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Saffron stigma, derived from Crocus sativus L., has long been revered in global traditional medicine and continues to hold significant market value. However, despite the extensive focus on saffron stigma, the therapeutic potential of other floral components remains underexplored, primarily due to limited insights into their complex molecular architectures and chemical diversity. To address this gap, we performed a comprehensive metabolomic analysis of various floral organs utilizing advanced analytical platforms, including GC-MS and UPLC-MS/MS. This in-depth profiling revealed a diverse array of 248 metabolites, encompassing amino acids, sugar derivatives, fatty acids, flavonoids, vitamins, polyamines, organic acids, and a broad spectrum of secondary metabolites. Distinct correlation patterns among these metabolites were identified through PCA and PLS-DA, highlighting unique metabolomic signatures inherent to each floral organ. We further integrated these metabolomic findings with our transcriptomic data, enabling a detailed understanding of the molecular and metabolic variations across different floral organs. The pronounced abundance of differentially expressed genes and metabolites in the stamen (424), leaf (345), tepal (196), stigma (177), and corm (133) underscores the intricate regulatory networks governing source-to-sink partitioning and dynamic metabolic processes. Notably, our study identified several bioactive compounds, including crocin, picrocrocin, crocetin, safranal, cannabielsoin, quercetin, prenylnaringenin, isorhamnetin, pelargonidin, kaempferol, and gallic acid, all of which exhibit potential therapeutic properties. In conclusion, this comprehensive analysis significantly enhances our understanding of the molecular mechanisms driving the biosynthesis of apocarotenoids, cannabinoids, anthocyanins, and flavonoids in saffron, thereby providing valuable insights and paving the way for future research in this area., (© 2024 Scandinavian Plant Physiology Society.)

Saffron spice owes its commercial appreciation to its specific apocarotenoids: crocins, picrocrocin, and safranal. In Crocus sativus, these compounds are biosynthesized from zeaxanthin through oxidative cleavage by the carotenoid cleavage dioxygenase 2 (CCD2). Transgenic tomato plants expressing CsCCD2 in the fruit, named Tomaffron, accumulate high levels of saffron apocarotenoids despite the low substrate availability for CsCCD2. In the present study, CsCCD2 has been introduced into Xantomato; this tomato variety accumulates high levels of zeaxanthin and β-carotene in ripe fruit due to a combination of four mutant alleles. Xantomato and Tomaffron genotypes have been combined to optimize apocarotenoid production. The best transgenic lines accumulated 15 and 14 times more crocins and picrocrocin than Tomaffron, alongside a fourfold increase in β-carotene compared to Xantomato, albeit at a cost in fruit yield. Segregation of the four mutations has been carried out to find the best combination for obtaining high levels of saffron apocarotenoids without adverse effects on fruit yield. Plants harboring the high-pigmented 3 (hp3) and BETA (B Sh ) mutations accumulated 6 and 15 times more crocins and picrocrocin than Tomaffron, without observable pleiotropic effects. Additionally, those high levels of saffron apocarotenoids were obtained in fruit accumulating high levels of both lycopene and β-carotene independently or in combination, suggesting a regulatory role for the apocarotenoids produced and indicating that it is possible to increase the levels of both types of healthy promoting molecules simultaneously., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The mechanisms by which low light accelerates starch macromolecules degradation by auxin and gibberellin (GA) in geophytes during sprouting remain largely unknown. This study investigated these mechanisms in saffron, grown under low light (50 μmol m -2  s -1 ) and optimal light (200 μmol m -2  s -1 ) during the sprouting phase. Low light reduced starch concentration in corms by 34.0 % and increased significantly sucrose levels in corms, leaves, and leaf sheaths by 19.2 %, 9.8 %, and 134.5 %, respectively. This was associated with a 33.3 % increase in GA 3 level and enhanced auxin signaling. Leaves synthesized IAA under low light, which was transported to the corms to promote GA synthesis, facilitating starch degradation through a 228.7 % increase in amylase activity. Exogenous applications of GA and IAA, as well as the use of their synthesis or transport inhibitors, confirmed the synergistic role of these phytohormones in starch metabolism. The unigenes associated with GA biosynthesis and auxin signaling were upregulated under low light, highlighting the IAA-GA module role in starch degradation. Moreover, increased respiration rate and invertase activity, crucial for ATP biosynthesis and the tricarboxylic acid cycle, were consistent with the upregulation of related unigenes, suggesting that auxin signaling accelerates starch degradation by promoting energy metabolism. Upregulated of auxin signaling (CsSAUR32) and starch metabolism (CsSnRK1) genes under low light suggests that auxin directly regulate starch degradation in saffron corms. This study elucidates that low light modulates auxin and GA interactions to accelerate starch degradation in saffron corms during sprouting, offering insights for optimizing agricultural practices under suboptimal light conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Crocus sativus L. is a perennial crop for its valuable active compounds. Plant-associated microbes impact on the quality and efficacy of medicinal herbs by promoting bioactive components accumulation. However, how microbes influence the accumulation of bioactive components in saffron have not been well studied. Here, the microbiome in C. sativus derived from 3 core production areas were deciphered by 16S rDNA sequencing and the relationship between endophytes and bioactive ingredients were further investigated. The main results are as follows: (1) Both Comamonadaceae and Burkholderiaceae were positively correlated with the content of bioactive components in the stigmas. (2) The synthesis of crocin was positively correlated with Xanthomonadaceae, negatively correlated with Lachnospiraceae and Prevotellaceae. Therefore, further investigation is required to determine whether Xanthomonadaceae plays an unknown function in the synthesis of crocin. These findings provide guidelines for disentangling the function of endophytes in the production of bioactive ingredients and thus for microbe-mediated breeding., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

So far, compliance with ISO 3632 standard specifications for top-quality saffron guarantees good agricultural and post-harvest production practices. Tracking early-stage oxidation remains challenging. Our study aims to address this issue by exploring the visible, fluorescence, and near-infrared spectra of category I saffron. Using a multi-spectral sensor, we tested fresh and artificially aged saffron in powder form. High autofluorescence intensities at 600-700 nm allowed calibration for the 'content of aged saffron'. Samples with minimum coloring strength (200-220 units) were classified as 70% aged, while those exceeding maximum aroma strength (50 units) as 100% aged. Consistent patterns across origin, age, and processing history indicated potential for objectively assessing early-oxidation markers. Further analyses uncovered multiple contributing fluorophores, including cis-apocarotenoids, correlated with FTIR-based aging markers. Our findings underscore that sensing autofluorescence of traded saffron presents an innovative quality diagnostic approach, paving new research pathways for assessing the remaining shelf-life along its supply chain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Crocus sativus L. is a both medicinal and food bulbous flower whose qualities are geographically characterized. However, identification involving different places of origin of such substances is currently limited to single-omics mediated content analysis. Integrated metabolomics and proteomics, 840 saffron samples from six countries (Spain, Greece, Iran, China, Japan, and India) were analyzed using the QuEChERS extraction method. A total of 77 differential metabolites and 14 differential proteins were identified. The limits of detection of the method were 1.33 to 8.33 μg kg -1 , and the recoveries were 85.56% to 105.18%. Using homology modeling and molecular docking, the Gln84, Lys195, Val182 and Pro184 sites of Crocetin glucosyltransferase 2 were found to be the targets of crocetin binding. By multivariate statistical analysis (PCA and PLS-DA), different saffron samples were clearly distinguished. The results provided the basis for the selection and identification of high quality saffron from different producing areas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare no conflicting financial interest., (Copyright © 2023. Published by Elsevier Ltd.)

Water stress is one of the most significant abiotic stresses that disrupts the osmotic balance of plants and consequently reduces their growth and performance. In recent years, it has been found that serotonin, as a signaling and regulatory molecule, can play important roles in the growth and development of plants and enhance their tolerance to abiotic stresses. Saffron is a plant known for its medicinal and culinary properties. Its distinct flavor, aroma, and vibrant color make it a sought-after ingredient in various cuisines and traditional medicines. The aim of this study is to investigate the possible effect of serotonin growth regulator on some morphophysiological and biochemical characteristics of saffron plant under water stress conditions. Water stress was applied using polyethylene glycol 6000 at a level of 30%, w/v. Serotonin was also applied exogenously at a concentration of 100 µM in both foliar and root applications. The experimental findings demonstrated that water stress had a detrimental impact on various growth and photosynthetic parameters including FW, DW, SH, RWC, photosynthetic pigments content, Pn, Fv/Fm, C and Ci. Under these conditions, H 2 O 2 content and ion leakage increased. The increase in the content of proline and sugars also confirmed that the saffron plant was placed in unfavorable growth conditions. Serotonin application in both foliar and root applications and especially root treatment under stressful conditions improved plant growth by activating enzymatic and non-enzymatic antioxidant systems. Overall, the exogenous application of serotonin increased the resistance of saffron plants to water stress., (© 2024. The Author(s).)

