Fortification and bioaccessibility of saffron apocarotenoids in potato tubers.

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.

Metabolic Engineering of Crocin Biosynthesis in Nicotiana Species.

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.

UGT709G1: a novel uridine diphosphate glycosyltransferase involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron (Crocus sativus).

Saffron, a spice derived from the dried red stigmas of Crocus sativus, is one of the oldest natural food additives. The flowers have long red stigmas, which store significant quantities of the glycosylated apocarotenoids crocins and picrocrocin. The apocarotenoid biosynthetic pathway in saffron starts with the oxidative cleavage of zeaxanthin, from which crocins and picrocrocin are derived. In the processed stigmas, picrocrocin is converted to safranal, giving saffron its typical aroma. By a targeted search for differentially expressed uridine diphosphate glycosyltransferases (UGTs) in Crocus transcriptomes, a novel apocarotenoid glucosyltransferase (UGT709G1) from saffron was identified. Biochemical analyses revealed that UGT709G1 showed a high catalytic efficiency toward 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene (HTCC), making it suited for the biosynthesis of picrocrocin, the precursor of safranal. The role of UGT709G1 in picrocrocin/safranal biosynthesis was supported by the absence or presence of gene expression in a screening for HTCC and picrocrocin production in different Crocus species and by a combined transient expression assay with CsCCD2L in Nicotiana benthamiana leaves. The identification of UGT709G1 completes one of the most highly valued specialized metabolic biosynthetic pathways in plants and provides novel perspectives on the industrial production of picrocrocin to be used as a flavor additive or as a pharmacological constituent.

Tissue-Specific Accumulation of Sulfur Compounds and Saponins in Different Parts of Garlic Cloves from Purple and White Ecotypes.

This study set out to determine the distribution of sulfur compounds and saponin metabolites in different parts of garlic cloves. Three fractions from purple and white garlic ecotypes were obtained: the tunic (SS), internal (IS) and external (ES) parts of the clove. Liquid Chromatography coupled to High Resolution Mass spectrometry (LC-HRMS), together with bioinformatics including Principal Component Analysis (PCA), Hierarchical Clustering (HCL) and correlation network analyses were carried out. Results showed that the distribution of these metabolites in the different parts of garlic bulbs was different for the purple and the white ecotypes, with the main difference being a slightly higher number of sulfur compounds in purple garlic. The SS fraction in purple garlic had a higher content of sulfur metabolites, while the ES in white garlic was more enriched by these compounds. The correlation network indicated that diallyl disulfide was the most relevant metabolite with regards to sulfur compound metabolism in garlic. The total number of saponins was almost 40-fold higher in purple garlic than in the white variety, with ES having the highest content. Interestingly, five saponins including desgalactotigonin-rhamnose, proto-desgalactotigonin, proto-desgalactotigonin-rhamnose, voghieroside D1, sativoside B1-rhamnose and sativoside R1 were exclusive to the purple variety. Data obtained from saponin analyses revealed a very different network between white and purple garlic, thus suggesting a very robust and tight coregulation of saponin metabolism in garlic. Findings in this study point to the possibility of using tunics from purple garlic in the food and medical industries, since it contains many functional compounds which can be exploited as ingredients.

Saffron: Its Phytochemistry, Developmental Processes, and Biotechnological Prospects.

The present state of knowledge concerning developmental processes and the secondary metabolism of saffron, Crocus sativus L. (Iridaceae), along with the genes involved in these processes so far known, is reviewed. Flowers and corms constitute the most valuable parts of saffron. Corm and flower development are two key aspects to be studied in saffron to increase the yield and quality of the spice, to raise its reproductive rate, and to implement new production systems. Important knowledge about the physiology of flowering and vegetative growth has been acquired in recent years, but there is still only limited information on molecular mechanisms controlling these processes. Although some genes involved in flower formation and meristem transition in other species have been isolated in saffron, the role of these genes in this species awaits further progress. Also, genes related with the synthesis pathway of abscisic acid and strigolactones, growth regulators related with bud endodormancy and apical dominance (paradormancy), have been isolated. However, the in-depth understanding of these processes as well as of corm development is far from being achieved. By contrast, saffron phytochemicals have been widely studied. The different flower tissues and the corm have been proved to be an important source of phytochemicals with pharmacological properties. The biotechnological prospects for saffron are here reviewed on the basis of the discovery of the enzymes involved in key aspects of saffron secondary metabolism, and we also analyze the possibility of transferring current knowledge about flowering and vegetative propagation in model species to the Crocus genus.

Screening for polyphenols, antioxidant and antimicrobial activitiesof extracts from eleven Helianthemum taxa (Cistaceae) used in folk medicine in south-eastern Spain.

