial toxicity parameters as well as histopathological alteration in brain tissues were measured. Our results demonstrated that Ara-C exposure significantly declines the brain enzymes activity (AChE and BChE), levels of antioxidant biomarkers (GSH), and mitochondrial functions, but markedly elevate the levels of oxidative stress biomarkers (MDA) and mitochondrial toxicity. Almost all of the previously mentioned parameters (especially mitochondrial toxicity) were retrieved by betanin, vitamin D, and thymoquinone compared to Ara-C group. These findings conclusively indicate that betanin, vitamin D, and thymoquinone administration provide adequate protection against Ara-C-induced neurotoxicity through modulations of oxidative, antioxidant activities, and mitochondrial protective (mitoprotective) effects.