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Metabolic dysfunction (MD) is a major health problem threatening the life quality of menopausal women. Saffron has been widely used in herb prescriptions for treating menopausal syndrome. However, the pharmacological effects and mechanisms of saffron are poorly understood. Here, we investigated the effect of crocin, the major ingredient of saffron and its active metabolite in blood, crocetin, on MD and lipid metabolism in ovariectomized (OVX) mice and 3T3-L1 adipocytes. The present study showed that intragastric treatment of crocin prevented weight gain, fat accumulation, and insulin resistance in OVX mice by increasing energy expenditure and fat oxidation. Mechanistically, crocin influenced adipose tissue homeostasis by regulating adipogenic and lipolytic factors, which was strongly associated with the restoration of the downregulated ERβ function in white adipose tissue (WAT). In vitro , crocetin facilitated lipid metabolism in an ERβ-dependent manner. Our results demonstrated the beneficial effects of crocetin/crocin-mediated intervention against metabolic dysfunction, revealing a prospective therapeutic application in menopausal women.

Saffron ( Crocus sativus L.) byproducts are considered as a cheap source of bioactive polyphenolics endowed with potential antioxidant effects. The saffron biowaste is utilized for extraction of flavonoid glycosides and their potential biological properties. The total amount of polyphenolics and polysaccharides was found to be higher in the tepal than in the stamen. The bioactive compounds quercetin-3- O -sophoroside (Q-3-sop) and kaempferol-3- O -sophoroside (K-3-sop) were analyzed using high-performance liquid chromatography equipped with a photodiode array detector (HPLC-PDA) and identified by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR). The antioxidant effects were studied using 2,2 diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), ferric ion reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC); Q-3-sop showed stronger antioxidant effects compared to K-3-sop, crocin-I, and crocin-II. Furthermore, Q-3-sop also inhibited cell apoptosis caused by H 2 O 2 by reducing the levels of cellular reactive oxygen species (ROS). In terms of cytogenetic effects, Q-3-sop revealed no cytogenic effects on onion root meristem cells but chromosomal aberration was observed at the highest dose (200 ppm). Thus, saffron byproducts and its flavonoids could be utilized as natural antioxidant agents with no cytogenetic effects.

Crocins are the primary coloring ingredients of saffron. The low-glycosylated members of this compound family, such as crocin-1 (crocetin mono-glucosyl ester) and crocin-2' (crocetin di-glucosyl ester), are rarely distributed in nature and attracting interest for their therapeutic uses. In the present study, a one-pot reaction system was used for efficient preparation of crocin-1 and crocin-2' with in situ regeneration of UDP-Glc by coupling Bs-GT with At-SuSy, a sucrose synthase from Arabidopsis thaliana . Noticeably, DMSO was used as a cosolvent and resulted in improvement of the solubility of the substrate crocetin and regulation of the selectivity of glycosylation. With periodic addition of crocetin, the biosynthesis of crocin-2' was performed with a high yield of 3.25 g/L in 2% DMSO aqueous solution, whereas crocin-1 (2.12 g/L) was selectively obtained in a 10% DMSO aqueous solution. The present study provided a simple approach for the biosynthesis of crocin-1 and crocin-2'.

Saffron, the dried stigmas of Crocus sativus L., is mainly used as a food coloring and flavoring agent. This agricultural product is used in traditional medicine for the treatment of several diseases including asthma, liver disease, menstruation disorders, and, of special interest in this review, metabolic syndrome. Saffron and its active components including crocin, crocetin, and safranal are potential therapeutic candidates for attenuating MetS complications including hypertension, hyperglycemia, obesity, and dyslipidemia. This review summarizes the protective role of saffron and its constituents in the pathogenesis of MetS for a better understanding and hence a better management of this disease.