Crocus sativus L. is a widely cultivated traditional plant for obtaining dried red stigmas known as "saffron," the most expensive spice in the world. The response of C. sativus to pre-sowing processing of corms with cold plasma (CP, 3 and 5 min), vacuum (3 min), and electromagnetic field (EMF, 5 min) was assessed to verify how such treatments affect plant performance and the quality and yield of herbal raw materials. The results show that applied physical stressors did not affect the viability of corms but caused stressor-dependent changes in the kinetics of sprouting, growth parameters, leaf trichome density, and secondary metabolite content in stigmas. The effect of CP treatment on plant growth and metabolite content was negative, but all stressors significantly (by 42-74%) increased the number of leaf trichomes. CP3 treatment significantly decreased the length and dry weight of flowers by 43% and 60%, respectively, while EMF treatment increased the length of flowers by 27%. However, longer CP treatment (5 min) delayed germination. Vacuum treatment improved the uniformity of germination by 28% but caused smaller changes in the content of stigma compounds compared with CP and EMF. Twenty-six compounds were identified in total in Crocus stigma samples by the HPLC-DAD method, including 23 crocins, rutin, picrocrocin, and safranal. Processing of Crocus corms with EMF showed the greatest efficiency in increasing the production of secondary metabolites in saffron. EMF increased the content of marker compounds in stigmas (crocin 4: from 8.95 to 431.17 mg/g; crocin 3: from 6.27 to 164.86 mg/g; picrocrocin: from 0.4 to 1.0 mg/g), although the observed effects on growth were neutral or slightly positive. The obtained findings indicate that treatment of C. sativus corms with EMF has the potential application for increasing the quality of saffron by enhancing the amounts of biologically active compounds.

The main constituents of saffron are the apocarotenoids crocins and crocetin, present in the stigmas. Numerous healthy properties, especially those related to the effects on the central nervous system, have been attributed to these compounds but the metabolites responsible for these effects are still unknown. Previous evidences in animal models suggest a role for the gut microbiota in the pharmacokinetics and the neuroprotective effects of these compounds. However, the interaction between these apocarotenoids and the gut microbiota has been poorly studied. In this article, we have thoroughly investigated the batch fermentation of crocin-1 and crocetin (10 μM) with human fecal samples of two donors at different incubation times (0-240 h) using a metabolomic approach. We corroborated a rapid transformation of crocin-1 which looses the glucose molecules through de-glycosylation reactions until its complete transformation into crocetin in 6 hours. A group of intermediate crocins with different degrees of glycosylation were detected in a very short time. Crocetin was further metabolized and new microbial metabolites produced by double-bond reduction and demethylation reactions were identified for the first time: dihydro and tetrahydro crocetins and di-demethyl crocetin. In addition, we detected changes in the levels of the short chain fatty acids valeric acid and hexanoic acid suggesting further structural modifications of crocetin or changes in the catabolic production of these compounds. This research is a pioneering study of the action of the human gut microbiota on the saffron apocarotenoids and goes one step further towards the discovery of metabolites potentially involved in the benefits of saffron.

Flowers of Crocus sativus L. are immensely important not only for arrangement of floral whorls but more because each floral organ is dominated by a different class of specialized compounds. Dried stigmas of C. sativus flowers form commercial saffron, and are known to accumulate unique apocarotenoids like crocin, picrocrocin and safranal. Inspite of being a high value crop, the molecular mechanism regulating flower development in Crocus remains largely unknown. Moreover, it would be very interesting to explore any co-regulatory mechanism which controls floral architecture and secondary metabolic pathways which exist in specific floral organs. Here we report transcriptome wide identification of MADS box genes in Crocus. A total of 39 full length MADS box genes were identified among which three belonged to type I and 36 to type II class. Phylogeny classified them into 11 sub-clusters. Expression pattern revealed some stigma up-regulated genes among which CstMADS19 encoding an AGAMOUS gene showed high expression. Transient over-expression of CstMADS19 in stigmas of Crocus resulted in increased crocin by enhancing expression of pathway genes. Yeast one hybrid assay demonstrated that CstMADS19 binds to promoters of phytoene synthase and carotenoid cleavage dioxygenase 2 genes. Yeast two hybrid and BiFC assays confirmed interaction of CstMADS19 with CstMADS26 which codes for a SEPALATA gene. Co-overexpression of CstMADS19 and CstMADS26 in Crocus stigmas enhanced crocin content more than was observed when genes were expressed individually. Collectively, these findings indicate that CstMADS19 functions as a positive regulator of stigma based apocarotenoid biosynthesis in Crocus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Saffron (Crocus sativus L.) is being embraced as the most important medicinal plant and the commercial source of saffron spice. Despite the beneficial economic and medicinal properties of saffron, the regulatory mechanism of the correlation of TFs and genes related to the biosynthesis of the apocarotenoids pathway is less obvious. Realizing these regulatory hierarchies of gene expression networks related to secondary metabolites production events is the main challenge owing to the complex and extensive interactions between the genetic behaviors. Recently, high throughput expression data have been highly feasible for constructing co-regulation networks to reveal the regulated processes and identifying novel candidate hub genes in response to complex processes of the biosynthesis of secondary metabolites. Herein, we performed Weighted Gene Co-expression Network Analysis (WGCNA), a systems biology method, to identify 11 regulated modules and hub TFs related to secondary metabolites. Three specialized modules were found in the apocarotenoids pathway. Several hub TFs were identified in notable modules, including MADS, C2H2, ERF, bZIP, HD-ZIP, and zinc finger protein MYB and HB, which were potentially associated with apocarotenoid biosynthesis. Furthermore, the expression levels of six hub TFs and six co-regulated genes of apocarotenoids were validated with RT-qPCR. The results confirmed that hub TFs specially MADS, C2H2, and ERF had a high correlation (P < 0.05) and a positive effect on genes under their control in apocarotenoid biosynthesis (CCD2, GLT2, and ADH) among different C. sativus ecotypes in which the metabolite contents were assayed. Promoter analysis of the co-expressed genes of the modules involved in apocarotenoids biosynthesis pathway suggested that not only are the genes co-expressed, but also share common regulatory motifs specially related to hub TFs of each module and that they may describe their common regulation. The result can be used to engineer valuable secondary metabolites of C. sativus by manipulating the hub regulatory TFs., (© 2024. The Author(s).)

