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.

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.

Traversing through the cell signaling pathways of neuroprotection by betanin: therapeutic relevance to Alzheimer's Disease and Parkinson's Disease.

Modulation of cell signaling pathways is the key area of research towards the treatment of neurodegenerative disorders. Altered Nrf2-Keap1-ARE (Nuclear factor erythroid-2-related factor 2-Kelch-like ECH-associated protein 1-Antioxidant responsive element) and SIRT1 (Sirtuin 1) cell signaling pathways are considered to play major role in the etiology and pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD). Strikingly, betanin, a betanidin 5-O-ß-D-glucoside compound is reported to show commendable anti-oxidative, anti-inflammatory and anti-apoptotic effects in several disease studies including AD and PD. The present review discusses the pre-clinical studies demonstrating the neuroprotective effects of betanin by virtue of its potential to ameliorate oxidative stress, neuroinflammation, abnormal protein aggregation and cell death. It highlights the direct linkage between the neuroprotective abilities of betanin and upregulation of the Nrf2-Keap1-ARE and SIRT1 signaling pathways. The review further hypothesizes the involvement of the betanin-Nrf2-ARE route in the inhibition of beta-amyloid aggregation through beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), one of the pivotal hallmarks of AD. The present review hereby for the first time elaborately discusses the reported neuroprotective abilities of betanin and decodes the Nrf2 and SIRT1 modulating potential of betanin as a primary mechanism of action behind, hence highlighting it as a novel drug candidate for the treatment of neurodegenerative diseases in the near future.

Betanin inhibits PI3K/AKT/mTOR/S6 signaling pathway, cell growth and death in osteosarcoma MG-63 cells.

It is possible to develop new chemopreventive compounds so that cancer cells can be targeted in an exclusive manner. Bioactive natural compounds have demonstrated to be efficient chemotherapeutic agents, safe and cost-effective. Majority of anti-cancer medications are derived from natural sources, particularly of plant origins. Betanin (betanidin-5-O-ß-glucoside) is the most common betacyanin with antioxidant, anti inflammatory and anticancer properties. The present study therefore investigated the effect of betanin onosteosarcoma MG-63 cells. The mechanistic pathway of inflammatory responses, cell proliferation and apoptosis were investigated. The MG-63 cells were treated with betanin for 24 h. Betanin actions on the appearance of cell arrangements, morphological changes, ROS induced Δψm , cell migration, cell adhesion and proliferative mechanistic marker expression of PI3K/AKT/mTOR/S6were analyzed. Betanin inhibited MG-63 cells at IC50 concentrations between 9.08 and 54.49 µM and induced apoptosis by triggering the ROS mechanism. Betanin inhibited proliferation and migration of MG-63 cells and induced DNA fragmentation. Betanin also modified the key mediator expression levels of PI3K/AKT/mTOR/S6 signaling pathways. Betanin can potentially be utilized in bone carcinoma therapeutics to inhibit, reverse or delay osteosarcoma.

Dissociation Mode of the O-H Bond in Betanidin, pKa-Clusterization Prediction, and Molecular Interactions via Shape Theory and DFT Methods.

Betanidin (Bd) is a nitrogenous metabolite with significant bioactive potential influenced by pH. Its free radical scavenging activity and deprotonation pathway are crucial to studying its physicochemical properties. Motivated by the published discrepancies about the best deprotonation routes in Bd, this work explores all possible pathways for proton extractions on that molecule, by using the direct approach method based on pKa. The complete space of exploration is supported by a linear relation with constant slope, where the pKa is written in terms of the associated deprotonated molecule energy. The deprotonation rounds 1, …, 6 define groups of parallel linear models with constant slope. The intercepts of the models just depend on the protonated energy for each round, and then the pKa can be trivially ordered and explained by the energy. We use the direct approximation method to obtain the value of pKa. We predict all possible outcomes based on a linear model of the energy and some related verified assumptions. We also include a new measure of similarity or dissimilarity between the protonated and deprotonated molecules, via a geometric-chemical descriptor called the Riemann-Mulliken distance (RMD). The RMD considers the cartesian coordinates of the atoms, the atomic mass, and the Mulliken charges. After exploring the complete set of permutations, we show that the successive deprotonation process does not inherit the local energy minimum and that the commutativity of the paths does not hold either. The resulting clusterization of pKa can be explained by the local acid and basic groups of the BD, and the successive deprotonation can be predicted by using the chemical explained linear models, which can avoid unnecessary optimizations. Another part of the research uses our own algorithm based on shape theory to determine the protein's active site automatically, and molecular dynamics confirmed the results of the molecular docking of Bd in protonated and anionic form with the enzyme aldose reductase (AR). Also, we calculate the descriptors associated with the SET and SPLET mechanisms.