Crocus sativus, commonly known as saffron or Kesar, is used in Ayurveda and other folk medicines for various purposes as an aphrodisiac, antispasmodic, and expectorant. Previous evidence suggested that Crocus sativus is linked to improving cognitive function in Alzheimer's disease (AD) patients. The aim of this study was to in vitro and in vivo investigate the mechanism(s) by which Crocus sativus exerts its positive effect against AD. The effect of Crocus sativus extract on Aβ load and related toxicity was evaluated. In vitro results showed that Crocus sativus extract increases the tightness of a cell-based blood-brain barrier (BBB) model and enhances transport of Aβ. Further in vivo studies confirmed the effect of Crocus sativus extract (50 mg/kg/day, added to mice diet) on the BBB tightness and function that was associated with reduced Aβ load and related pathological changes in 5XFAD mice used as an AD model. Reduced Aβ load could be explained, at least in part, by Crocus sativus extract effect to enhance Aβ clearance pathways including BBB clearance, enzymatic degradation and ApoE clearance pathway. Furthermore, Crocus sativus extract upregulated synaptic proteins and reduced neuroinflammation associated with Aβ pathology in the brains of 5XFAD mice. Crocin, a major active constituent of Crocus sativus and known for its antioxidant and anti-inflammatory effect, was also tested separately in vivo in 5XFAD mice. Crocin (10 mg/kg/day) was able to reduce Aβ load but to a lesser extent when compared to Crocus sativus extract. Collectively, findings from this study support the positive effect of Crocus sativus against AD by reducing Aβ pathological manifestations.

Inhibition of lactate dehydrogenase (LDH) represents an innovative approach to tackle cancer because this peculiar glycolytic metabolism is characteristic of most invasive tumor cells. An investigation into the biological properties of saffron extracts led to the discover of their LDH-inhibition properties. In particular, the most important saffron components, crocetin, was found to inhibit LDH (IC 50 = 54.9 ± 4.7 μM). This carotenoid was independently produced by chemical synthesis, and its LDH-inhibition properties manifested via its antiproliferative activity against two glycolytic cancer cell lines (A549 and HeLa, IC 50 = 114.0 ± 8.0 and 113.0 ± 11.1 μM, respectively). The results described in this article suggest that saffron may be a helpful alimentary component in the prevention of cancer that potentially contributes to the efficacy of approved cancer therapies.

Alzheimer's disease (AD) is the most common form of dementia, in which the death of brain cells causes memory loss and cognitive decline. Several factors are thought to play roles in the development and course of AD. Existing medical therapies only modestly alleviate and delay cognitive symptoms. Current research has been focused on developing antibodies to remove the aggregates of amyloid-β (Aβ) and tau protein. This approach has achieved removal of Aβ; however, no cognitive improvement in AD patients has been reported. The biological properties of saffron, the dry stigma of the plant Crocus sativus L., and particularly its main constituent crocin, have been studied extensively for many conditions including dementia and traumatic brain injury. Crocin is a unique antioxidant because it is a water-soluble carotenoid. Crocin has shown potential to improve learning and memory as well as protect brain cells. A search of the studies on saffron and crocin that have been published in recent years for their impact on AD as well as crocin's effects on Aβ and tau protein has been conducted. This review demonstrates that crocin exhibits multifunctional protective activities in the brain and could be a promising agent applied as a supplement or drug for prevention or treatment of AD.

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

The cellular transport and bioactivity of the second major saffron apocarotenoid, picrocrocin, was examined in parallel to that of the major group, crocetin sugar esters, in aqueous extracts. The transport of pure picrocrocin was investigated in comparison to that of other saffron apocarotenoids, trans-crocetin (di-β-D-gentiobiosyl) ester and crocetin using the Caco-2 cell model coupled with an in vitro digestion procedure. RP-HPLC-DAD was employed to quantify the bioaccessible and bioavailable amounts of individual apocarotenoids. Picrocrocin and crocetin sugar esters though highly bioaccessible (75% and 60%, respectively) were transported at minute quantities (0.2% and 0.5%, respectively; 10-fold lower than crocetin). Picrocrocin did not protect against oxidant-induced DNA damage in U937, human monocytic blood cells at the concentration investigated, however, it reduced the proliferation of human adenocarcinoma and hepatocarcinoma cells. Our findings may be useful for the requirements of food legislation regarding saffron preparations, in which both apocarotenoid groups coexist.