Saffron, the "golden spice" derived from Crocus sativus L., is renowned for its richness in secondary metabolites such as crocin and safranal, contributing to its unique properties. Facing challenges like decreasing global production, optimizing cultivation techniques becomes imperative for enhanced yields. Although the impact of factors like planting density, planting depth, spacing, and corm size on saffron growth has been studied, the interaction between corm size and planting depth remains underexplored. This study systematically investigates the interactive effects of corm size and planting depth on saffron growth and yield, providing evidence-based guidelines for optimizing cultivation. A factorial experiment, employing a completely randomized design, was conducted to assess the influence of corm size (05-10g, 10.1-15g, 15.1-20g) and planting depth (10cm, 15cm, 20cm) on saffron yield. Uniform-sized corms were obtained, and a suitable soil mixture was prepared for cultivation. Morphological and agronomic parameters were measured, and statistical analyses were performed using ANOVA and Tukey's HSD test. The study revealed that planting depth significantly affected saffron emergence. The corms sown under 15cm depth showed 100% emergence regardless of corm size (either 05-10g, 10.1-15g, 15.1-20g) followed by 10cm depth corms. Corm dry weight exhibited a complex interaction, where larger corms benefited from deeper planting, while intermediate-sized corms thrived at shallower depths. Similar patterns were observed in shoot fresh weight and dry weight. Specifically, the largest corm size (t3, 15.1-20g) produced the greatest fresh-weight biomass at the deepest planting depth of 20cm (T3), while intermediate-sized corms (t2, 10.1-15g) were superior at the shallowest 10cm depth (T1). The total plant biomass demonstrated that larger corms excelled in deeper planting, while intermediate-sized corms were optimal at moderate depths. This research highlights the intricate interplay between corm size and planting depth in influencing saffron growth. Larger corms generally promote higher biomass, but the interaction with planting depth is crucial. Understanding these dynamics can aid farmers in tailoring cultivation practices for optimal saffron yields. The study emphasizes the need for a coordinated approach to corm selection and depth placement, providing valuable insights for sustainable saffron production and economic growth., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sarwar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The increasing demand for saffron metabolites in various commercial industries, including medicine, food, cosmetics, and dyeing, is driven by the discovery of their diverse applications. Saffron, derived from Crocus sativus stigmas, is the most expensive spice, and there is a need to explore additional sources to meet global consumption demands. In this study, we focused on yellow-flowering crocuses and examined their tepals to identify saffron-like compounds., Results: Through metabolomic and transcriptomic approaches, our investigation provides valuable insights into the biosynthesis of compounds in yellow-tepal crocuses that are similar to those found in saffron. The results of our study support the potential use of yellow-tepal crocuses as a source of various crocins (crocetin glycosylated derivatives) and flavonoids., Conclusions: Our findings suggest that yellow-tepal crocuses have the potential to serve as a viable excessive source of some saffron metabolites. The identification of crocins and flavonoids in these crocuses highlights their suitability for meeting the demands of various industries that utilize saffron compounds. Further exploration and utilization of yellow-tepal crocuses could contribute to addressing the growing global demand for saffron-related products., (© 2024. The Author(s).)

Background: Cultivation of Crocus sativus (saffron) faces challenges due to inconsistent flowering patterns and variations in yield. Flowering takes place in a graded way with smaller corms unable to produce flowers. Enhancing the productivity requires a comprehensive understanding of the underlying genetic mechanisms that govern this size-based flowering initiation and commitment. Therefore, samples enriched with non-flowering and flowering apical buds from small (< 6 g) and large (> 14 g) corms were sequenced., Methods and Results: Apical bud enriched samples from small and large corms were collected immediately after dormancy break in July. RNA sequencing was performed using Illumina Novaseq 6000 to access the gene expression profiles associated with size dependent flowering. De novo transcriptome assembly and analysis using flowering committed buds from large corms at post-dormancy and their comparison with vegetative shoot primordia from small corms pointed out the major role of starch and sucrose metabolism, Auxin and ABA hormonal regulation. Many genes with known dual responses in flowering development and circadian rhythm like Flowering locus T and Cryptochrome 1 along with a transcript showing homology with small auxin upregulated RNA (SAUR) exhibited induced expression in flowering buds. Thorough prediction of Crocus sativus non-coding RNA repertoire has been carried out for the first time. Enolase was found to be acting as a major hub with protein-protein interaction analysis using Arabidopsis counterparts., Conclusion: Transcripts belong to key pathways including phenylpropanoid biosynthesis, hormone signaling and carbon metabolism were found significantly modulated. KEGG assessment and protein-protein interaction analysis confirm the expression data. Findings unravel the genetic determinants driving the size dependent flowering in Crocus sativus., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Nicotine is a psychostimulant that induces neurochemical and behavioral changes upon chronic administration, leading to neurodegenerative conditions associated with smoking. As of now, no preventive or therapeutic strategies are known to counteract nicotine‑induced neurodegeneration. In this study, we explore the neuroprotective effects of crocin, a bioactive agent commonly found in saffron - a spice derived from the flower of Crocus sativus - using a rat model. The dose‑dependent effects of crocin were evaluated in nicotine‑induced neurodegeneration and compared with a control group. Neurobehavioral changes, assessed through the elevated plus maze, the open field test, the forced swim test, and the Morris water maze, as well as oxidative stress in the hippocampus, were evaluated. Interestingly, nicotine administration resulted in depression, anxiety, and abnormal motor and cognitive functions, while crocin treatment protected the rat brain from these abnormalities. The beneficial effects of crocin were associated with reduced oxidative stress biomarkers such as malondialdehyde, along with increases in superoxide dismutase, glutathione peroxidase, and glutathione reductase activities. These results demonstrate that crocin can mitigate nicotine‑induced neurodegeneration by reducing oxidative stress, potentially offering a protective measure against neurodegenerative effects in smokers.

Aims: Geophytic plants have evolved to develop underground storage organs (USO) in the active growing season to withstand harsh environments as well as to coordinate growth and reproduction when conditions are favourable. Saffron is an autumn flowering geophyte and an expensive spice crop restricted to certain geographical locations in the world. Saffron, being sterile, does not produce seeds and thus propagates only through corms, the quality of which determines its yield. Corm development in saffron is unexplored and the underlying molecular mechanism is still elusive. In this study, we performed an extensive characterisation of the transcriptional dynamics in the source (leaf) and sink (corm) tissues during corm development in saffron., Key Results: Via morphological and transcriptome studies, we identified molecular factors regulating corm development process in saffron, which defined corm development into three stages: the initiation stage demonstrates enhanced vegetative growth aboveground and swelling of shoot base belowground due to active cell division & carbohydrate storage; the bulking stage comprises of increased source and sink strength, active photosynthesis, circadian gating and starch accumulation; the maturation stage represents reduced source and sink strength, lowered photosynthesis, sugar transport, starch synthesis and cell cycle arrest., Utility: The global view of transcriptional changes in source and sink identifies similar and new molecular factors involved in the saffron corm development process compared to USO formation in other geophytes and provides a valuable resource for dissecting the molecular network underlying the corm development. We propose a hypothetical model based on data analysis, of how molecular factors via environmental cues can regulate the corm development process in saffron., (© 2024 Scandinavian Plant Physiology Society.)

Crocus sativus has emerged as an important crop because it is the only commercial source of saffron that contains unique apocarotenoids. Saffron is composed of dried stigmas of Crocus flower and constitutes the most priced spice of the world. Crocus floral organs are dominated by different classes of metabolites. While stigmas are characterized by the presence of apocarotenoids, tepals are rich in flavonoids and anthocyanins. Therefore, an intricate regulatory network might play a role in allowing different compounds to dominate in different organs. Work so far done on Crocus is focussed on apocarotenoid metabolism and its regulation. There are no reports describing the regulation of flavonoids and anthocyanins in Crocus tepals. In this context, we identified an R2R3 transcription factor, CstMYB16, which resembles subgroup 4 (SG4) repressors of Arabidopsis. CstMYB16 is nuclear localized and acts as a repressor. Overexpression of CstMYB16 in Crocus downregulated anthocyanin biosynthesis. The C2/EAR motif was responsible for the repressor activity of CstMYB16. CstMYB16 binds to the promoter of the anthocyanin biosynthetic pathway gene (LDOX) and reduces its expression. CstMYB16 also physically interacts with CstPIF4, which in turn is regulated by temperature and circadian clock. Thus, CstPIF4 integrates these signals and forms a repressor complex with CstMYB16, which is involved in the negative regulation of anthocyanin biosynthesis in Crocus. Independent of CstPIF4, CstMYB16 also represses CstPAP1 expression, which is a component of the MYB-bHLH-WD40 (MBW) complex and positively controls anthocyanin biosynthesis. This is the first report on identifying and describing regulators of anthocyanin biosynthesis in Crocus., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The accumulation of relatively higher dose of zinc oxide nanoparticles in brain was reported to produce neurotoxicity. Indeed, nanoparticles have a high ability to penetrate biological membranes and be uptaken by cells, which may cause cell disorders and physiological dysfunctions. The aim of the current study was to evaluate, whether oral administration of saffron extract, in rats, can protect from neurotoxicity and behavioural disturbances induced by chronic administration of ZnO-NPs. Daily oral administration of ZnO-NPs was performed for 21 consecutive days to induce oxidative stress-like situation. Then after the saffron extract was concomitantly administrated in several rat groups to overcome the nanotoxicological effect induced by ZnO-NPs. In the frontal cortex, the hippocampus and the cerebellum, ZnO-NPs induced a H 2 O 2 -oxydative stress-like effect reflected in reduced enzymatic activities of catalase, superoxide dismutase and glutathione S-transferase, and decreased acetylcholinesterase activity. In addition, increased levels of proinflammatory interleukins IL-6 and IL-1-⍺ occurred in the hippocampus, reveal the existence of brain inflammation. The concomitant administration of saffron extract to animals exposed to ZnO-NPs prevented the enhanced anxiety-related to the behaviour in the elevated plus-maze test, the open field test and preserved spatial learning abilities in the Morris water maze. Moreover, animals exposed to ZnO-NPs and saffron showed abnormal activity of several antioxidant enzymes as well as acetylcholinesterase activity, an effect that may underly the preserved anxiety-like behaviour and spatial learning abilities observed in these animals. Saffron extract has a potential beneficial therapeutic effect: antioxidant, anti-inflammatory and neuroprotective agent., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

