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An analytical approach employing headspace sorptive extraction coupled with gas chromatography-mass spectrometry (HSSE-GC-MS) has been successfully developed for the determination of apocarotenoid volatiles arising from the enzymatic activity of carotenoid cleavage enzymes (CCDs) in Escherichia coli. The GjCCD4a enzyme derived from gardenia, known for its cleavage specificity at 7,8 and 7',8' double bonds across diverse carotenoid substrates, was utilized as a reference enzyme, using ß-carotene as the substrate for the enzymatic activity assays. Optimal headspace conditions for analysis were established following a 5 hours induction period of the recombinant GjCCD4a protein within E. coli cells, engineered to produce ß-carotene. The analytical method demonstrated linearity, with correlation coefficient (R2 > 0.95) in calibration, while achieving detection and quantification limits conducive to the accurate determination of ß-cyclocitral. Notably, this methodological framework significantly reduced both the handling complexity and sample processing time in comparison to conventional liquid chromatography methods employed for the detection of cleavage products and determination of CCD activities. The proposed HSSE-GC-MS approach not only enhances the efficiency of apocarotenoid analysis but also provides a sensitive means for unraveling the intricate enzymatic processes associated with CCD-mediated carotenoid cleavage in a bacterial model system.
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Dioxigenases , Escherichia coli , Cromatografia Gasosa-Espectrometria de Massas , Cromatografia Gasosa-Espectrometria de Massas/métodos , Escherichia coli/enzimologia , Dioxigenases/metabolismo , Dioxigenases/química , beta Caroteno/análise , beta Caroteno/química , beta Caroteno/metabolismo , Carotenoides/química , Carotenoides/análise , Carotenoides/metabolismo , Aldeídos , DiterpenosRESUMO
Ginger, valued for its culinary and medicinal properties, suffers substantial production loss-up to 90 %-due to fungal soft rot. To combat this, we have developed an environmentally sustainable antifungal polysaccharide gel derived from a water-soluble Schiff base of O-carboxymethyl chitosan (CMC) and cinnamaldehyde (CIN). Terpene incorporation was confirmed via various characterization techniques, including Fourier transform infrared (FT-IR), pH-dependent release, solubility, thermogravimetric analysis, and UV-vis spectra. Results showed successful grafting of CIN onto the polysaccharide, at a CIN:CMC ratio of 120 mg/g. In vitro evaluation demonstrated significant antifungal activity against F. oxysporum, with a MIC value of 159.25 µg/mL. Application of the CMC=CIN gel to ginger rhizomes inhibited spore germination in all evaluated wounds, enhancing gloss and appearance. These findings validate the efficacy of this novel, environmentally friendly gel in preventing ginger loss caused by fungal infections.
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Saffron (Crocus sativus L.), a historically significant crop valued for its nutraceutical properties, has been poorly explored from a phytosanitary perspective. This study conducted a thorough examination of viruses affecting saffron samples from Spanish cultivars, using high-throughput sequencing alongside a systematic survey of transcriptomic datasets from Crocus sativus at the Sequence Read Archive. Our analysis unveiled a broad diversity and abundance, identifying 17 viruses across the 52 analyzed libraries, some of which were highly prevalent. This includes known saffron-infecting viruses and previously unreported ones. In addition, we discovered 7 novel viruses from the Alphaflexiviridae, Betaflexiviridae, Potyviridae, Solemoviridae, and Geminiviridae families, with some present in libraries from various locations. These findings indicate that the saffron-associated virome is more complex than previously reported, emphasizing the potential of phytosanitary analysis to enhance saffron productivity.