Intact Crocus sativus petals were studied for the first time by high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy, revealing the presence of kinsenoside (2) and goodyeroside A (3), together with 3-hydroxy-γ-butyrolactone (4). These findings were confirmed by HR-NMR analysis of the ethanol extract of fresh petals and showed that, even though carried out rapidly, partial hydrolysis of glucopyranosyloxybutanolides occurs during extraction. On the other hand, kaempferol 3-O-sophoroside (1), which is "NMR-silent" in intact petals, is present in extracts. These results suggest to evaluate the utilization of saffron petals for phytopharmaceutical and nutraceutical purposes to exploit a waste product of massive production of commercial saffron and point to the application of HR-MAS NMR for monitoring bioactive compounds directly on intact petals, avoiding the extraction procedure and the consequent hydrolysis reaction.

Comprehensive two-dimensional gas chromatography hyphenated with accurate mass time-of-flight mass spectrometry (GC × GC-accTOFMS) was applied for improved analytical accuracy of saffron analysis, by using retention indices in the two-dimensional separation. This constitutes 3 dimensions of identification. In addition to accTOFMS specificity, and first dimension retention indices ((1)I), a simple method involving direct multiple injections with stepwise isothermal temperature programming is described for construction of isovolatility curves for reference alkane series in GC × GC. This gives access to calculated second dimension retention indices ((2)I). Reliability of the calculated (2)I was evaluated by using a Grob test mixture, and saturated alkanes, revealing good correlation between previously reported I values from the literature, with R(2) correlation being 0.9997. This essentially recognizes the retention property of peaks in the GC × GC 2D space as being reducible to a retention index in each dimension, which should be a valuable tool supporting identification. The benefit of (2)I data, in supplementing (1)I and MS library matching, was clearly demonstrated by the progressive reduction of the number of possible compound matches for peaks observed in saffron. 114 analytes were assessed according to (1)I and (2)I values within ±20 index unit of reference values, and by MS spectrum matching above a match statistic of 750 (including mass accuracy of the molecular ion <20 ppm) and their possible identities derived. The described method provides a new avenue to utilize the full capability of the two-dimensional separation (GC × GC), in combination with MS library matching in complex sample analysis, to provide improved component identification.

Tyrosinase inhibitors have been used as whitening or antihyperpigment agents because of their ability to suppress dermal-melanin production. In the present study, screening and kinetic evaluation of various small molecules were performed on mushroom tyrosinase (MT) using surface plasmon resonance. The binding constant KD (M) values obtained for tannic acid, phloroglucinol, saffron, catechol, and pyrogallol are 1.213 × 10(-4), 7.136 × 10(-5), 3.111 × 10(-5), 1.557 × 10(-5), and 7.981 × 10(-6) M, respectively. Pyrogallol has been found to display high affinity for MT, whereas catechol, saffron, and phloroglucinol have been found to bind with low affinity. MT shows considerable changes in the secondary structure in the presence of inhibitors. The study reveals the Biacore/SPR sensor's ability in the rapid identification and characterization of inhibitors for MT. The methodology described here can be used to rapidly screen and optimize various lead compounds for other enzymes and elucidate structure function inter-relationships between various enzymes.

The aim of this work was to propose a high-performance liquid chromatography with diode array detection (HPLC-DAD) method for determining the three main compounds responsible for determining the quality of saffron (crocetin esters, picrocrocin, and safranal) by preparing an aqueous extract according to the ISO 3632 standard to solve the difficulty that this standard has for aroma and taste determination by ultraviolet-visible spectroscopy. Toward this aim, laboratory-isolated picrocrocin, a safranal standard with a purity of ≥ 88%, trans-crocetin di(β-D-gentiobiosyl) ester (trans-4-GG) and trans-crocetin (β-D-glucosyl)-(β-D-gentiobiosyl) ester (trans-3-Gg) standards, both with a purity of ≥ 99%, and 50 different saffron spice samples from Italy, Iran, Greece, and Spain were used in the intralaboratory validation of the HPLC method. The analytical method proposed was adequate in terms of linearity, selectivity, sensitivity, and accuracy for determining the three foremost parameters that define the quality of saffron using only a saffron solution prepared according to the ISO 3632 standard.