Paraquat is a green liquid toxin that is used in agriculture and can induce multi-organ including lung injury. Various pharmacological effects of Crocus sativus (C. sativus) were indicated in previous studies. In this research, the effects of C. sativus extract and pioglitazone on inhaled paraquat-induced lung inflammation, oxidative stress, pathological changes, and tracheal responsiveness were studied in rats. Eight groups of rats (n = 7 in each) including control (Ctrl), untreated paraquat aerosol exposed group (54 mg/m 3 , 8 times in alternate days), paraquat treated groups with dexamethasone (0.03 mg/kg/day, Dexa) as positive control, two doses of C. sativus extract (20 and 80 mg/kg/day, CS-20 and CS-80), pioglitazone (5 and 10 mg/kg/day, Pio-5 and Pio-10), and the combination of CS-20 + Pio-5 were studied. Total and differential WBC, levels of oxidant and antioxidant biomarkers in the BALF, lung tissue cytokine levels, tracheal responsiveness (TR), and pathological changes were measured. The levels of IFN-γ, IL-10, SOD, CAT, thiol, and EC 50 were reduced, but MDA level, total and differential WBC count in the BALF and lung pathological changes were increased in the paraquat group (all, p < 0.001). The levels of IFN-γ, IL-10, SOD, CAT, thiol and EC 50 were increased but BALF MDA level, lung pathological changes, total and differential WBC counts were reduced in all treated groups. The effects of C. sativus high dose and combination groups on measured parameters were equal or even higher than dexamethasone (p < 0.05 to p < 0.001). The effects of the combination of CS-20 + Pio-5 on most variables were significantly higher than CS-20 and Pio-5 alone (p < 0.05 to p < 0.001). C. sativus treatment improved inhaled paraquat-induced lung injury similar to dexamethasone and showed a synergistic effect with pioglitazone, suggesting possible PPAR-γ receptor-mediated effects of the plant., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Liver fibrosis refers to a complex inflammatory response caused by multiple factors, which is a known cause of liver cirrhosis and even liver cancer. As a valuable medicine food homology herb, saffron has been widely used in the world. Saffron is commonly used in liver-related diseases and has rich therapeutic and health benefits. The therapeutic effect is satisfactory, but its mechanism is still unclear. In order to clarify these problems, we planned to determine the pharmacological effects and mechanisms of saffron extract in preventing and treating liver fibrosis through network pharmacology analysis combined with in vivo validation experiments. Through UPLC-Q-Exactive-MS analysis, a total of fifty-six nutrients and active ingredients were identified, and nine of them were screened to predict their therapeutic targets for liver fibrosis. Then, network pharmacology analysis was applied to identify 321 targets for saffron extract to alleviate liver fibrosis. Functional and pathway enrichment analysis showed that the putative targets of saffron for the treatment of hepatic fibrosis are mainly involved in the calcium signaling pathway, the HIF-1 signaling pathway, endocrine resistance, the PI3K/Akt signaling pathway, lipid and atherosclerosis, and the cAMP signaling pathway. Based on the CCl 4 -induced liver fibrosis mice model, we experimentally confirmed that saffron extract can alleviate the severity and pathological changes during the progression of liver fibrosis. RT-PCR and Western blotting analysis confirmed that saffron treatment can prevent the CCl 4 -induced upregulation of HIF-1α, VEGFA, AKT, and PI3K, suggesting that saffron may regulate AKT/HIF-1α/VEGF and alleviate liver fibrosis.

Autism is a neurobehavioral disease that induces cognitive and behavioral alterations, usually accompanied by oxidative stress in the brain. Crocus sativus (saffron) and its active ingredient, crocin, have potent antioxidative effects that may benefit autistic behaviors. This study aimed to determine the effects of saffron extract and crocin against brain oxidative stress and behavioral, motor, and cognitive deficits in an animal model of autism in male offspring rats. 14 female rats were randomly divided into the saline and valproic acid (VPA) groups. Then, they were placed with mature male rats to mate and produce offspring. VPA (500 mg/kg, i.p.) was injected on day 12.5 of pregnancy (gestational day, GD 12.5) to induce an experimental model of autism. 48 male pups were left undisturbed for 29 days. First-round behavioral tests (before treatments) were performed on 30-33 post-natal days (PND), followed by 28 days of treatment (PND 34-61) with saffron (30 mg/kg, IP), crocin (15 or 30 mg/kg, i.p.), or saline (2 ml/kg, i.p.). The second round of behavioral tests (after treatments) was performed on PND 62-65 to assess the effects of the treatments on behavioral and cognitive features. In the end, animals were sacrificed under deep anesthesia, and their brains were dissected to evaluate the brain oxidative stress parameters, including malondialdehyde (MDA), glutathione (GSH), and catalase (CAT). VPA injection into female rats increased anxiety-like behaviors, enhanced pain threshold, impaired motor functions, disturbed balance power, increased MDA, and decreased GSH and CAT in their male offspring. 28 days of treatment with saffron or crocin significantly ameliorated behavioral abnormalities, reduced MDA, and increased GSH and CAT levels. Brain oxidative stress has been implicated in the pathophysiology of autistic-like behaviors. Saffron and crocin ameliorate anxiety-like behaviors, pain responses, motor functions, and brain oxidative stress parameters in an experimental model of autism. Saffron and crocin may hold promise as herbal-based pharmacological treatments for individuals with autism. However, further histological evidence is needed to confirm their efficacy., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Tumor necrosis factor-α (TNF-α), an inflammatory cytokine, is produced by monocytes and macrophages. It is known as a 'double-edged sword' because it is responsible for advantageous and disadvantageous events in the body system. The unfavorable incident includes inflammation, which induces some diseases such as rheumatoid arthritis, obesity, cancer, and diabetes. Many medicinal plants have been found to prevent inflammation, such as saffron (Crocus sativus L.) and black seed (Nigella sativa). Therefore, the purpose of this review was to assess the pharmacological effects of saffron and black seed on TNF-α and diseases related to its imbalance. Different databases without time limitations were investigated up to 2022, including PubMed, Scopus, Medline, and Web of Science. All the original articles (in vitro, in vivo, and clinical studies) were collected on the effects of black seed and saffron on TNF-α. Black seed and saffron have therapeutic effects against many disorders, such as hepatotoxicity, cancer, ischemia, and non-alcoholic fatty liver, by decreasing TNF-α levels based on their anti-inflammatory, anticancer, and antioxidant properties. Saffron and black seed can treat a variety of diseases by suppressing TNF-α and exhibiting a variety of activities such as neuroprotective, gastroprotective, immunomodulatory, antimicrobial, analgesic, antitussive, bronchodilator, antidiabetic activity, anticancer, and antioxidant effects. To uncover the beneficial underlying mechanisms of black seed and saffron, more clinical trials and phytochemical research are required. Also, these two plants affect other inflammatory cytokines, hormones, and enzymes, implying that they could be used to treat a variety of diseases., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Polycystic ovary syndrome (PCOS) is a hormonal disorder that affects women of reproductive age, characterized by a range of symptoms, including irregular menstrual cycles, excess male hormones (androgens), metabolic abnormalities such as hyperinsulinemia, hyperlipidemia, and metabolic disturbances like glucose imbalance. Botanical supplements are perceived first and safe choice over available regimens to regulate PCOS. There are several reports available stating that apocarotenoids, carotenoids, and whole extracts of Crocus sativus were identified to have a potential role in the management of women health. This study aimed to propose a network pharmacology-based method to determine the potential therapeutic pathways of phytoconstituents (apocarotenoids and carotenoids) of UHPLC-PDA standardized stigma-based Crocus sativus extract (CSE) for the management of PCOS. Furthermore, to validate the potential targets and signaling pathways, these apocarotenoids, and carotenoids were screened for molecular docking and in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions. The information regarding PCOS-related genes was retrieved from the PCOS knowledge database (PCOSKB), resulting in an established network between putative targets of PCOS and Crocus sativus extract phytochemicals to prevail the mechanism of action. Based on the screening conditions, 4 prominent targets namely, serine/threonine kinase 1 (AKT1), signal transducer and activator of transcription (STAT3), mitogen-activated protein kinase 3 (MAPK3), and mitogen-activated protein kinase 1 (MAPK1), were identified through network analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested that MAP kinase and serine-threonine pathways were found prominent targets in PCOS. Further, a molecular docking study shows that crocetin, picrocrocin, and safranal had the best binding affinity for the identified targets. In silico ADMET results revealed that carotenoids and apocarotenoids were found to have the maximum bioavailability and were able to cross the blood-brain barrier without any toxic effects. The combined results revealed that the apocarotenoids and carotenoids of Crocus sativus extract could act on various targets to regulate multiple pathways related to PCOS., Competing Interests: The authors have no funding and conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.)