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Crocus , Doenças das Plantas , Crocus/genética , Doenças das Plantas/virologia , Vírus de Plantas/genética , Vírus de Plantas/classificação , Vírus de Plantas/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Viroma/genética , Perfilação da Expressão Gênica , Transcriptoma , Filogenia , Genoma Viral , Potyviridae/genética , Potyviridae/isolamento & purificação , Flexiviridae/genética , Flexiviridae/classificação , Flexiviridae/isolamento & purificaçãoRESUMO
Crocins are bioactive glucosylated apocarotenoids that confer a yellow pigmentation. In addition to their coloring ability, crocins offer potential health benefits because of their antioxidant and anti-inflammatory properties. These compounds are present in the flowers and fruits of a few plant species, including saffron, gardenia, Buddleja and Verbascum species. Saffron extracts have been used for the formulation of functional foods. However, there is no evidence of the use of the other plants producing crocins in the food industry. This study evaluated the effect of the addition of ground dry flowers of two Verbascum species, with antioxidant activity, as well as dry fruit powder, from a recently engineered tomato plant producing fruits that accumulate high levels of crocins, as functional ingredients during the processing of rice, wheat cous-cous and maize noodles, providing a yellow pigmentation. Correlation analyses revealed that the increased antioxidant activity in the three food matrices was due to the presence of crocins, which showed no toxicity. Furthermore, in vitro digestion showed that crocins were more bioaccessible from rice than from cous-cous or maize noodles, inferring the importance of the food matrix in bio accessibility. The obtained results showed the commercial potential of Verbascum's flowers, as a source of crocins, natural pigments with antioxidant activities.
Assuntos
Antioxidantes , Carotenoides , Flores , Extratos Vegetais , Verbascum , Flores/química , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Carotenoides/química , Carotenoides/farmacologia , Antioxidantes/farmacologia , Antioxidantes/química , Verbascum/química , Pigmentos Biológicos/química , Pigmentos Biológicos/farmacologia , Humanos , Frutas/químicaRESUMO
Mushroom waste can account for up to 50% of the total mushroom mass. Spent mushroom substrate, misshapen mushrooms, and mushroom stems are examples of mushroom byproducts. In ancient cultures, fungi were prized for their medicinal properties. Aqueous extracts containing high levels of ß-glucans as functional components capable of providing prebiotic polysaccharides and improved texture to foods have been widely used and new methods have been tested to improve extraction yields. Similarly, the addition of insoluble polysaccharides controls the glycemic index, counteracting the effects of increasingly high-calorie diets. Numerous studies support these benefits in vitro, but evidence in vivo is scarce. Nonetheless, many authors have created a variety of functional foods, ranging from yogurt to noodles. In this review, we focus on the pharmacological properties of edible mushroom by-products, and the possible risks derived from its consumption. By incorporating these by-products into human or animal feed formulations, mushroom producers will be able to fully optimize crop use and pave the way for the industry to move toward a zero-waste paradigm.
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Agaricales , Animais , Humanos , Agaricales/química , Polissacarídeos , Ração AnimalRESUMO
Saffron has many pharmacological properties in addition to being a frequently used food seasoning. Crocin and picrocrocin which accumulate in saffron stigma, are responsible for these pharmacological properties. These natural products have health-promoting effects for the prevention and treatment of numerous diseases, including age-related cognitive and memory disfunction. Currently, crocin and picrocrocin are obtained from saffron, considered as the spice with the highest price in the market. To develop an efficient and low-cost approach to producing these compounds with high bioactivity, biosynthetic genes isolated from saffron can be exploited in the metabolic engineering of heterologous hosts and the production of crocins in productive crop plants. Recently, we engineered tomato fruit producing crocins (Tomafran). In this study, we demonstrated that crocin-rich extract, encapsulated in chitosan or in exosomes may function as a neuroprotective strategy. Crocins contained in the Tomafran extracts and much lower doses in chitosan nanoparticles or exosomes were enough to rescue the neuroblastoma cell line SH-SY5Y after damage caused by okadaic acid. Our results confirm the neuroprotective effect of Tomafran and its exosomes that may be useful for the delay or prevention of neurodegenerative disorders such as Alzheimer's disease.
