Isolation, Identification, and Determination of the Virulence of the Causal Agents of Corm Rot of Saffron (Crocus sativus L.) in Valle de Uco, Argentina.

Saffron (Crocus sativus L.) presents an attractive opportunity for diversifying production and adding value, particularly for small-scale growers and family-based agriculture. However, the agamic propagation of the crop through corms raises concerns regarding disease dispersion. During the summers of 2013 and 2015, symptoms of corm rot were observed in saffron crops in La Consulta, Valle de Uco, Argentina. These symptoms manifested in the form of wilting plants and red-coloured areas on the surface of the corms, in some cases affecting deeper regions. This study aimed to isolate and identify the causal agent responsible for saffron corm rot while also comparing the virulence of four strains isolated on saffron plants. Consistent isolation of Fusarium spp. colonies from affected corms confirmed its association with the disease. The obtained isolates were inoculated into healthy corms, and the reproduction of symptoms was confirmed, as well as subsequent pathogen re-isolation. Morphological and molecular characterisation of the strains was performed using rDNA gene sequencing. Furthermore, disease progression was assessed with fitting epidemiological models to empirical data, which served as estimators of fungal strain aggressiveness. The results conclusively identified Fusarium oxysporum Schltdl. as the causal agent of corm rot, and variations in virulence were observed among the strains on the host plant. After basic molecular and pathological studies, it is postulated that the fungal strains possibly belong to the forma specialis gladioli, but further studies are necessary to confirm that. The present study provides findings that highlight the importance of early detection and the preservation of pathogen-free fields to sustain saffron cultivation. These findings may constitute the initial step for future projects aimed at understanding the epidemiology of the disease better, determining the species/races of the pathogen, and developing effective management strategies.

Effect of Crocetin on Basal Lipolysis in 3T3-L1 Adipocytes.

Crocetin (CCT) is a natural saffron-derived apocarotenoid that possesses healthy properties such as anti-adipogenic, anti-inflammatory, and antioxidant activities. Lipolysis is enhanced in obesity and correlates with a pro-inflammatory, pro-oxidant state. In this context, we aimed to investigate whether CCT affects lipolysis. To evaluate CCT's possible lipolytic effect, 3T3-L1 adipocytes were treated with CCT10µM at day 5 post-differentiation. Glycerol content and antioxidant activity were assessed using colorimetric assays. Gene expression was measured using qRT-PCR to evaluate the effect of CCT on key lipolytic enzymes and on nitric oxide synthase (NOS) expression. Total lipid accumulation was assessed using Oil Red O staining. CCT10µM decreased glycerol release from 3T3-L1 adipocytes and downregulated adipose tissue triglyceride lipase (ATGL) and perilipin-1, but not hormone-sensitive lipase (HSL), suggesting an anti-lipolytic effect. CCT increased catalase (CAT) and superoxide dismutase (SOD) activity, thus showing an antioxidant effect. In addition, CCT exhibited an anti-inflammatory profile, i.e., diminished inducible NOS (NOS2) and resistin expression, while enhanced the expression of adiponectin. CCT10µM also decreased intracellular fat and C/EBPα expression (a transcription factor involved in adipogenesis), thus revealing an anti-adipogenic effect. These findings point to CCT as a promising biocompound for improving lipid mobilisation in obesity.

Effect of Soil Composition on Secondary Metabolites of Moroccan Saffron (Crocus sativus L.).

Climate and soil are important factors that affect the quality of saffron. Saffron quality is determined by the marked content of secondary metabolites. The objective of this work was to study the effect of soil physicochemical properties on the secondary metabolites of saffron. Our study concerned the analysis of saffron samples by high-performance liquid chromatography-detection by diode array (HPLC-DAD). Soil samples were analyzed by physicochemical methods, ED-XRF fluorescence and X-ray diffraction to determine the different types of clays. Saffron samples grown in loam-clay-sand soils contained high values of crocins and kaempferol 3-sophoroside 7-glucoside but low values of safranal. In addition, saffron samples grown in soils rich in organic matter, phosphorus and potassium contained high values of crocins and kaempferol 3-sophoroside 7-glucoside but low values of safranal. This original approach was carried out for the first time in our study, both by ED-XRF fluorescence and by X-ray diffraction, to determine what elements affect the quality of saffron. Thus, we concluded that clays containing low amounts of iron could have a positive effect on the coloring strength of saffron.

Morphological, molecular and phytochemical variations induced by colchicine and EMS chemical mutagens in Crocus sativus L.

Crocus sativus L., also known as saffron, is one of the most important medicinal and spice plants throughout the world. The plant is a rich source of apocarotenoids such as crocetin esters, picrocrocin, and safranal. The purpose of this study was to investigate the effect of colchicine and ethyl methanesulfonate (EMS) mutagens on possible inducing new variation in C. sativus. Accordingly, corms were exposed to EMS (0.1% and 0.2%) and colchicine (0.05% and 0.025%) for three incubation times. The lowest survival rate of corms was related to EMS treatments. The relative expression of ALDH, BGL, and CCD2 genes under 0.025% colchicine treatment for 12 h revealed a 2 fold increase compared with the control. The flow cytometric measurements (FCM) of the nuclear DNA content of the colchicine-treated plants did not reveal any significant changes in 2C DNA content. The results, manifest the potential of mutagens to create new variations in the plant.