Saffron treatment is a broad-spectrum therapy used for several retinal diseases, and its effectiveness depends on a particular molecular composition (REPRON ® saffron). Its production requires specific crops and procedures that, together with low yields, make this spice expensive. To reduce costs, the use of hydroponic crops is gradually increasing. In this study, we tested the protective properties of a hydroponic saffron (sH) batch in models of retinal pigmented epithelium (RPE) degeneration. ARPE-19 cells were pretreated with 40 µg/mL saffron and exposed to different types of damage: excess light and retinol (LE + RET) or oxidative stress (H 2 O 2 ). After analyzing the composition of all saffron types with spectroscopy, we performed cell viability and immunofluorescence analysis for both protocols. We compared the sH results with those of a validated batch of saffron REPRON ® (sR) and those of a saffron non-REPRON ® (sNR) batch. sH and sR, which we found had the same chemical composition, were more effective than sNR in increasing cell survival and attenuating the morphological changes related to apoptosis. In conclusion, hydroponic culturing is a suitable strategy to produce high-quality saffron to reduce costs and increase the accessibility of this promising treatment for retinal degeneration.

Background: Some patients with locally advanced/metastatic non-small cell lung cancer (NSCLC) respond poorly to anti-programmed cell death protein 1 (PD-1)/anti-programmed death-ligand 1 (PD-L1) treatments. Combination with other agents may improve the outcomes. This open-label, multicenter, phase 1b trial investigated the combination of sitravatinib, a spectrum-selective tyrosine kinase inhibitor, plus anti-PD-1 antibody tislelizumab., Methods: Patients with locally advanced/metastatic NSCLC were enrolled (Cohorts A, B, F, H, and I; N=22-24 per cohort). Cohorts A and F included patients previously treated with systemic therapy, with anti-PD-(L)1-resistant/refractory non-squamous (cohort A) or squamous (cohort F) disease. Cohort B included patients previously treated with systemic therapy, with anti-PD-(L)1-naïve non-squamous disease. Cohorts H and I included patients without prior systemic therapy for metastatic disease, no prior anti-PD-(L)1/immunotherapy, with PD-L1-positive non-squamous (cohort H) or squamous (cohort I) histology. Patients received sitravatinib 120 mg orally one time per day plus tislelizumab 200 mg intravenously every 3 weeks, until study withdrawal, disease progression, unacceptable toxicity, or death. The primary endpoint was safety/tolerability among all treated patients (N=122). Secondary endpoints included investigator-assessed tumor responses and progression-free survival (PFS)., Results: Median follow-up was 10.9 months (range: 0.4-30.6). Treatment-related adverse events (TRAEs) occurred in 98.4% of the patients, with ≥Grade 3 TRAEs in 51.6%. TRAEs led to discontinuation of either drug in 23.0% of the patients. Overall response rate was 8.7% (n/N: 2/23; 95% CI: 1.1% to 28.0%), 18.2% (4/22; 95% CI: 5.2% to 40.3%), 23.8% (5/21; 95% CI: 8.2% to 47.2%), 57.1% (12/21; 95% CI: 34.0% to 78.2%), and 30.4% (7/23; 95% CI: 13.2% to 52.9%) in cohorts A, F, B, H, and I, respectively. Median duration of response was not reached in cohort A and ranged from 6.9 to 17.9 months across other cohorts. Disease control was achieved in 78.3-90.9% of the patients. Median PFS ranged from 4.2 (cohort A) to 11.1 months (cohort H)., Conclusions: In patients with locally advanced/metastatic NSCLC, sitravatinib plus tislelizumab was tolerable for most patients, with no new safety signals and overall safety profiles consistent with known profiles of these agents. Objective responses were observed in all cohorts, including in patients naïve to systemic and anti-PD-(L)1 treatments, or with anti-PD-(L)1 resistant/refractory disease. Results support further investigation in selected NSCLC populations., Trial Registration Number: NCT03666143., Competing Interests: Competing interests: QZ reports honoraria for lectures/presentations from AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, MSD, Pfizer, Roche, and Sanofi. DD reports institutional research funding for clinical trials participation from BeiGene, Bristol-Myers Squibb, EpimAb, Harbour BioMed, Maxinovel, MSD, Olema Pharmaceuticals, Pfizer, PhamAbcine, and Roche. MV reports honoraria from MSD and has served as an advisory board member for AstraZeneca. HL, JZhang, and YP are employees of BeiGene. Y-LW reports institutional grants from AstraZeneca, Bristol-Myers Squibb, and Pfizer; and honoraria for lectures/presentations from AstraZeneca, Bristol-Myers Squibb, Eli Lilly, Hengrui, MSD, Pfizer, Roche, and Sanofi. All other authors declare no competing interests., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Sofosbuvir is a novel drug candidate for the treatment of hepatitis C viral infection; however, vision loss is one of its growing adverse effects. Saffron is a natural biomolecule with a high antioxidant potential that has been efficiently used in some diseases caused by oxidative stress. This study evaluated Sofosbuvir's neurodegenerative effect on the retina of albino rat and examined the potential protective role of saffron aqueous extract. Twenty-one adult male albino rats were randomly divided into three groups: Control, Sofosbuvir-treated (41.1 mg/kg /day for 6 weeks), and Sofosbuvir + Saffron co-treated groups. Retinal specimens were biochemically analyzed for malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) levels. In addition, light and transmission electron microscopic examination, as well as immunohistochemical staining for Caspase-3, COX-2, and GFAP were performed. Sofosbuvir treatment caused a significant increase in retinal MDA, IL-6, and TNF-α levels coupling with a significant decrease in retinal total antioxidant capacity level. Histopathological findings revealed disturbed retinal architecture, detached pigment epithelium, vacuolated photoreceptors, in addition to a significant decrease in the thicknesses of both outer and inner nuclear layers, and the number of ganglionic cells. Ultrastructural examination revealed extensive degenerative changes in all retinal layers. Caspase-3, COX-2, and GFAP immunohistochemical expressions were significantly increased. Meanwhile, concomitant treatment with Saffron significantly improved retinal redox status, inflammation, histological, and ultrastructural parameters. Saffron may protect the retina from the hazardous effects of Sofosbuvir. Saffron could be used as an adjuvant therapy to protect patients receiving Sofosbuvir from retinal damage., (© 2022 American Association for Anatomy.)