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Crocins are glucosylated apocarotenoids present in flowers and fruits of a few plant species, including saffron, gardenia, and Buddleja. The biosynthesis of crocins in these plants has been unraveled, and the enzymes engineered for the production of crocins in heterologous systems. Mullein (Verbascum sp.) has been identified as a new source of crocins and picrocrocin. In this work, we have identified eight enzymes involved in the cleavage of carotenoids in two Verbascum species, V. giganteum and V. sinuatum. Four of them were homologous to the previously identified BdCCD4.1 and BdCCD4.3 from Buddleja, involved in the biosynthesis of crocins. These enzymes were analyzed for apocarotenogenic activity in bacteria and Nicotiana benthamiana plants using a virus-driven system. Metabolic analyses of bacterial extracts and N. benthamiana leaves showed the efficient activity of these enzymes to produce crocins using ß-carotene and zeaxanthin as substrates. Accumulations of 0.17% of crocins in N. benthamiana dry leaves were reached in only 2 weeks using a recombinant virus expressing VgCCD4.1, similar to the amounts previously produced using the canonical saffron CsCCD2L. The identification of these enzymes, which display a particularly broad substrate spectrum, opens new avenues for apocarotenoid biotechnological production.
Assuntos
Crocus , Cicloexenos , Glucosídeos , Terpenos , Verbascum , Verbascum/metabolismo , Crocus/genética , Crocus/química , Vitamina A/metabolismo , Carotenoides/metabolismoRESUMO
Paulownia tomentosa is an economically important fast-growing tree, and its flowers and fruits are a rich source of biologically active secondary metabolites. In addition, the flowers of P. tomentosa are distinguished by a strong aroma and are also excellent nectariferous plants. The flowers are pale lilac and characterized by the presence of yellow nectar guides, whose color changes during the development of the flower, representing reliable signals to pollinators while enhancing reproductive success. The chemical analyses of the nectar guides revealed the presence of carotenoids as the pigments responsible for the observed coloration, with ß-carotene levels determining the color changes observed after anthesis, with a reduction at anthesis and further increase and accumulation in post anthesis. To understand how ß-carotene accumulation was controlled in the nectar guides, the expression of genes related to carotenoid biosynthesis and metabolism was analyzed. Carotenogenic gene expression was not associated with the observed changes in ß-carotene during flower development. However, the expression of a gene encoding a carotenoid cleavage dioxygenase, CCD4-4, was co-related with the levels of ß-carotene in the nectar guides. In addition, CCD4-4 cleavage ß-carotene at C9-C10 and C9'-C10' positions, resulting in the generation of ß-ionone, which was detected in flowers at anthesis. The obtained results indicated a developmental stage specific regulation of apocarotenoid formation through ß-carotene cleavage, resulting in color changes and volatile production as key traits for plant-pollinator interactions. DATA AVAILABILITY: Data will be made available on request.
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Dioxigenases , beta Caroteno , beta Caroteno/metabolismo , Dioxigenases/genética , Néctar de Plantas , Odorantes , Carotenoides/metabolismo , Flores/genéticaRESUMO
In this study, chitosan-coated biogenic silver nanoparticles (AgNP-CH) were obtained through green chemistry by recycling wheat crop leaf residues. The nanoparticles were characterized by UV-VIS spectroscopy, and total reflectance-Fourier transform infrared spectroscopy confirmed the nanoparticle formation, and the incorporation of chitosan surrounding silver nanoparticles. The size and morphology of nanoparticles were evaluated by microscopy techniques, showing a size range of 2-10 nm, with spherical shape and narrow distribution. The antifungal assay indicated a higher antimicrobial activity showing values of minimum inhibitory concentrations of 41.7 µg/mL against Fusarium oxysporum, and 208.37 µg/mL for Aspergillus niger, A. versicolor and A. brasiliensis. Finally, non-phytotoxic effects were observed in germination assays at early plant stage of development, and an increase in chlorophyll levels were observed at the doses tested with AgNP-CH. Thus, the use of AgNP-CH could be a potential alternative for the prevention of fungal infections in cereals in the early stages of wheat crop development.