Changes that may be expected in crocetin esters (crocins) upon digestion were examined in saffron aqueous extracts for the first time. Chemical characterization of total and individual crocins and other bioactive compounds was achieved by UV-vis spectrophotometry, RP-HPLC-DAD, and LC-ESI-MS. Antioxidant activity was evaluated using in vitro assays and the comet assay. The observed loss for both total and trans-crocins was higher in saffron (∼50%) than in gardenia extracts (∼30%), which were also examined for comparison. Loss was lower than that reported for hydrophobic carotenoids. cis-Isomers were less affected, leading to the hypothesis that trans/cis isomerization may occur in parallel to degradation reactions. Monitoring changes in the extracts at oral, gastric, or intestinal phases, separately, verified this view pointing out the critical effect of pH, temperature, and duration of process but not of digestive enzymes. No isomerization and less degradation (<20% loss) was evidenced when pure trans-crocetin (di-β-D-gentiobiosyl) ester was subjected to gastric or intestinal conditions.

A method based on sequence-characterized amplified regions (SCARs) was developed from random amplified polymorphic DNA markers (RAPDs) specific for Arnica montana L., Bixa orellana L., Calendula officinalis L., Carthamus tinctorius L., Crocus vernus L. (Hill), Curcuma longa L., and Hemerocallis sp. to detect these common bulking agents in commercial saffron (Crocus sativus). The method enabled the unequivocal detection of low amounts (up to 1%) of each adulterant, allowing the preemptive rejection of suspect samples. Its enforcement limits the number of samples to be subjected to further evaluation with pharmacognostic or phytochemical analyses, especially when multiple batches have to be evaluated in a short time. The dimension of the amplicons is suitable for the analysis of degraded DNA obtained from dried, stored, processed, and finely ground commercial material. Proper SCAR markers may represent a fast, sensitive, reliable, and low-cost screening method for the authentication of dried commercial saffron material.

Inhibitors of acetylcholine breakdown by acetylcholinesterase (AChE) constitute the main therapeutic modality for Alzheimer's disease. In the search for natural products with inhibitory action on AChE, this study investigated the activity of saffron extract and its constituents by in vitro enzymatic and molecular docking studies. Saffron has been used in traditional medicine against Alzheimer's disease. Saffron extract showed moderate AChE inhibitory activity (up to 30%), but IC(50) values of crocetin, dimethylcrocetin, and safranal were 96.33, 107.1, and 21.09 μM, respectively. Kinetic analysis showed mixed-type inhibition, which was verified by in silico docking studies. Safranal interacts only with the binding site of the AChE, but crocetin and dimethylcrocetin bind simultaneously to the catalytic and peripheral anionic sites. These results reinforce previous findings about the beneficial action of saffron against Alzheimer's disease and may be of value for the development of novel therapeutic agents based on carotenoid-based dual binding inhibitors.

Metabolic profiling of natural products is used to map correlated concentration variances of known and unknown secondary metabolites in extracts. NMR-spectroscopy is in this respect regarded as a convenient and reproducible technique with the ability to detect a wide range of small organic compounds. Two-dimensional J-resolved NMR-spectra are used in this context to resolve overlapping signals by separating the effect of J-coupling from the effect of chemical shifts. Often one-dimensional projections of these data are used as input for standard multivariate statistical methods, and only the intensity variances along the chemical shift axis are taken into account. Here, we describe the use of parallel factor analysis (PARAFAC) as a tool to preprocess a set of two-dimensional J-resolved spectra with the aim of keeping the J-coupling information intact. PARAFAC is a mathematical decomposition method that fits three-way experimental data to a model whose parameters in this case reflect concentrations and individual component spectra along the chemical shift axis and corresponding profiles along the J-coupling axis. A set of saffron samples, directly extracted with methanol-d(4), were used as a model system to evaluate the feasibility and merits of the method. To successfully use PARAFAC, the two-dimensional spectra (n = 96) had to be aligned and processed in narrow windows (0.04 ppm wide) along the chemical shift axis. Selection of windows and number of components for each PARAFAC-model was done automatically by evaluating amount of explained variance and core consistency values. Score plots showing the distribution of objects in relation to each other, and loading plots in the form of two-dimensional pseudospectra with the same appearance as the original J-resolved spectra but with positive and negative contributions are presented. Loadings are interpreted not only in terms of signals with different chemical shifts but also the associated J-coupling profiles.