Ethnopharmacological Relevance: Saffron, the dried stigma of Crocus sativus L., has a long history of use in the treatment of depression in traditional Chinese medicine and Islamic medicine. The unique aroma of saffron, primarily derived from its volatile oil, has been widely used by folk to mitigate anxiety and depression via sniffing because the aroma of saffron has a pleasant and invigorating effect., Aim of the Study: This study aimed to investigate the antidepressant effect and the underlying mechanism of saffron essential oil (SEO) in mice exposed to chronic unpredictable mild stress (CUMS)., Materials and Methods: In this study, compounds of SEO were identified using gas chromatography-mass spectrometry analysis, while network pharmacology was used to predict potential active compounds, antidepressant targets, and related signaling pathways of SEO. The CUMS depression model was further used to explore the therapeutic effect and possible mechanism of SEO. During the modeling period, mice were regularly administered fluoxetine (3.6 mg/kg, i.g.) or diluted SEO (2%, 4%, and 6% SEO, inhalation). The antidepressant and neuroprotective effects of SEO were evaluated by behavior tests (the open field test, the sucrose preference test, the tail suspension test, and the forced swimming test), hematoxylin-eosin staining, and Nissl staining. The enzyme-linked immunosorbent assay kits were used to measure dopamine (DA), 5-serotonin (5-HT), brain-derived neurotrophic factor (BDNF), and γ-aminobutyric acid (GABA) levels in serum. The relative abundance of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B in the hippocampus was determined using western blot (WB)., Results: According to the network pharmacology analysis, seven active SEO compounds mediated 113 targets related to depression treatment, most of which were enriched in the 5-HT synapse, calcium signaling pathway, and cAMP signaling pathway. In vivo experiments indicated that fluoxetine and SEO improved depression-like behaviors in depressed mice. The levels of 5-HT, DA, BDNF, and GABA in serum increased significantly. Histopathological examinations revealed that fluoxetine and SEO ameliorated neuronal damage in the hippocampus. WB analysis showed that the relative expressions of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B were significantly higher in the fluoxetine and SEO groups than in the CUMS group., Conclusion: Overall, these findings suggest that SEO significantly alleviates the depressive symptoms in CUMS exposed mice and partially restores hippocampal neuronal damage. Meanwhile, the best efficacy was observed in 4% SEO. Furthermore, the antidepressant mechanism of SEO is primarily dependent on the regulation of the MAPK-CREB1-BDNF signaling pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The prevalence of both Alzheimer's disease (AD) and diabetes mellitus is increasing with the societies' aging and has become an essential social concern worldwide. Accumulation of amyloid plaques and neurofibrillary tangles (NFTs) of tau proteins in the brain are hallmarks of AD. Diabetes is an underlying risk factor for AD. Insulin resistance has been proposed to be involved in amyloid-beta (Aβ) aggregation in the brain. It seems that diabetic conditions can result in AD pathology by setting off a cascade of processes, including inflammation, mitochondrial dysfunction, and ROS and advanced glycation end products (AGEs) synthesis. Due to the several side effects of chemical drugs and their high cost, using herbal medicine has recently attracted attention for the treatment of diabetes and AD. Saffron and its active ingredients have been used for its anti-inflammatory, anti-oxidant, anti-diabetic, and anti-AD properties. Therefore, in the present review paper, we take account of the clinical, in vivo and in vitro evidence regarding the anti-diabetic and anti-AD effects of saffron and discuss the preventive or postponing properties of saffron or its components on AD development via its anti-diabetic effects., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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 β-carotene isomerase showing high specificity for the C9-C10 double bond catalyzing the interconversion of all- trans - into 9- cis -β-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 -β-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.

Background: People with inflammatory bowel disease (IBD) including ulcerative colitis are at risk for colorectal cancer. Despite available effective drugs used to treat IBD, many patients fail or lose response over time with some displaying drug-induced adverse events. Saffron (Crocus sativus) has been reported to have anti-inflammatory properties. Its protective role in IBD has not been explored extensively., Aim: To establish whether saffron treatment alleviates inflammation in experimental colitis., Methods: Colitis was induced in C57BL/6 mice with 3% DSS and treated with either saffron doses (7.5, 15, 20, 25 mg/kg body weight) or vehicle through daily gavage. On day 11, mice were euthanized and analyzed for gross and microscopic inflammation. Distal colon segments were collected for mRNA and protein expression of HO-1 protein and GPX2, (the downstream targets of NRF-2). Nrf-2 translocation from cytosol to nucleus was confirmed by immunofluorescence, and further Nrf-2 protein expression in nuclear and cytosolic fraction of colon was analyzed by immunoblot. Immune cells were isolated from the lamina propria of mouse colon for flow cytometry-based immunophenotyping. Colitis was also induced in C57BL/6 Ahr knockout and wild type mice to explore the involvement of Ahr-dependent pathways in saffron's protective effect(s). The therapeutic effect of saffron was further validated in another TNBS model of colitis., Results: Saffron 20 mg/kg body weight showed improved colon gross and histology features and led to better body weight, colon length, histology score, and reduced disease activity index (DAI). Saffron significantly decreased pro-inflammatory macrophages (M1), while increasing anti-inflammatory macrophages (M2) and IL10 + dendritic cells. Saffron treatment also enhanced CD3 + T and CD3 + CD8 + T cells followed by increase in different CD3 + CD4 + T cells subsets like CD25 + T cells, FoxP3 + CD25 + regulatory T cells, and CD4 + FOXP3 + CD25-regulatory T cells. Immunoblot analysis showed a significant increase in HO-1/GPX2 protein expression. With saffron treatment, Nrf-2 translocation into nucleus from cytosol also supports the involvement of Nrf-2 and its downstream targets in the protective effect of saffron. Further, we demonstrated that saffron in part exert anti-inflammatory effect through activation of aryl hydrocarbon receptor (AhR)-nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways., Conclusion: These data demonstrate saffron's therapeutic potential and its protective role in part via Ahr/Nrf-2 pathways and regulatory innate and adaptive immune cells., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: Isolating high-quality RNA is a basic requirement while performing high throughput sequencing, microarray, and various other molecular investigations. However, it has been quite challenging to isolate RNA with absolute purity from plants like Crocus sativus that are rich in secondary metabolites, polysaccharides, and other interfering compounds which often irreversibly co-precipitate with the RNA. While many methods have been proposed for RNA extraction including CTAB, TriZol, and SDS-based methods, which invariably yield less and poor quality RNA and hence it necessitated the isolation of high-quality RNA suitable for high throughput applications., Results: In the present study we made certain adjustments to the available protocols including modifications in the extraction buffer itself and the procedure employed. Our method led to the isolation of clear and non-dispersive total RNA with an RNA Integrity Number (RIN) value greater than 7.5. The quality of the RNA was further assessed by qPCR-based amplification of mRNA and mature miRNAs such as Cs-MIR166c and Cs-MIR396a., (© 2022. The Author(s).)

Saffron is a unique plant in many aspects, and its cellular processes are regulated at multiple levels. The genetic makeup in the form of eight chromosome triplets (2n = 3x = 24) with a haploid genetic content (genome size) of 3.45 Gbp is decoded into different types of RNA by transcription. The RNA then translates into peptides and functional proteins, sometimes involving post-translational modifications too. The interactions of the genome, transcriptome, proteome and other regulatory molecules ultimately result in the complex set of primary and secondary metabolites of saffron metabolome. These complex interactions manifest in the form of a set of traits 'phenome' peculiar to saffron. The phenome responds to the environmental changes occurring in and around saffron and modify its response in respect of growth, development, disease response, stigma quality, apocarotenoid biosynthesis, and other processes. Understanding these complex relations between different yet interconnected biological activities is quite challenging in saffron where classical genetics has a very limited role owing to its sterility, and the absence of a whole-genome sequence. Omics-based technologies are immensely helpful in overcoming these limitations and developing a better understanding of saffron biology. In addition to creating a comprehensive picture of the molecular mechanisms involved in apocarotenoid synthesis, stigma biogenesis, corm activity, and flower development, omics-technologies will ultimately lead to the engineering of saffron plants with improved phenome., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Reactive oxygen species (ROS) behave as signaling molecules and induce biosynthesis of many secondary metabolites, including apocarotenoids, which play critical roles in stress tolerance through radical scavenging. However, the mechanism that regulates ROS responsive apocarotenoid metabolism and subsequent stress response is unknown. In this study, an R2R3-MYB transcription factor (CstMYB14) was identified from Crocus sativus L., which acts as a regulator of apocarotenoid biosynthesis. CstMYB14 expression increases in response to H 2 O 2 in a concentration and time-dependent manner. CstMYB14 localizes to the nucleus and acts as a transcriptional activator. Over-expression of CstMYB14 in Crocus stigmas enhanced apocarotenoid biosynthesis. Yeast-one-hybrid demonstrated binding of CstMYB14 to promoters of two apocarotenoid pathway genes (phytoene synthase and carotenoid cleavage dioxygenase 2). Nicotiana benthamiana plants overexpressing CstMYB14 showed better growth and higher stress tolerance than wild type plants. Higher antioxidant activity in CstMYB14-Ox plants indicated that stress tolerance might be due to ROS scavenging. These results establish a molecular link between ROS signaling, apocarotenoid metabolism and stress tolerance. Further, CstMYB14 is shown to act as a key regulator which modulates ROS responsive biosynthesis of apocarotenoids which in turn impart stress tolerance through ROS scavenging., (© 2022 Scandinavian Plant Physiology Society.)