Isolation of High-Purity Betanin from Red Beet and Elucidation of Its Antioxidant Activity against Peroxynitrite: An in vitro Study.

Reactive oxygen species and reactive nitrogen species (RNS) are damaging for many biomolecules. Peroxynitrite (ONOO-) is the most toxic molecular species among RNS. Betalains are known to possess ONOO- scavenging ability. Betanin, a betalain isolated from red beet, possesses antioxidant, anti-inflammatory, and antitumor activities; however, detailed studies of this isolated pigment have not been conducted, owing to its instability under physiological conditions. This study aimed to isolate highly purified betanin from red beetroots using an improved purification method involving deproteinization and citric acid co-precipitation and evaluated its antioxidant activities. The purified betanin thus obtained had a significantly lower isobetanin content than the commercially available betanin dyes. The antioxidant activity of purified betanin examined in the 2,2-diphenyl-1-picrylhydrazyl assay, the direct ONOO- reaction, ONOO--dependent DNA damage, and lipid peroxidation reactions revealed that betanin possessed higher antioxidant capacity than general antioxidants such as ascorbic acid and quercetin. Furthermore, betanin showed indirect and direct cytoprotective effects against H2O2 and ONOO- cytotoxicity, respectively, in cultured mouse fibroblasts. To the best of our knowledge, this is the first study to demonstrate the cytoprotective effects of betanin against ONOO- toxicity. The highly purified betanin obtained in this study will aid in further exploring its physiological functions.

A Comparative Study of Ethanol and Citric Acid Solutions for Extracting Betalains and Total Phenolic Content from Freeze-Dried Beetroot Powder.

This research compares the extraction of betalains (betacyanin and betaxanthin) and total phenolic content using citric acid and aqueous-ethanol solutions. The aim is to find an environmentally sustainable alternative solvent for extracting these compounds from dried beetroot powder. Using citric acid solution as a solvent offers several benefits over ethanol. Citric acid is a weak organic acid found naturally in citrus fruits, making it a safe and environmentally friendly choice for certain extraction processes. Moreover, the use of citric acid as solvent offers biodegradability, non-toxicity, non-flammability, and is cost effective. A full factorial design and response surface methodology (RSM) were employed to assess the effects of extraction parameters (extraction time (5-30 min), extraction temperature (20, 30, 40 °C), pH of citric acid solution (3, 4, 5) and ethanol concentration (10, 20, 30% v/v)). The yield was determined spectrophotometrically and expressed as mg/g of dry powder. The results showed that citric acid solution yielded 85-90% of the ethanolic extract under identical conditions. The maximum yields of betacyanin, betaxanthin, and total phenolic content in citric acid solution were 3.98 ± 0.21 mg/g dry powder, 3.64 ± 0.26 mg/g dry powder, and 8.28 ± 0.34 mg/g dry powder, respectively, while aqueous-ethanol yielded 4.38 ± 0.17 mg/g dry powder, 3.95 ± 0.22 mg/g dry powder, and 8.45 ± 0.45 mg/g dry powder. Optimisation resulted in maximum extraction yields of 90% for betalains and 85% for total phenolic content. The study demonstrates the potential of citric acid as a viable alternative to polar organic solvents for extracting phytochemicals from plant material, providing comparable results to aqueous-ethanol. Artificial Neural Network (ANN) models outperformed RSM in predicting extraction yields. Overall, this research highlights the importance of exploring bio-solvents to enhance the environmental sustainability of phytochemical extraction.

Characterization and Application of Guar Gum/Polyvinyl Alcohol-Based Food Packaging Films Containing Betacyanins from Pokeweed (Phytolacca acinosa Roxb.) Berries and Silver Nanoparticles.