ETHNOPHARMACOLOGICAL RELEVANCE: The Helianthemum genus contains approximately one hundred taxa. Some of them are important medicinal plants used in several countries for many different purposes. However, studies addressing the phytochemistry of many of these species or their biological activities are currently nonexistent. AIM OF THE STUDY: To perform a comparative analysis of the qualitative composition of secondary metabolites and biological activities of the leaves of the most commonly distributed and used Spanish Helianthemum taxa in order to find a relationship between chemotype, biological activity and uses. MATERIALS AND METHODS: water and 80% methanolic extracts derived from 11 different Helianthemum taxa were analyzed for their phytochemical composition using standard methods. Furthermore, HPLC analysis was performed for the estimation of gallic acid, ellagic acid, tannins and flavonols as marker compounds. The antioxidant activity was measured by employing the scavenging activity on DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate) radicals. The 80% methanolic extracts were evaluated for antibacterial (Gram-positive Staphylococcus aureus, Enterococcus faecalis and Listeria monocytogenes and Gram-negative Escherichia coli and Salmonella enterica) activity using the micro dilution technique. RESULTS: The total polyphenolic content and antioxidant capabilities differed significantly among the Helianthemum leaf extracts. A strong correlation between total phenolic content, antioxidant properties and antimicrobial activity was found. The polyphenolic profile was specific for each taxon. Whereas Helianthemum alypoides, Helianthemum cinereum subsp. rotundifolium, Helianthemum hirtum, Helianthemum asperum, and Helianthemum marifolium subsp. marifolium were characterized by the presence of gallic acid, egallic derivatives and ellagitannins; the polyphenolic profile of Helianthemum apenninum subsp. cavanillesianum, Helianthemum syriacum and Helianthemum polygonoides was mostly based on flavonoids. Helianthemum cinereum, Helianthemum alypoides and Helianthemum marifolium consistently presented the strongest radical scavenging activity (in water extracts EC50 ranges from 29.88 to 38.1 against DPPH and from 8.11 to 20.8 against ABTS, and in 80% MeOH extracts from 25.3 to 31.8 against DPPH and from 6.15 to 8.6 against ABTS), total phenol content (>117mg GAE/l) and antimicrobial activities. CONCLUSION: The Helianthemum taxa used in folk medicine did not cluster in a unique section, being equally distributed in two out of the four sections analysed. There was not a clear relationship between the chemotype, based on the polyphenolic composition of the taxa, and their taxonomical classification. However, the composition of the methanolic and water extracts from the leaves of plants belonging to the Helianthemum genus was strongly related to their medicinal uses.

Analysis of free and esterified sterols in edible oils by online reversed phase liquid chromatography-gas chromatography (RPLC-GC) using the through oven transfer adsorption desorption (TOTAD) interface.

An online reversed phase liquid chromatography-gas chromatography (RPLC-GC) method is proposed to quantify free, total and esterified sterols of edible oils. To determine free sterols the diluted oils are injected into the liquid chromatograph, where free sterols are separated from triglycerides and the sterol fraction is automatically transferred to the gas chromatograph to be analysed. To determine total sterols the samples were saponified with potassium hydroxide in ethanolic solution and the unsaponifiable fraction was extracted with diethyl ether. The extract was then analysed by RPLC-GC, avoiding the laborious thin layer chromatography step used in the Official European Union (EU) Method. The relative standard deviations (RSDs) from the absolute peak area varied from 7.6% to 15.8%. Limits of detection (LODs) were less than 8.5 mg/kg. No variability in retention time was observed. The method was applied to the determination of total sterols in edible oil samples and the results were compared with those obtained with the Official EU Method. Good agreement was found between both methods, except in the case of campesterol.

Saponins from the Spanish saffron Crocus sativus are efficient adjuvants for protein-based vaccines.

Protein and peptide-based vaccines provide rigorously formulated antigens. However, these purified products are only weakly immunogenic by themselves and therefore require the addition of immunostimulatory components or adjuvants in the vaccine formulation. Various compounds derived from pathogens, minerals or plants, possess pro-inflammatory properties which allow them to act as adjuvants and contribute to the induction of an effective immune response. The results presented here demonstrate the adjuvant properties of novel saponins derived from the Spanish saffron Crocus sativus. In vivo immunization studies and tumor protection experiments unambiguously establish the value of saffron saponins as candidate adjuvants. These saponins were indeed able to increase both humoral and cellular immune responses to protein-based vaccines, ultimately providing a significant degree of protection against tumor challenge when administered in combination with a tumor antigen. This preclinical study provides an in depth immunological characterization of a new saponin as a vaccine adjuvant, and encourages its further development for use in vaccine formulations.