The chemical compositions of 15 saffron samples from 11 countries (Morocco, India, Italy, Spain, Germany, Switzerland, Iran, Lithuania, Ukraine, Australia, and Azerbaijan) were evaluated. The samples were analyzed regarding the impact of environmental factors on the composition of apocarotenoids and phenolic constituents. Quantification of saffron metabolites was carried out using high-performance liquid chromatography. It was found that the high content of chlorogenic acid (0.2 mg/g, Ukraine) and ferulic acid (0.28 mg/g, India) was controlled by the duration of solar radiation during plant development. The accumulation of caffeic acid (the higher content 4.88 mg/g, Ukraine) in stigmas depended on the average air temperature. In contrast, the total crocins content according to the correlation analysis depended on the duration of solar radiation, the solar UV index, and the soil type. Rutin was found in all samples (0.83-8.74 mg/g). The highest amount of crocins (average 382.45 mg/g) accumulated in saffron from Italy and Ukraine. Crocins, picrocrocin, safranal, and rutin can further serve as saffron quality markers. All validation parameters were satisfactory and high-performance liquid chromatography methods could be successfully applied for the composition assessment of saffron metabolites. Saffron extracts showed the highest antibacterial activity against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli (MICs 62.5-125 µg/ml)., (© 2022 Deutsche Pharmazeutische Gesellschaft.)

Saffron is derived from the stigmas of the flower Crocus sativus L. The drying process is the most important post-harvest step for converting C. sativus stigmas into saffron. The aim of this review is to evaluate saffron's post-harvest conditions in the development of volatile compounds and its aroma descriptors. It describes saffron's compound generation by enzymatic pathways and degradation reactions. Saffron quality is described by their metabolite's solubility and the determination of picrocrocin, crocins, and safranal. The drying process induce various modifications in terms of color, flavor and aroma, which take place in the spice. It affects the aromatic species chemical profile. In the food industry, saffron is employed for its sensory attributes, such as coloring, related mainly to crocins (mono-glycosyl esters or di-glycosyl polyene).

Saffron is a valuable plant and one of the most expensive spices worldwide. Nowadays, there is a tendency to produce this crop in indoor plant production systems. However, the production of saffron is restricted by the need for the reproduction of high-quality corms. In this study, we investigated the effect of different ratios of red (R) and blue (B) light spectra (including 100% B (monochromatic B), 75%, 50%, 40%, 25% B, and 0% B (monochromatic R) on the photosynthetic performance and biomass partitioning as well as morphological and biochemical characteristics of saffron. The growth of flower, root, and corm was improved by increasing the proportion of B to R light. B-grown plants were characterized by the highest photosynthetic functionality with efficient electron transport and lower energy dissipation when compared to R-grown plants. B light directed biomass toward the corms and floral organs, while R light directed it toward the leaves. In saffron, the weight of a daughter corm is of great importance since it determines the yield of the next year. As the ratio of B to R light increased, the daughter corms also became heavier, at the cost of reducing their number, though increasing the proportion of B-enhanced antioxidant capacity as well as the activity of ascorbate peroxidase and catalase while superoxide dismutase activity was enhanced in R-grown plants. In conclusion, B light increased the production of high-quality daughter corms and altered biomass partitioning towards harvestable organs (corms and flowers) in saffron plants.

Background: Saffron or stigmas of Crocus sativus L. is one of the most valuable food products with interesting health-promoting properties. C. sativus has been widely used as a coloring and flavoring agent. Stigmas secondary metabolites showed potent cytotoxic effects in previous reports., Methods: The present study investigated the chemical composition and the cytotoxic effect of Ukrainian saffron crude extracts and individual compounds against melanoma IGR39, triple-negative breast cancer MDA-MB-231, and glioblastoma U-87 cell lines in vitro using MTT assay. Several bioactivity in vitro assays were performed. The chemical profile of the water and hydroethanolic (70%, v/v) crude extracts of saffron stigmas was elucidated by HPLC-DAD analysis., Results: Seven compounds were identified including crocin, picrocrocin, safranal, rutin, apigenin, caffeic acid, ferulic acid. Crocin, picrocrocin, safranal, rutin, and apigenin were the major active constituents of Ukrainian C. sativus stigmas. The hydroethanolic extract significantly reduced the viability of MDA-MB-231 and IGR39 cells and the effect was more potent in comparison with the water extract. However, the water extract was almost 5.6 times more active against the U-87 cell line (EC 50 of the water extract against U-87 was 0.15 ± 0.02 mg/mL, and EC 50 of the hydroethanolic extract was 0.83 ± 0.03 mg/mL). The pure compounds, apigenin, and caffeic acid also showed high cytotoxic activity against breast cancer, melanoma, and glioblastoma cell lines. The screening of the biological activities of stigmas water extract (up to 100 μg/mL) including anti-allergic, anti-virus, anti-neuraminidase, and anti-inflammatory effects revealed its inhibitory activity against neuraminidase enzyme by 41%., Conclusions: The presented results revealed the qualitative and quantitative chemical composition and biological activity of Crocus sativus stigmas from Ukraine as a source of natural anticancer and neuraminidase inhibitory agents. The results of the extracts' bioactivity suggested future potential applications of saffron as a natural remedy against several cancers., (© 2021. The Author(s).)

The metabolite profiling of saffron ( Crocus sativus L.) from several countries was measured by using ultra-performance liquid chromatography combined with high resolution mass spectrometry (UPLC-HR MS). Multivariate statistical analysis was employed to distinguish among the several samples of C. sativus L. from Greece, Italy, Morocco, Iran, India, Afghanistan and Kashmir. The results of this study showed that the phytochemical content in the samples of C. sativus L. were obviously diverse in the different countries of origin. The metabolomics approach was deemed to be the most suitable in order to evaluate the enormous array of putative metabolites among the saffron samples studied, and was able to provide a comparative phytochemical screening of these samples. Several markers have been identified that aided the differentiation of a group from its counterparts. This can be important for the selection of the appropriate saffron sample, in view of its health-promoting effect which occurs through the modulation of various biological and physiological processes.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia in the old age population, making it a worldwide concern. Unfortunately, few drugs have been presented for treatment of mild and moderate AD. To meet this need, more effective anti-AD agents are emerging. Accumulating evidence supports the beneficial roles of natural-based products in brain function, neurotransmission, neurogenesis, synaptogenesis, and the prevention of amyloid fibrillation and neuronal injury. Several in vitro , preclinical, and clinical studies suggest that saffron (its bioactive compounds) is a potential nutraceutical with antioxidant, radical scavenging, anti-inflammatory, hypolipidemic, hypotensive, neuroendocrine, and neuroprotective effects. It has also been proposed that saffron may delay the onset of AD, prevent its progression or help to attenuate the symptoms of the disease. Therefore, we performed a comprehensive search on this plant and its derivatives for AD treatment. Saffron and its active constituents interfere with AD by improving learning behavior, spatial memory, and cognitive function; protecting against neuronal loss; inhibiting beta-amyloid aggregation and neurotoxicity; preventing senile plaques and neurofibrillary tangle (NFT) formation; suppressing the acetylcholinesterase (AChE) activity; and reducing neuroinflammation. Given conclusive scientific findings, saffron and its derivatives might counter neurodegenerative diseases through multiple pathways. Further clinical trials are expected to confirm the neuroprotective properties of this herb and also to translate such findings to improve patients' outcomes., (© 2021 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.)