Partial Characterization of the Impact of Saffron on the Sensory and Physicochemical Quality Traits of Dry-Cured Ham.

This study determined the effect of adding three concentrations of saffron (A: high, B: medium, and C: low) on vacuum-packaged dry-cured ham slices. The pH and the color coordinates were assessed at 0, 7, 14, 28 and 60 days of storage, and sensorial quality (visual appearance, odor and flavor) and safranal content were analyzed at 7, 14, 28 and 60 days. Saffron concentration did not significantly affect the pH or color (except in a* (redness) and b* (yellowness) at day 28; p < 0.001). Storage period affected pH values (p < 0.001) in all groups with a significant decline from day 28 (p < 0.05); the color coordinates showed a high stability (only L* (lightness) varied in the C group samples; p < 0.01). Sensorial quality did not vary with the time in any group. Significant differences were found among groups in visual appearance (p < 0.05) and flavor (p < 0.001) at day 14 and in odor at day 14, 28, and 60. In general, the C group samples obtained the highest scores. Safranal content varied significantly with the time in a different way in each group, with differences among groups at day 14 and 60 (p < 0.001).

Pathogenic Microenvironment from Diabetic-Obese Visceral and Subcutaneous Adipocytes Activating Differentiation of Human Healthy Preadipocytes Increases Intracellular Fat, Effect of the Apocarotenoid Crocetin.

In diabetes mellitus type 2 (DM2), developed obesity is referred to as diabesity. Implementation of a healthy diet, such as the Mediterranean, prevents diabesity. Saffron is frequently used in this diet because of its bioactive components, such as crocetin (CCT), exhibit healthful properties. It is well known that obesity, defined as an excessive accumulation of fat, leads to cardiometabolic pathology through adiposopathy or hypertrophic growth of adipose tissue (AT).This is related to an impaired adipogenic process or death of adipocytes by obesogenic signals. We aimed to evaluate the effect of the pathogenic microenvironment and CCT, activating differentiation of healthy preadipocytes (PA). For this, we used human cryopreserved PA from visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) depots obtained from healthy and obese-DM2 donors. We studied the effect of a metabolically detrimental (diabesogenic) environment, generated by obese-DM2 adipocytes from VAT (VdDM) or SAT (SdDM), on the viability and accumulation of intracellular fat of adipocytes differentiated from healthy PA, in the presence or absence of CCT (1 or 10 µM). Intracellular fat was quantified by Oil Red O staining. Cytotoxicity was measured using the MTT assay. Our results showed that diabesogenic conditions induce cytotoxicity and provide a proadipogenic environment only for visceral PA. CCT at 10 µM acted as an antiadipogenic and cytoprotective compound.

Determination of Saffron Volatiles by HS-SBSE-GC in Flavored Cured Ham.

At present, the development of new agri-food products, including flavored meat products presented in ready-to-eat vacuum packs, is encouraged. The addition of ingredients used as flavoring agents creates the need to be able to determine the volatile compounds responsible for their characteristic aroma. The aim of this study is to propose, develop, and validate a new method that uses headspace-stir bar sorptive extraction-gas chromatography/mass spectrometry (HS-SBSE-GC/MS) to determine the saffron aroma in cured ham flavored with this spice. Results showed that safranal was the main volatile compound that could be identified and quantified in cured ham flavored with saffron. This analytical method was adequate in terms of linearity, selectivity, sensitivity, and accuracy. To our knowledge, this is the first time that an HS-SBSE-GC/MS method for determining the saffron aroma of flavored cured ham has been developed and validated, and it is of interest to agri-food industries.

Effect of Crocus sativus L. Stigmas Microwave Dehydration on Picrocrocin, Safranal and Crocetin Esters.

The dehydration process is the basis to obtain high quality saffron and to preserve it for a long time. This process modifies saffron's main metabolites that define its quality, and are responsible for the characteristic color, taste, and aroma of the spice. In this work, the effect of microwave dehydration on saffron main metabolites (picrocrocin, safranal and crocetin esters) from Crocus sativus L. stigmas at three determinate powers and different time lapses was evaluated. The results showed that this dehydration process obtained similar or lower crocetin esters content, and after three months of storage, higher concentration was shown in treatments at 440 W for 36 s, 55 s, and 73 s; at 616 W for 90 s; and at 800 W for 20 s. Picrocrocin content was lower and safranal content was higher in all treatments compared to the control both before and after storage. Regarding to commercial quality, microwave dehydration obtained Category I of saffron according to International Standard Organization (ISO) 3632. After three months of storage, treatments at 616 W for 83 s and 800 W for 60 s obtained lower categories. The results obtained suggest that microwave dehydration is a suitable process for obtaining high quality saffron, 800 W with 6 lapses of 20 s being the best conditions studied.