The kinetics of picrocrocin degradation in aqueous extracts of saffron upon thermal treatment from 5 to 70 °C have been studied, together with the degradation of purified picrocrocin in water at 100 °C. The best fits to experimental data were found for a second-order kinetics model. Picrocrocin showed high stability with half-life periods (t(1/2)) ranging from >3400 h at 5 °C in saffron extracts to 9 h in the experiments with purified picrocrocin at 100 °C. In saffron extracts, the evolution of the rate constant (k) with temperature showed maximum values at 35 °C, and filtration of the extracts contributed to picrocrocin stability. In the case of purified picrocrocin, the generation of safranal in the first 5 h (yield up to 7.4%) was confirmed. Spectrometric parameters used in saffron quality control (E(1cm)(1%) 257 nm and ΔΕ(pic)) were not appropriate for documenting the evolution of picrocrocin.

The feasibility of Raman spectroscopy for predicting the content of crocetin esters (crocins), and coloring strength was assessed. 114 samples from Greece, Iran, Italy and Spain were divided into two sets: a calibration set with 49 samples and a validation one with 65 samples. Calibration models for crocetin esters (r 0.97, RMSEC 0.92, RMSEP 0.97, RPD 3.46) and coloring strength (r 0.95, RMSEC 12.2, RMSEP 11.3, RPD 2.59) were built in the spectral region 1700-955 cm(-1) using partial least-squares (PLS) regression. The calibration models were validated using cross-validation, leaving one sample out (r 0.97, RMSECV 1.09 for crocetin esters and r 0.93, RMSECV 14.5 for coloring strength). The crocetin esters content as determined by liquid chromatography fluctuated between 18.8 and 31.7 mg/100 g saffron. The corresponding values, as calculated using the Raman method, fluctuated between 19.2 and 32.0 mg/100 g saffron. The coloring strength determined by the reference method ranged from 177.0 to 296.7 units, while with the Raman method the values were between 186.8 and 297.6 units. The results, as compared to the reference methods (liquid chromatography and UV-vis spectrophotometry), show that the proposed methodology gives data with acceptable accuracy. The proposed models can be used as a tool for rapid screening of quality in saffron samples.

In this paper, 345 saffron samples were analyzed from different countries to study their picrocrocin content using different analytical techniques. The E1cm 1% of 257 nm results from all samples are inflated in comparison by the high-performance liquid chromatography (HPLC) data, because of the interferences with the crocetin ester pool and especially with those with a lower trans/cis relation. A picrocrocin range update is proposed for International Organization for Standardization (ISO) 3632 normative because category III should be incremented up to 50 units, while category II should be incremented up to 60 units. More accurate data are achieved when the deltaEpic measurement is carried out. Consequently, improvements to the ISO method are suggested. Fourier transform (FT)-near-infrared spectrometry analysis has also been carried out, showing excellent results from the calibration with HPLC data. This spectrophotometric technique could be used by saffron enterprises to obtain quick and more accurate data for picrocrocin determination.

Data for antiradical properties of saffron extract and its bioactive constituents (crocins, crocetin) are limited and poorly discussed in comparison with those of extracts containing potent scavengers. Further examination was sought using the Folin-Ciocalteu (F-C) reagent and various free radical species produced in cell-free or cell model systems. Oregano and turmeric methanol extracts, rich in well established scavengers, and also crocetin, rosmarinic acid, and curcumin, representing the major types of constituents in the three studied extracts, were used as "reference". On the same weight basis, saffron extract activity was found to be rather negligible in all cell-free systems with regard to that found for reference ones. On the contrary, in the human monocyte system, saffron extracts or free crocetin were found to reduce ROS production as effectively as the phenolic antioxidants. Our findings point out that saffron extracts exhibit a remarkable intracellular antioxidant activity that cannot be revealed using assays repeatedly applied to the evaluation of phenolic-type antioxidants.