The dehydration process is a prerequisite to preserve saffron for a long time. According to this process, saffron shows differences in the main compounds responsible for its quality (colour, taste, aroma, and flavonol content). At present, the freeze-drying method obtains dried products with the highest quality. Viruses can modify the physiology and metabolism of plants, being able to affect the activities of several enzymes. For this reason, the main compounds of saffron have been analyzed under two different dehydrating processes, freeze-drying and dark-drying, considering their infection status with the Saffron latent virus (SaLV). Results showed that the picrocrocin and safranal content enables to differ dark-dried samples from freeze-dried ones. Besides, the kaempferol-3-O-sophoroside-7-O-glucoside content allows differentiating between SaLV-infected (SaLV + ) and uninfected (SaLV - ) saffron samples. Moreover, our data suggest that the freeze-drying would decrease crocins content, and dark-drying can nullify the adverse effect of SaLV on crocins content., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Saffron stigmas are widely used as food additives and as traditional medicine in Iran and many other countries. The unique taste, flavor and pharmaceutical properties of saffron stigmas are due to the presence of three apocarotenoids secondary metabolites crocin, picrocrocin and safranal. There is limited knowledge about the effect of environmental stresses on the metabolism of apocarotenoids in saffron. We analyzed the content of crocin and picrocrocin and the expression of key genes of apocarotenoid biosynthesis pathways (CsCCD2, CsCCD4, CsUGT2, CsCHY-β and CsLCYB) in saffron plants exposed to moderate (90 mM) and high (150 mM) salt (NaCl) concentrations. Measuring ion concentrations in leaves showed an increased accumulation of Na + and decreased uptake of K + in salt treated compared to control plants indicating an effective salt stress. HPLC analysis of apocarotenoids revealed that crocin production was significantly halted (P < 0.05) with increasing salt concentration while picrocrocin level did not change with moderate salt but significantly dropped by high salt concentration. Real-time PCR analysis revealed a progressive decrease in transcript levels of CsUGT2 and CsLCYB genes with increasing salt concentration (P < 0.05). The expression of CsCCD2 and CsCHY-β tolerated moderate salt concentration but significantly downregulated with high salt concentration. CsCCD4 however responded differently to salt concentration being decreased with moderate salt but increased at higher salt concentration. Our result suggested that salt stress had an adverse effect on the production of saffron apocarotenoids and it is likely influencing the quality of saffron stigma produced.

Crocus sativus L. belongs to the Iridaceae family and it is commonly known as saffron. The different cultures together with the geoclimatic characteristics of the territory determine a different chemical composition that characterizes the final product. This is why a complete knowledge of this product is fundamental, from which more than 150 chemical compounds have been extracted from, but only about one third of them have been identified. The chemical composition of saffron has been studied in relation to its efficacy in coping with neurodegenerative retinal diseases. Accordingly, experimental results provide evidence of a strict correlation between chemical composition and neuroprotective capacity. We found that saffron's ability to cope with retinal neurodegeneration is related to: (1) the presence of specific crocins and (2) the contribution of other saffron components. We summarize previous evidence and provide original data showing that results obtained both "in vivo" and "in vitro" lead to the same conclusion.

Oxidative stress-mediated neurodegeneration is responsible for 12% mortality around the globe. Alzheimer's Disease (AD) and Parkinson's Disease (PD) are the most prevalent neurodegenerative diseases, associated with modulation of acetylcholine levels and amyloid beta accumulation & dopamine level and alpha-synuclein oligomerization, respectively. Therefore, a better understanding of their pathological mechanisms reveals novel target proteins and encourages exploitation of suitable lead molecules. In the present study, targets for AD and PD were sought not only to suppress the pathological condition but to restore the normal physiological function. In this view, activation of retinoic acid receptor alpha can be formulated as a novel target to improve choline acetyltransferase transcription that works together with acetylcholine esterase and beta-secretase 1 inhibition against AD. Likewise, inhibition of Polo-like kinase 2 fails to phosphorylate alpha-synuclein and motivates efficient autophagic clearance. Therefore, PLK2 inhibition, together with L-DOPA supplementation and monoamine oxidase B inhibition widens the therapeutic options for PD. As oxidative stress is the major factor for neurodegeneration, AMPK activation stabilizes energy metabolism and Sirtuin 1 (histone deacetylase 1) activation enhances AMPK, PGC1a and Nrf gene expressions. Phytochemical extracts from saffron stigma were broadly appreciated on memory enhancement and cognition. However, the exact mechanism was not established. Therefore, this inspires the exploitation of phytochemicals in saffron stigma extract using in-silico tools, to anticipate lead molecules that interact with various neurodegeneration associated protein targets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Saffron is a highly adulterated spice due to its limited production and high costs. Non-saffron plant material is coloured with synthetic dyes to produce counterfeit saffron. Continuous monitoring of the synthetic dyes used in counterfeit saffron is essential because some dyes are not safe for human consumption. In the present study, 104 commercial saffron samples from 16 countries were screened, and 20 samples were found to contain dyes. Thin-Layer Chromatography (TLC) analysis showed adulteration with magenta- and pink- coloured dyes (18 and 2 samples, respectively), which did not correspond to any of the dyes reported earlier. Mass spectrometry analysis identified the magenta-coloured dye as magenta III or new fuchsin and the pink-coloured dye as rhodamine B. Magenta III is regarded as possibly carcinogenic, and rhodamine B was reported to cause local sarcomas. Both of these synthetic dyes are reported as adulterants in saffron for the first time., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Saffron is the world highest-priced spice because its production requires intensive hand labour. Reduce saffron production costs require containerised plant production under controlled conditions and expand the flowering period. Controlling the flowering process and identify the factors involved in saffron flowering is crucial to introduce technical improvements. The research carried out so far in saffron has allowed an extensive knowledge of the influence of temperature on the flower induction, but the molecular mechanisms controlling flowering induction processes are largely unknown. The present study is the first conducted to isolate and characterize a regulator gene of saffron floral induction the Short Vegetative Phase (SVP) gene, which represses the floral initiation genes in the temperature response pathway, which involved in saffron flower induction. The results obtained from both phylogenetic analysis and T-coffee alignment confirms that the isolated sequence belongs to the SVP gene clades of MADS-box gene family. Gene expression analysis in different developmental stages revealed the highest expression of SVP transcript (CsSVP) during the dormancy and the vegetative stages, but decrease when flower development initiated and it was the least in late September when flower primordia are developed. Furthermore, its expression increased in the apical bud when corms are storage at 9-10 ºC, thus inhibiting flower induction. Additionally, comparison of the CsSVP transcript in apical buds from big and small corms, differing in their flowering capacity, indicates that the CsSVP transcript is present only in vegetative buds. Taken together, these results suggested inhibitory role of the SVP gene.

Colored apocarotenoids accumulate at high concentrations in few plant species, where display a role in attraction of pollinators and seed dispersers. Among these apocarotenoids, crocins accumulate at high concentrations in the stigma of saffron and are responsible for the organoleptic and medicinal properties of this spice. Phytoene synthase and Orange protein are key for carotenoid biosynthesis and accumulation. We previously isolated four phytoene synthase genes from saffron with differential roles in carotenoid and apocarotenoid biosynthesis. However, the implications of Orange genes in the regulation of apocarotenoid accumulation are unknown. Here, we have identified two Orange genes from saffron, with different expression patterns. CsOr-a was mainly expressed in vegetative tissues and was induced by light and repressed by heat stress. Both CsOr-a and CsOr-b were expressed in stigmas but showed a different profile during the development of this tissue. The interactions of CsOr-a and CsOr-b were tested with all the four phytoene synthase proteins from saffron and with CsCCD2. None interactions were detected with CCD2 neither with the phytoene synthase 2, involved in apocarotenoid biosynthesis in saffron. The obtained results provide evidence of different mechanisms regulating the phytoene synthase enzymes in saffron by Orange for carotenoid and apocarotenoid accumulation in saffron.

Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast ( Saccharomyces cerevisiae ), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.plantcell;31/11/2789/FX1F1fx1., (© 2019 American Society of Plant Biologists. All rights reserved.)

Saffron is appreciated by its colour, taste, and aroma. To examine the effect of abiotic and biotic stress on these main properties, in the span of 2014-2016, saffron stigmas were collected from major different saffron cultivation areas of Iran and saffron quality was estimated. The quality of saffron was assessed by ultraviolet-visible spectroscopy following the ISO3632:2011 standard. However, the composition and concentration of crocetin esters, picrocrocin, safranal, and kaempferols, the most critical compounds determining the properties and quality of saffron can vary with the geographical origin and virus effects, being more accurate High-Performance Liquid Chromatography and Diode Array Detection (HPLC-DAD) methods were used to analyze saffron quality. Using HPLC-DAD we analyzed saffron plants grown at various conditions (considering altitude, temperature, and precipitation/rainfall) and in presence/absence of virus infections; we found that edaphoclimatic and cultivation conditions significantly determine the quality of the spice and the presence of virus modifies the content of its metabolites., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)