Food packaging films were prepared by using guar gum/polyvinyl alcohol (GP) as the film matrix, 2% Ag nanoparticles (AgNPs) as reinforcing filler and antimicrobial agent, and 1%, 2% and 3% pokeweed betacyanins (PB) as the colorant and antioxidant agent. The structures and color-changing, barrier, mechanical, thermal and antioxidant/antibacterial properties of different films were measured. The results show that the PB were pH-sensitive pigments with pink, purple and yellow colors at pH 3-8, pH 9-11 and pH 12, respectively. PB improved the compatibility of guar gum and polyvinyl alcohol through hydrogen bonds. The films with PB showed a color-changing capacity under ammonia vapor and good color stability in chilled storage. AgNPs and PB elevated the barrier capacity of GP film to light, water vapor and oxygen gas. Meanwhile, AgNPs and PB improved the stiffness, thermal stability and antioxidant/antibacterial activity of GP film. The film with AgNPs and 3% PB showed the highest barrier capacity, stiffness, thermal stability and antioxidant/antimicrobial activity. In shrimp spoilage test, the films with AgNPs and 2% and 3% PB indicated shrimp freshness through film color changes. The results reveal the potential use of the prepared films in active and smart packaging.

Influence of High-Pressure Homogenization on the Physicochemical Properties and Betalain Pigments of Red Beetroot (Beta vulgaris L.) Juice.

High-pressure homogenization (HPH) is considered an innovative and modern method of processing and preserving liquid and semi-liquid foods. The aim of this research was to examine the impact of HPH processing on the content of betalain pigments and physicochemical properties of beetroot juice. Combinations of the following HPH parameters were tested: the pressure used (50, 100, 140 MPa), the number of cycles (1 and 3) and the applied cooling or no cooling. The physicochemical analysis of the obtained beetroot juices was based on the determination of the extract, acidity, turbidity, viscosity and color values. Use of higher pressures and a greater number of cycles reduces the turbidity (NTU) of the juice. Moreover, in order to maintain the highest possible extract content and a slight color change of the beetroot juice, it was crucial to perform sample cooling after the HPH process. The quantitative and qualitative profiles of betalains have been also determined in the juices. In terms of the content of betacyanins and betaxanthins, the highest values were found in untreated juice at 75.3 mg and 24.8 mg per 100 mL, respectively. The high-pressure homogenization process resulted in a decrease in the content of betacyanins in the range of 8.5-20.2% and of betaxanthins in the range of 6.5-15.0%, depending on the parameters used. Studies have shown that that the number of cycles was irrelevant, but an increase in pressure from 50 MPa to 100 or 140 MPa had a negative effect on pigment content. Additionally, juice cooling significantly limits the degradation of betalains in beetroot juice.

Betanin mitigates scopolamine-induced cognitive impairment by restoring cholinergic function, boosting brain antioxidative status, and increasing BDNF level in the zebrafish model.

Betalains obtained from Beta vulgaris (family Caryophyllales) are regularly consumed as part of the regular diet with medicinal benefits due to antioxidant and anti-inflammatory properties. The objective of this article was to evaluate betanin's neuroprotective properties in a scopolamine-induced zebrafish paradigm. Betanin (BET) (50, 100, and 200 mg/L), and donepezil (10 mg/L) were delivered to zebrafish in a treatment tank once a day for 8 days, while memory impairment was produced by scopolamine (100 µM), which was given 60 min before behavioral assessments. The treatment dosages were determined based on acute toxicity studies. The existence of betacyanin and betaxanthins of BET was tested using liquid chromatography-mass spectrometry (LC-MS). The Y-maze task was used to examine the novelty and spatial memory, while the novel tank diving test was used to assess anxiety-like behavior (NTT). The activities of acetylcholinesterase (AChE) and the oxidative stress sensitivity in zebrafish brains were examined. Also, brain-derived neurotrophic factor (BDNF) level is quantified by an ELISA kit. Scopolamine-induced rises in AChE activity, memory loss, anxiety, and brain oxidant capacity were all reduced by BET. These results suggest that BET (50 and 100 mg/L) has a therapeutic ability to treat brain oxidative stress and cognitive deficits in amnesic zebrafish.

Anti-Amyloid ß Aggregation Activity and Cell Viability Effect of Betacyanins from Red Pitahaya (Hylocereus polyrhizus) for Alzheimer's Disease.