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Quitosana , Nanopartículas Metálicas , Antifúngicos/química , Nanopartículas Metálicas/química , Quitosana/química , Prata/farmacologia , Prata/química , Triticum/metabolismo , Aspergillus niger/metabolismo , Sementes/metabolismo , Extratos Vegetais/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Carotenoids are C40 isoprenoids with well-established roles in photosynthesis, pollination, photoprotection, and hormone biosynthesis. The enzymatic or ROS-induced cleavage of carotenoids generates a group of compounds named apocarotenoids, with an increasing interest by virtue of their metabolic, physiological, and ecological activities. Both classes are used industrially in a variety of fields as colorants, supplements, and bio-actives. Crocins and picrocrocin, two saffron apocarotenoids, are examples of high-value pigments utilized in the food, feed, and pharmaceutical industries. In this study, a unique construct was achieved, namely O6, which contains CsCCD2L, UGT74AD1, and UGT709G1 genes responsible for the biosynthesis of saffron apocarotenoids driven by a patatin promoter for the generation of potato tubers producing crocins and picrocrocin. Different tuber potatoes accumulated crocins and picrocrocin ranging from 19.41-360 to 105-800 µg/g DW, respectively, with crocetin, crocin 1 [(crocetin-(ß-D-glucosyl)-ester)] and crocin 2 [(crocetin)-(ß-D-glucosyl)-(ß-D-glucosyl)-ester)] being the main compounds detected. The pattern of carotenoids and apocarotenoids were distinct between wild type and transgenic tubers and were related to changes in the expression of the pathway genes, especially from PSY2, CCD1, and CCD4. In addition, the engineered tubers showed higher antioxidant capacity, up to almost 4-fold more than the wild type, which is a promising sign for the potential health advantages of these lines. In order to better investigate these aspects, different cooking methods were applied, and each process displayed a significant impact on the retention of apocarotenoids. More in detail, the in vitro bioaccessibility of these metabolites was found to be higher in boiled potatoes (97.23%) compared to raw, baked, and fried ones (80.97, 78.96, and 76.18%, respectively). Overall, this work shows that potatoes can be engineered to accumulate saffron apocarotenoids that, when consumed, can potentially offer better health benefits. Moreover, the high bioaccessibility of these compounds revealed that potato is an excellent way to deliver crocins and picrocrocin, while also helping to improve its nutritional value.
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Chromoplasts and chloroplasts contain carotenoid pigments as all-trans- and cis-isomers, which function as accessory light-harvesting pigments, antioxidant and photoprotective agents, and precursors of signaling molecules and plant hormones. The carotenoid pathway involves the participation of different carotenoid isomerases. Among them, D27 is a ß-carotene isomerase showing high specificity for the C9-C10 double bond catalyzing the interconversion of all-trans- into 9-cis-ß-carotene, the precursor of strigolactones. We have identified one D27 (CsD27-1) and two D27-like (CsD27-2 and CsD27-3) genes in saffron, with CsD27-1 and CsD27-3, clearly differing in their expression patterns; specifically, CsD27-1 was mainly expressed in the undeveloped stigma and roots, where it is induced by Rhizobium colonization. On the contrary, CsD27-2 and CsD27-3 were mainly expressed in leaves, with a preferential expression of CsD27-3 in this tissue. In vivo assays show that CsD27-1 catalyzes the isomerization of all-trans- to 9-cis-ß-carotene, and could be involved in the isomerization of zeaxanthin, while CsD27-3 catalyzes the isomerization of all-trans- to cis-ζ-carotene and all-trans- to cis-neurosporene. Our data show that CsD27-1 and CsD27-3 enzymes are both involved in carotenoid isomerization, with CsD27-1 being specific to chromoplast/amyloplast-containing tissue, and CsD27-3 more specific to chloroplast-containing tissues. Additionally, we show that CsD27-1 is co-expressed with CCD7 and CCD8 mycorrhized roots, whereas CsD27-3 is expressed at higher levels than CRTISO and Z-ISO and showed circadian regulation in leaves. Overall, our data extend the knowledge about carotenoid isomerization and their implications in several physiological and ecological processes.