Crocetin, a unique carotenoid with potent antioxidative and anti-inflammatory activities, is a major ingredient of saffron used as an important spice and food colorant in various parts of the world. In the present study, the effects of crocetin on platelet activity and thrombosis formation were systematically investigated. Crocetin showed a dose-dependent inhibition of platelet aggregation induced by ADP, collagen, but not by arachidonic acid (AA). Crocetin significantly attenuated dense granule release, while neither platelets adhesion to collagen nor cyclic AMP level was altered by crocetin. Pretreatment with crocetin was confirmed to partially inhibit Ca (2+) mobilization via reducing both intracellular Ca (2+) release and extracellular Ca (2+) influx. Besides that, crocetin prolonged the occlusive time in electrical stimulation-induced carotid arterial thrombosis. These findings suggest that the favorable impacts of crocetin on platelet activity and thrombosis formation may be related to the inhibition of Ca (2+) elevation in stimulated platelets.

The purpose of this research was to study the effect of centrifugal ultrafiltration (UF) on the composition of aqueous extracts of saffron spice. The contents of seven crocetin esters, picrocrocin, and two kaempferol glycosides were analyzed by UV-vis and HPLC in the filtrate and retentate fractions from 16 centrifugal filter devices with regenerated cellulose (RC) and polyethersulfone (PES) membranes ranging from 1-100 kDa nominal molecular weight cutoff (MWCO). The separation of crocetin esters from picrocrocin and their concentration with centrifugal UF have been demonstrated. A great heterogeneity of results regarding devices with equal MWCO was found that could not be related to the membrane material or manufacturer. Four devices of 5 and 10 kDa MWCO, three of which had RC membranes, showed the best results. The device having the lowest MWCO also showed a potential to obtain picrocrocin without crocetin esters and could be considered in successive UF steps. The less polar crocetin esters were rejected better than the others.

The aim of this work was the development of multivariate models able to determine the content of the main crocetin esters and picrocrocin from spectrophotometric data that could be used for routine quality control of saffron. These compounds were determined with HPLC in Spanish saffron, and their absorbance spectra from 190 to 700 nm were simultaneously monitored. Partial least-squares regression (PLSR) models have been obtained and applied to the determination of individual crocetin esters, to the sum of crocetin esters, and to picrocrocin. A modification of the Kennard-Stone algorithm was used to divide the pool of samples into calibration and validation subsets. The best predictions were obtained with the sum of crocetin esters model, followed by the model for cis-crocetin (beta- D-glucosyl)-(beta- D-gentiobiosyl) ester, trans-crocetin di-(beta- D-gentiobiosyl) ester, and trans-crocetin (beta- D-glucosyl)-(beta- D-gentiobiosyl) ester, whereas the worst predictions were found with the picrocrocin and trans-crocetin (beta- D-gentiobiosyl) ester models. These models may enhance quality control in saffron enterprises.

Kinetics of individual crocetin ester degradation in aqueous extracts of saffron upon thermal treatment in the dark has been studied. Special attention has been paid to the comparison between saffron extracts and aqueous solutions of a crocetin ester rich fraction, with a lower stability of the latter observed. The degradation reaction was the same for all crocetin esters whether they were in saffron extracts or whether they were purified, although it was affected by external factors that modified their kinetic and thermodynamic parameters, making some of them less stable than others.

Crocus sativus stigmas are one of the widely known spices (saffron) and consist of unusually polar carotenoids. Alzheimer's disease is characterized pathologically by deposition of amyloid beta-peptide (Abeta) fibrils. Oxidation is thought to promote Abeta fibril formation and deposition. To identify agents inhibiting the pathogenesis of Alzheimer's disease, we examined in vitro the antioxidant properties of extract of C. sativus stigmas and its effect on Abeta(1-40) fibrillogenesis. The antioxidant properties were determined by measuring the ferric-reducing antioxidant power and Trolox-equivalent antioxidant capacity, while its effects on Abeta-aggregation and fibrillogenesis were studied by thioflavine T-based fluorescence assay and by DNA binding shift assay. The water:methanol (50:50, v/v) extract of C. sativus stigmas possesses good antioxidant properties, higher than those of tomatoes and carrots, and inhibited Abeta fibrillogenesis in a concentration and time-dependent manner. The main carotenoid constituent, trans-crocin-4, the digentibiosyl ester of crocetin, inhibited Abeta fibrillogenesis at lower concentrations than dimethylcrocetin, revealing that the action of the carotenoid is enhanced by the presence of the sugars. Our findings suggest the possible use of C. sativus stigma constituents for inhibition of aggregation and deposition of Abeta in the human brain.