Betacyanin-rich extract from red beet (Beta vulgaris) was recently reported to inhibit amyloid ß (Aß) aggregation, a main pathological event in Alzheimer's disease. However, the anti-Aß aggregation effect of individual betacyanin isolates has not been reported before. This study investigated the anti-Aß aggregation activity and cytotoxicity of betacyanins from red pitahaya or red dragon fruit (Hylocereus polyrhizus). Betacyanin fraction (IC50 = 16.02 ± 1.15 µg/mL) and individual betacyanin isolates exhibited anti-Aß aggregation activity in a concentration-dependent manner using a thioflavin T fluorescence assay. The highest to lowest IC50 was in the order of betanin (426.30 ± 29.55 µM), phyllocactin (175.22 ± 1.52 µM), and hylocerenin (131.73 ± 5.58 µM), following a trend of increase in functional groups of carboxyl, hydroxyl, and/or carbonyl. Further, the betacyanin fraction of 135.78 µg/mL and below, which were concentrations with an anti-Aß aggregation effect, were validated as non-neurotoxic based on an in vitro cytotoxicity assay using human neuroblastoma (SH-SY5Y) cells. These findings highlight the potential neuroprotective activity of betacyanins for Alzheimer's disease.

[Natural pigments in fruit and vegetable juices: the content of anthocyanins, carotenoids and betalaines].

The color of the juice is determined by the color of the corresponding fruit or vegetable from which the juice is made. The color of a fruit or vegetable, in turn, is determined by the presence of natural coloring pigments - secondary plant metabolites, which include mainly anthocyanins, carotenoids and betalains. These substances, in addition to bright colors, give the juices properties that largely provide a positive effect on health. The quantitative content of these pigments in juices (especially in commercially produced juices, the most commonly consumed by the population at present) is important for understanding of the contribution of that juices in real intake of these bioactive compounds with diet. The purpose of the work was to study the content of anthocyanins, carotenoids and betalaines in juices a nd nectars (cherry, pomegranate, red grapes, tomato, carrot, peach and vegetable juices containing red beetroot) widely represented on the Russian market. Material and methods. The content of natural coloring pigments was determined by HPLC: anthocyanins - according to GOST 32709-2014 "Juice products. Methods for the determination of anthocyanins¼, carotenoids - in accordance with R 4.1.1672-03 "Guidelines for methods of quality control and safety of biologically active food supplements", betalains by revised IFU method of analysis No 71 (rev. 2023) "Anthocyanins and Betalains by HPLC". The results of measurements in 66 samples selected from Russian retail chains were analyzed. Results. The highest content of anthocyanins (in terms of cyanidin-3-O-glucoside) was found in cherry nectars - an average of 11.4 mg/100 cm3, lower values were obtained for red grape juices (an average of 2.5 mg/100 cm3) and pomegranate juices (0.9 mg/100 cm3). In tomato juices, in addition to lycopene (7.0-14.1 mg/100 cm3), ß-carotene was found in an amount of 0.3- 1.2 mg/100 cm3. In carrot juices, the content of ß-carotene was at the level of 5.7-12.5 mg/100 cm3, in peach nectars - 0.14- 0.38 mg/100 cm3. The highest concentrations of betalains were found in directly pressed red beet juice at a level of 156.2 mg/100 cm3, with a predominance of betacyanins (99.4 mg/100 cm3) over betaxanthins (56.8 mg/100 cm3). The content of betacyanins decreased to 51.5 mg/100 cm3 in directly pressed lacto-fermented juice and to 2-3 mg/100 cm3 in multi-vegetable juices; betaxanthins were not detected in these samples. Conclusion. The study showed high levels of natural coloring substances - anthocyanins, carotenoids and in some extent betalains in juice products. A serving (200 cm3) of cherry nectar can provide up to 100% of an adequate daily intake of anthocyanins, a serving of red grape juice and a serving of pomegranate juice can provide up to 20% and up to 10%, respectively. The content of ß-carotene in a serving of carrot juice is several times higher than the daily requirement for adults; a serving of peach nectar contains up to 10% of the daily requirement for ß-carotene. Tomato juice is rich in lycopene, this carotenoid content in a serving is several times higher than the adequate daily intake, while the content of ß-carotene is also at a high level - up to 50% of the daily requirement for this substance. Despite the fact that direct data on the adequate daily intake of betalains have not yet been established, relatively high concentrations of betalains, especially in directly pressed red beet juices, determine their significant potential in increasing the nutritive value of the diet through beet-based juices intake.

Efficient assembly of long DNA fragments and multiple genes with improved nickase-based cloning and Cre/loxP recombination.