Assuntos
Crocus , zeta Caroteno , Antioxidantes , Carotenoides/metabolismo , Crocus/genética , Crocus/metabolismo , Isomerases/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Zeaxantinas , beta Caroteno/metabolismo , zeta Caroteno/metabolismoRESUMO
Crocins are high-value compounds with industrial and food applications. Saffron is currently the main source of these soluble pigments, but its high market price hinders its use by sectors, such as pharmaceutics. Enzymes involved in the production of these compounds have been identified in saffron, Buddleja, and gardenia. In this study, the enzyme from Buddleja, BdCCD4.1, was constitutively expressed in Nicotiana glauca, a tobacco species with carotenoid-pigmented petals. The transgenic lines produced significant levels of crocins in their leaves and petals. However, the accumulation of crocins was, in general, higher in the leaves than in the petals, reaching almost 302 µg/g DW. The production of crocins was associated with decreased levels of endogenous carotenoids, mainly ß-carotene. The stability of crocins in leaf and petal tissues was evaluated after three years of storage, showing an average reduction of 58.06 ± 2.20% in the petals, and 78.37 ± 5.08% in the leaves. This study illustrates the use of BdCCD4.1 as an effective tool for crocin production in N. glauca and how the tissue has an important impact on the stability of produced high-value metabolites during storage.
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Crocins and picrocrocin are high-value hydrophilic pigments produced in saffron and used commercially in the food and pharmaceutical industries. These apocarotenoids are derived from the oxidative cleavage of zeaxanthin by specific carotenoid cleavage dioxygenases. The pathway for crocins and picrocrocin biosynthesis was introduced into tomato using fruit specific and constitutive promoters and resulted in 14.48 mg/g of crocins and 2.92 mg/g of picrocrocin in the tomato DW, without compromising plant growth. The strategy involved expression of CsCCD2L to produce crocetin dialdehyde and 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene, and of glycosyltransferases UGT709G1 and CsUGT2 for picrocrocin and crocins production, respectively. Metabolic analyses of the engineered fruits revealed picrocrocin and crocetin-(ß-D-gentiobiosyl)-(ß-D-glucosyl)-ester, as the predominant crocin molecule, as well as safranal, at the expense of the usual tomato carotenoids. The results showed the highest crocins content ever obtained by metabolic engineering in heterologous systems. In addition, the engineered tomatoes showed higher antioxidant capacity and were able to protect against neurological disorders in a Caenorhabditis elegans model of Alzheimer's disease. Therefore, these new developed tomatoes could be exploited as a new platform to produce economically competitive saffron apocarotenoids with health-promoting properties.
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Crocins are high-value soluble pigments that are used as colorants and supplements, their presence in nature is extremely limited and, consequently, the high cost of these metabolites hinders their use by other sectors, such as the pharmaceutical and cosmetic industries. The carotenoid cleavage dioxygenase 2L (CsCCD2L) is the key enzyme in the biosynthetic pathway of crocins in Crocus sativus. In this study, CsCCD2L was introduced into Nicotiana tabacum and Nicotiana glauca for the production of crocins. In addition, a chimeric construct containing the Brevundimonas sp. ß-carotene hydroxylase (BrCrtZ), the Arabidopsis thaliana ORANGE mutant gene (AtOrMut), and CsCCD2L was also introduced into N. tabacum. Quantitative and qualitative studies on carotenoids and apocarotenoids in the transgenic plants expressing CsCCD2L alone showed higher crocin level accumulation in N. glauca transgenic plants, reaching almost 400 µg/g DW in leaves, while in N. tabacum 36 µg/g DW was obtained. In contrast, N. tabacum plants coexpressing CsCCD2L, BrCrtZ, and AtOrMut accumulated, 3.5-fold compared to N. tabacum plants only expressing CsCCD2L. Crocins with three and four sugar molecules were the main molecular species in both host systems. Our results demonstrate that the production of saffron apocarotenoids is feasible in engineered Nicotiana species and establishes a basis for the development of strategies that may ultimately lead to the commercial exploitation of these valuable pigments for multiple applications.