Generation of volatiles by thermal treatments has been studied in saffron spice for two reasons: (a) to determine volatile profile changes during simulated aging processes and (b) to study the volatile generation pathway. During the aging process, while the amounts of C10 compounds such as safranal and HTCC increase, the amounts of C9 compounds such as isophorone and 2,6,6-trimethylcyclohexane-1,4-dione decrease. A new compound tentatively identified as 4,5,6,7-tetrahydro-7,7-dimethyl-5-oxo-3H-isobenzofuranone seems to play a very important role in the aging process. The importance of this compound, structurally similar to dihydroactindiolide, was also confirmed when the saffron volatile fraction was analyzed via the degradation of the linear chain of crocetin and crocetin esters and is reported for the first time in this paper. Thermal degradation studies of zeaxanthin, crocetin, and trans and cis crocetin esters isomers allowed us to propose different mechanisms which explain saffron volatile generation depending on the crocetin ester isomer structure.

Examination of the crocin bleaching assay performance and in-house validation were focused on probe and test compound characteristics, conditions for peroxyl radical generation, reaction monitoring, and expression of results. HPLC and spectrometric examination showed that any authentic commercial saffron (origin, grade) can be used for probe preparation given that (a) interferences, such as tocopherols, are removed, (b) working solution concentration is adequately adjusted, and (c) stock probe solution changes during storage are not neglected. As suggested by log P values, calculated for a great number of radical scavengers (AHs), any AH more polar than Trolox (common reference compound) can be tested in the aqueous environment of the assay. AH activities order obeyed principles of structure-activity relationships. The assay was robust toward preheating of the azo-initiator (2,2'-azobis(2-aminopropane) dihydrochloride). Reaction monitoring through periodic UV-vis spectra recording was very informative. An alternative expression of results as "percent inhibition of crocin bleaching value", % Inh = [(DeltaA(0) - DeltaA)/DeltaA(0))] x 100, is proposed for [AH]/[crocin] = 1, instead of the so far used k(rel) values. The above findings also lead to analysis cost and time reduction.

Crocetin esters present in saffron (Crocus sativus L.) stigmas and in Gardenia jasminoides Ellis fruit are the compounds responsible for their color. Of the fifteen crocetin esters identified in this study, five new compounds were tentatively identified: trans and cis isomers of crocetin (beta-D-triglucoside)-(beta-D-gentibiosyl) ester, trans and cis isomers of crocetin (beta-D-neapolitanose)-(beta-D-glucosyl) ester, and cis crocetin (beta-D-neapolitanose)-(beta-D-gentibiosyl) ester. The most relevant differences between both species were a low content of the trans crocetin (beta-D-glucosyl)-(beta-D-gentibiosyl) ester, the absence of trans crocetin di-(beta-D-glucosyl) ester in gardenia, and its higher content of trans crocetin (beta-D-gentibiosyl) ester and cis crocetin di-(beta-D-gentibiosyl) ester. With the same chromatographic method it was possible to identify, in a single run, ten glycosidic compounds in saffron extracts with a UV/vis pattern similar to that of picrocrocin; among them, 5-hydroxy-7,7-dimethyl-4,5,6,7-tetrahydro-3H-isobenzofuranone 5-O-beta-D-gentibioside and 4-hydroxymethyl-3,5,5-trimethyl-cyclohexen-2-one 4-O-beta-D-gentibioside were tentatively identified for the first time in saffron. Of these ten glycosides, only the O-beta-D-gentibiosyl ester of 2-methyl-6-oxo-2,4-hepta-2,4-dienoic acid was found in gardenia samples, but it was possible to identify the iridoid glycoside, geniposide.