Development and validation of an HPLC-DAD method for determination of oleuropein and other bioactive compounds in olive leaf by-products.

BACKGROUND: Oil mills could benefit by preparing their own aqueous extracts from olive leaves. Accordingly, the present study aimed to measure the bioactive compounds richness of such extracts, especially oleuropein. A water-based microwave extraction procedure was developed and a selective and precise high-performance liquid chromatography with diode array detection (HPLC-DAD) method was validated for the determination of oleuropein and others bioactive compounds from olive leaves. RESULTS: The water solubility of oleuropein was determined to be 9.5 g L-1 . The extraction procedure was optimized in terms of power, olive leaf weight/water volume ratio and time of extraction, and the results revealed that 2 mg mL-1 and a microwave irradiation at 800 W for 30 s resulted in the greatest efficiency. Oleuropein was determined by the new validation method, which showed good linearity (r2 = 0.996), precision (% relative standard deviation < 10%), recovery (118.6%), and limits of detection (17.48 mg L-1 ) and quantification (21.54 mg L-1 ). Good correlation (r2 = 0.979) was obtained between oleuropein of the olive leaf extracts determined by HPLC-DAD and by UV-visible spectrophotometry. CONCLUSION: A simple extraction method was developed and validated to obtain aqueous extract from olive leaves by microwave extraction, determining for the first time oleuropein water solubility. Validation of the method showed that oleuropein in olive leaves could be quantified when it is at least 1% of dry weight by means of HPLC-DAD. UV-visible spectrophotometry can be useful in oil mills because it enables the content of oleuropein and other bioactive compounds content to be determined in situ in such leaf aqueous extracts. © 2020 Society of Chemical Industry.

Impact of two different dehydration methods on saffron quality, concerning the prevalence of Saffron latent virus (SaLV) in Iran.

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.

Crocetin Isolated from the Natural Food Colorant Saffron Reduces Intracellular Fat in 3T3-L1 Adipocytes.

Saffron, as a food colorant, has been displaced by low-cost synthetic dyes. These have unhealthy properties; thus, their replacement with natural food colorants is an emerging trend. Obesity is a worldwide health problem due to its associated comorbidities. Crocetin esters (crocins) are responsible for the red saffron color. Crocetin (CCT) exhibits healthful properties. We aimed to broaden the existing knowledge on the health properties of CCT isolated from saffron, to facilitate its consideration as a healthy natural food colorant in the future. We evaluated the ability of CCT (1 and 5 µM) to reduce lipid accumulation during the differentiation of 3T3-L1 preadipocytes. Intracellular fat was quantified by Oil Red O staining. CTT cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The number and size of lipid droplets were analyzed using WimLipid software. The expression of adipogenic genes (CCAAT/enhancer-binding protein (C/EBPß, C/EBPδ, C/EBPα), and peroxisome proliferator-activated receptor γ (PPARγ)) was analyzed using quantitative real-time PCR (qRT-PCR). CCT 5 µM decreased intracellular fat by 22.6%, without affecting viability or lipid droplet generation, via a decrease in C/EBPα expression, implicated in lipid accumulation. Thus, CCT is a potential candidate to be included in dietary therapies aimed at reversing adipose tissue accumulation in obesity.

Bioactivity and Bioavailability of the Major Metabolites of Crocus sativus L. Flower.

Crocus sativus L. has been cultivated throughout history to obtain its flowers, whose dried stigmas give rise to the spice known as saffron. Crocetin esters, picrocrocin, and safranal are the main metabolites of this spice, which possess a great bioactivity, although the mechanisms of action and its bioavailability are still to be solved. The rest of the flower is composed by style, tepals, and stamens that have other compounds, such as kaempferol and delphinidin, which have an important antioxidant capacity, and these can be applied in foods, phytopharmaceuticals, and cosmetics. The aim of this work was to provide an updated and critical review of the research on the main compounds of Crocus sativus L. flower, including the adequate analytical methods for their identification and quantification, with a focus on their bioactivity and bioavailability.

The effects of geographical origin and virus infection on the saffron (Crocus sativus L.) quality.

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.

Kinetics of polyphenol content of dry flowers and floral bio-residues of saffron at different temperatures and relative humidity conditions.

The cultivation of Crocus sativus L. is valued for its dried stigmas, but the rest of the parts of its flowers are increasingly important. Saffron flowers (SF) are natural sources of antioxidant compounds. Kaempferols and anthocyanins are the main compounds of the high-phenolic content of SF. This work studies the evolution of flavonols and anthocyanins of dry SF and floral bio-residues of saffron (FBR) and their kinetics at different temperatures and relative humidity (RH) conditions. There was a degradation process of anthocyanins that fitted a second-order kinetic model and kaempferols showed better fit in a first-order kinetics model. The best storage conditions for anthocyanins studied in SF and FBR was 25 °C and 23% RH. The main kaempferol (Kaempferol 3-O-sophoroside) was no deteriorated in FBR. These results could contribute to the using SF and FBR as food and active ingredients in cosmetic industry, as well as development of new food products.