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The current status of controversy regarding the use of certain preservatives in cosmetic products makes it necessary to seek new ecological alternatives that are free of adverse effects on users. In our study, two different natural terpenes Carvacrol and Eugenol were encapsulated in chitosan nanoparticles in different ratios of Chitosan:terpene. The nanoparticles were characterized by DLS and TEM showing a maximum particle size of 100 nm. The chemical structure, thermal properties, and release profile of terpenes were evaluated showing a successful protection of terpene in Chitosan matrix. Two different release profile were observed showing a faster release profile in the case of Eugenol. Antimicrobial properties of nanoparticles were evaluated against typical microbial contaminants found in cosmetic products, showing higher antimicrobial properties with chitosan encapsulation of terpenes. Furthermore, natural moisturizing cream inoculated with beforementioned microorganisms was formulated with Carvacrol-chitosan nanoparticles and Eugenol-chitosan nanoparticles to evaluate the preservative efficiency, indicating a highest preservative efficiency with the use of Eugenol-chitosan nanoparticles.
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Anti-Infecciosos , Quitosana , Cosméticos , Nanopartículas , Antibacterianos , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Quitosana/química , Cosméticos/química , Cimenos , Eugenol/química , Eugenol/farmacologia , Nanopartículas/química , Conservantes FarmacêuticosRESUMO
The current status of controversy regarding the use of certain preservatives in cosmetic products makes it necessary to seek new ecological alternatives that are free of adverse effects on users. In our study, the natural terpene thymoquinone was encapsulated in chitosan nanoparticles. The nanoparticles were characterized by DLS and TEM, showing a particle size of 20 nm. The chemical structure, thermal properties, and release profile of thymoquinone were evaluated and showed a successful stabilization and sustained release of terpenes. The antimicrobial properties of the nanoparticles were evaluated against typical microbial contaminants found in cosmetic products, showing high antimicrobial properties. Furthermore, natural moisturizing cream inoculated with the aforementioned microorganisms was formulated with thymoquinone-chitosan nanoparticles to evaluate the preservative efficiency, indicating its promising use as a preservative in cosmetics.
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Benzoquinonas , Produtos Biológicos , Quitosana , Cosméticos , Nanopartículas , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Benzoquinonas/administração & dosagem , Benzoquinonas/química , Produtos Biológicos/química , Fenômenos Químicos , Quitosana/química , Cosméticos/química , Composição de Medicamentos , Concentração de Íons de Hidrogênio , Nanopartículas/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , TermogravimetriaRESUMO
In this study, garlic essential oil (GEO) has been encapsulated in chitosan nanoparticles (NPCH) with sodium tripolyphosphate (TPP). Fourier transform infrared (FT-IR) spectroscopy, UV-vis spectrophotometry, thermogravimetric analysis (TGA) and X-ray diffraction (XRD) techniques were applied to characterize GEO-NPCH. The obtained nanoparticles exhibited a regular distribution and spherical shape with size range of 200-400 nm as revealed by scanning electron microscopy (SEM). The maximum encapsulation efficiency (EE) and loading capacity (LC) of GEO-loaded chitosan nanoparticles were about 32.8% and 19.8% respectively. Nanoparticle formulations of GEO were found to have antifungal activity against Aspergillus versicolor, A. niger and Fusarium oxysporum. In addition, they showed growth promoting effects by increasing emergence, shoot and root fresh weight on wheat, oat and barley.