Functional genomics, synthetic biology and metabolic engineering require efficient tools to deliver long DNA fragments or multiple gene constructs. Although numerous DNA assembly methods exist, most are complicated, time-consuming and expensive. Here, we developed a simple and flexible strategy, unique nucleotide sequence-guided nicking endonuclease (UNiE)-mediated DNA assembly (UNiEDA), for efficient cloning of long DNAs and multigene stacking. In this system, a set of unique 15-nt 3' single-strand overhangs were designed and produced by nicking endonucleases (nickases) in vectors and insert sequences. We introduced UNiEDA into our modified Cre/loxP recombination-mediated TransGene Stacking II (TGSII) system to generate an improved multigene stacking system we call TGSII-UNiE. Using TGSII-UNiE, we achieved efficient cloning of long DNA fragments of different sizes and assembly of multiple gene cassettes. Finally, we engineered and validated the biosynthesis of betanin in wild tobacco (Nicotiana benthamiana) leaves and transgenic rice (Oryza sativa) using multigene stacking constructs based on TGSII-UNiE. In conclusion, UNiEDA is an efficient, convenient and low-cost method for DNA cloning and multigene stacking, and the TGSII-UNiE system has important application prospects for plant functional genomics, genetic engineering and synthetic biology research.

Betanin as a multipath oxidative stress and inflammation modulator: a beetroot pigment with protective effects on cardiovascular disease pathogenesis.

Oxidative stress is a common physiopathological condition enrolled in risk factors for cardiovascular diseases. Individuals in such a redox imbalance status present endothelial dysfunctions and inflammation, reaching the onset of heart disease. Phytochemicals are able to attenuate the main mechanisms of oxidative stress and inflammation and should be considered as supportive therapies to manage risk factors for cardiovascular diseases. Beetroot (Beta vulgaris L.) is a rich source of bioactive compounds, including betanin (betanidin-5-O-ß-glucoside), a pigment displaying the potential to alleviate oxidative stress and inflammantion, as previously demonstrated in preclinical trials. Betanin resists gastrointestinal digestion, is absorbed by the epithelial cells of intestinal mucosa and reaches the plasma in its active form. Betanin displays free-radical scavenger ability through hydrogen or electron donation, preserving lipid structures and LDL particles while inducing the transcription of antioxidant genes through the nuclear factor erythroid-2-related factor 2 and, simultaneously, suppressing the pro-inflammatory nuclear factor kappa-B pathways. This review discusses the anti-radical and gene regulatory cardioprotective activities of betanin in the pathophysiology of endothelial damage and atherogenesis, the main conditions for cardiovascular disease. In addition, betanin influences on these multipath cellular signals and aiding in reducing cardiovascular disorders is proposed.

Betanin alleviates oxidative stress through the Nrf2 signaling pathway in the liver of STZ-induced diabetic rats.

BACKGROUND: Continuing hyperglycemia causes and exacerbate oxidative stress. Betanin as the principal pigment of red beet root has antioxidant, anti-inflammatory, and anti-diabetic properties. The purpose of this study was to investigate the potency of betanin on antioxidant defense in STZ-induced diabetic rats' livers. METHODS: STZ at a single dose of 60 mg/kg body weight was intraperitoneally injected and betanin (10, 20, and 40 mg/kg/day) was administered orally for 28 days. Malondialdehyde (MDA), total antioxidant capacity (TAC), protein carbonyl (PC) levels, and the enzyme activity of superoxide dismutase (SOD), catalases and glutathione peroxidases (GPx) were evaluated in the liver. Furthermore, gene expression of Nrf2 and mentioned antioxidant enzymes were measured by Real-time PCR. RESULTS: Betanin (10 and 20 mg/kg) significantly reduced PC levels and increased antioxidant enzyme activity in diabetic rats compared to the control diabetic group (P < 0.01). In comparison to the diabetic control group, all studied genes expression in diabetic rats were increased significantly with betanin at doses of 10 and 20 mg/kg (P < 0.02). The increase in gene expression at 20 mg/kg of betanin was significantly stronger than others (P < 0.015) except for the catalase (P = 0.201), that was almost the same. Moreover, treatment of diabetic rats with 20 mg/kg of betanin could significantly increase TAC levels (P < 0.05) and decrease MDA levels (P < 0.001) compared to diabetic control group. CONCLUSIONS: Betanin could increase the antioxidant capacity of liver tissue associated with the Nrf2-mediated pathway in a dose-dependent manner.