Near-infrared reflectance spectroscopy has been applied for the first time to saffron spice to determine the chemical composition and geographical origin of 111 samples from the there main producers' countries: Iran, Greece, and Spain. The validation procedures with the results obtained by UV-vis and HPLC-DAD measurements demonstrated that this technique is appropriate to determine the following parameters: moisture and volatile content, coloring strength, (250 nm), and (330 nm), established on the ISO 3632 Technical Specification Normative and used to certify saffron quality in the international market. Also, it can be used to estimate the content of the five main crocetin glycosides, the compounds responsible for saffron color, the best correlations being for trans-crocetin di-(beta-D-gentibiosyl) ester (R2= 0.93), trans-crocetin (beta-D-glucosyl)-(beta-D-gentibiosyl) (R2= 0.94), and picrocrocin (R2= 0.92), the compound accepted as responsible for saffron bitterness. Finally, a discriminant analysis among the three geographical origins reveals that Iranian samples are the most different, whereas Greek and Spanish samples are more similar. All of these results reveal that NIRS spectroscopy has an enormous potential for its application to saffron quality control as the results are obtained in 2 min and without any sample manipulation.

Safranal is the compound most responsible for the aroma of saffron spice and is, together with the suite of crocin pigments, the major determinant of the product quality. The content of safranal and pigments in saffron is determined by the method of postharvest treatment of the Crocus stigmas. A range of drying treatments involving different temperatures, with or without air flow, was applied to stigmas from three harvest dates. Dual solvent extractions combined with quantitative measurement using GC and HPLC-UV-vis techniques were used to analyze the secondary metabolite contents of the products. It was demonstrated that these methods overcame the previously reported problems in measuring the concentration of both pigments and safranal in saffron caused by the very different polarities and thus solubilities of these compounds. The results showed that a brief (20 min) initial period at a relatively high temperature (between 80 and 92 degrees C) followed by continued drying at a lower temperature (43 degrees C) produced saffron with a safranal content up to 25 times that of saffron dried only at lower temperatures. Evidence was provided suggesting that drying with significant air flow reduced the safranal concentration. The results, moreover, indicated that high-temperature treatment had allowed greater retention of crocin pigments than in saffron dried at intermediate temperatures (46-58 degrees C). The biochemical implications of the various treatments are discussed in relation to the potential for optimizing color and fragrance quality in the product.

A dehydration postharvesting treatment is necessary to convert Crocus sativus L. stigmas into saffron spice. Three different dehydration treatments were evaluated: dehydration at room temperature; dehydration with hot air at different temperatures (70, 90, and 110 degrees C); and dehydration following traditional processing in Castille-La Mancha (Spain) with three different heating sources (vineshoot charcoal, gas cooker, and electric coil). The time (between 28 and 55 min) and mean temperature (between 54 and 83 degrees C) conditions for traditional dehydration were established for the first time. The highest coloring strength was obtained when saffron was submitted to higher temperatures and lower times. These findings may be supported by the fact that samples dehydrated at high temperature were more porous than those dehydrated at room temperature, as was observed by scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The higher the temperature during the process, the higher the proportion of trans-crocetin di-(beta-D-gentibiosyl) ester, although trans-crocetin (beta-D-glucosyl)-(beta-D-gentibiosyl) and trans-crocetin di-(beta-D-glucosyl) ester decrease while cis-crocins did not change significantly. A thermal aging process reveals that the trans-crocetin di-(beta-D-gentibiosyl) ester increases when saffron is resubmitted to a heating treatment before it is decomposed by the extreme conditions. The picrocrocin extinction during the aging process does not imply a consistent generation of safranal.

Safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde) is the main component of saffron's essential oil. It was obtained using microsimultaneous hydro distillation-extraction (MSDE) and by ultrasound-assisted extraction (USE), which is a mild method. 4-Hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC) is a precursor of safranal and was obtained in considerable amounts only by USE. Five C(13)-norisoprenoids were found in saffron for the first time. Using a gas chromatography technique, safranal and HTCC were quantified from Greek saffron samples. The quantity of safranal isolated by MSDE ranged between 288.1 and 687.9 mg/100 g of saffron, whereas in the case of USE safranal and HTCC ranged between 40.7 and 647.7 mg/100 g of saffron and between 41.7 and 397.7 mg/100 g of saffron, respectively. Freeze-drying was also tested as an alternative drying method. Over years of storage at 4 degrees C the quantity of safranal remained mostly constant while the quantity of HTCC decreased over the same periods.