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Biogenic-silver nanoparticles emerge as new nanosilver platforms that allow us to obtain silver nanoparticles via "green chemistry". In our study, biogenic-silver nanoparticles were obtained from Iris tuberosa leaf extract. Nanoparticles were characterized by a UV-vis spectroscopy, dynamical light scattering technique. The transmission electron microscope revealed spheric and irregular nanoparticles with 5 to 50 nm in diameter. Antimicrobial properties were evaluated against typical microbial contaminants found in cosmetic products, showing high antimicrobial properties. Furthermore, natural moisturizing cream was formulated with biogenic-silver nanoparticles to evaluate the preservative efficiency through a challenge test, indicating its promising use as preservative in cosmetics.
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Anti-Infecciosos/química , Cosméticos/química , Iris/química , Nanopartículas Metálicas/química , Conservantes Farmacêuticos/química , Prata/químicaRESUMO
Crocetin is an apocarotenoid formed from the oxidative cleavage of zeaxanthin, by the carotenoid cleavage enzymes CCD2 (in Crocus species) and specific CCD4 enzymes in Buddleja davidii and Gardenia jasminoides. Crocetin accumulates in the stigma of saffron in the form of glucosides and crocins, which contain one to five glucose molecules. Crocetin glycosylation was hypothesized to involve at least two enzymes from superfamily 1 UDP-sugar dependent glycosyltransferases. One of them, UGT74AD1, produces crocins with one and two glucose molecules, which are substrates for a second UGT, which could belong to the UGT79, 91, or 94 families. An in silico search of Crocus transcriptomes revealed six candidate UGT genes from family 91. The transcript profiles of one of them, UGT91P3, matched the metabolite profile of crocin accumulation, and were co-expressed with UGT74AD1. In addition, both UGTs interact in a two-hybrid assay. Recombinant UGT91P3 produced mostly crocins with four and five glucose molecules in vitro, and in a combined transient expression assay with CCD2 and UGT74AD1 enzymes in Nicotiana benthamiana. These results suggest a role of UGT91P3 in the biosynthesis of highly glucosylated crocins in saffron, and that it represents the last missing gene in crocins biosynthesis.
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Carotenoides/metabolismo , Crocus/enzimologia , Perfilação da Expressão Gênica/métodos , Glicosiltransferases/genética , Vias Biossintéticas , Simulação por Computador , Crocus/química , Crocus/genética , Regulação da Expressão Gênica de Plantas , Glicosilação , Proteínas de Plantas/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Crocetin biosynthesis in Buddleja davidii flowers proceeds through a zeaxanthin cleavage pathway catalyzed by two carotenoid cleavage dioxygenases (BdCCD4.1 and BdCCD4.3), followed by oxidation and glucosylation reactions that lead to the production of crocins. We isolated and analyzed the expression of 12 genes from the carotenoid pathway in B. davidii flowers and identified four candidate genes involved in the biosynthesis of crocins (BdALDH, BdUGT74BC1, BdUGT74BC2, and BdUGT94AA3). In addition, we characterized the profile of crocins and their carotenoid precursors, following their accumulation during flower development. Overall, seven different crocins, crocetin, and picrocrocin were identified in this study. The accumulation of these apocarotenoids parallels tissue development, reaching the highest concentration when the flower is fully open. Notably, the pathway was regulated mainly at the transcript level, with expression patterns of a large group of carotenoid precursor and apocarotenoid genes (BdPSY2, BdPDS2, BdZDS, BdLCY2, BdBCH, BdALDH, and BdUGT Genes) mimicking the accumulation of crocins. Finally, we used comparative correlation network analysis to study how the synthesis of these valuable apocarotenoids diverges among B. davidii, Gardenia jasminoides, and Crocus sativus, highlighting distinctive differences which could be the basis of the differential accumulation of crocins in the three species.