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The saffron composition is being widely studied for authenticity and traceability, but very few works have been carried out to investigate the relationship between the chemical and physico-chemical properties of saffron solutes and their technological functionality in colloidal systems. This study aims at evaluating the surface properties of saffron extracts obtained using solvents of different polarities to achieve extracts with different compositions in terms of the pattern and content of polar and medium polarity crocins. The air–water surface was evaluated alone and in the presence of Tween 20 at different surfactant–extract ratios. Saffron extracts were able to decrease the surface tension of the aqueous phase, indicating the presence of surface-active compounds. In the mixed saffron extract–Tween 20 systems, competitive adsorption at the air–water interface occurred when the surfactant was present at a low concentration, while at concentrations higher than the CMC, Tween 20 hindered the adsorption of the extract surface-active compounds. The results highlight the interesting technological functionality of saffron extracts for applications in colloidal systems. To better exploit their use in the design and development of formulated foods, nutraceutics and pharma products, further studies are needed to unravel the relationship between the composition of saffron extracts and corresponding surface activity. [ABSTRACT FROM AUTHOR]

Experimental evidence indicates that the noncompetitive N‐methyl‐D‐aspartate (NMDA) receptor antagonists ketamine and MK‐801 induce schizophrenia‐like symptoms in rodents, including cognitive deficits. Crocins are among the active components of the plant Crocus sativus L. and were found to be effective in different models of psychiatric disorders comprising schizophrenia. The present study was designed to evaluate the efficacy of the joint administration of sub‐effective doses of crocins with those of the atypical antipsychotics clozapine and risperidone in alleviating nonspatial recognition and emotional memory deficits induced either by ketamine (3 mg/kg) or MK‐801 (0.1 mg/kg) in the rat. To this end, the object recognition and the step‐through passive avoidance tests were used. Co‐administration of sub‐effective doses of crocins (5 mg/kg) with those of clozapine (0.1 mg/kg) or risperidone (0.03 mg/kg) counteracted nonspatial recognition and emotional memory deficits induced by NMDA receptor antagonists. The current findings suggest that this combinatorial treatment was efficacious in attenuating cognitive impairments related to the blockade of the NMDA receptor. In addition, the present results support the potential of crocins as an adjunctive drug for the therapy of schizophrenia. [ABSTRACT FROM AUTHOR]

Most research on saffron has focused on its composition and beneficial effects, while the culinary perspective to enhance its gastronomic potential remains unexplored. This study aims to define the transfer of the main compounds responsible for color, flavor, and aromatic properties, evaluating three critical variables: temperature (60 ◦C, 80 ◦C and 100 ◦C), infusion time (ranging from 10 to 30 min), and the composition of the medium (water, oil, and water/oil). Samples were analyzed using the LC-QTOF MS/MS and ISO 3632-1:2011 methods. The major compounds were crocins, including trans-crocin and picrocrocin. Among the flavonoids, kaempferol 3-O-sophoroside stands out. Regarding extraction conditions, crocins, glycoside flavonoids, and picrocrocin were enhanced in water, the former in 100% water and at low temperatures, while picrocrocin proved to be the most stable compound with extraction favored at high temperatures. The variable with the greatest incidence of picrocrocin isolation seemed to be the concentration of water since water/oil compositions reported higher concentrations. Safranal and kaempferol were enriched in the oil phase and at lower temperatures. This study provides a chemical interpretation for the appropriate gastronomic use of saffron according to its versatility. Finally, the determination of safranal using the ISO method did not correlate with that obtained using chromatography. [ABSTRACT FROM AUTHOR]

Plants are an important source of essential bioactive compounds that not only have a beneficial role in human health and nutrition but also act as drivers for shaping gut microbiome. However, the mechanism of their functional attributes is not fully understood despite their significance. One such important plant is Crocus sativus, also known as saffron, which possesses huge medicinal, nutritional, and industrial applications like food and cosmetics. The importance of this plant is grossly attributed to its incredible bioactive constituents such as crocins, crocetin, safranal, picrocrocin, and glycosides. These bioactive compounds possess a wide range of therapeutic activities against multiple human ailments. Since a huge number of studies have revealed negative unwanted side effects of modern-day drugs, the scientific communities at the global level are investigating a large number of medicinal plants to explore natural products as the best alternatives. Taken into consideration, the available research findings indicate that saffron has a huge scope to be further explored to establish alternative natural-product-based drugs for health benefits. In this review, we are providing an update on the role of bioactive compounds of saffron as therapeutic agents (human disorders and antimicrobial activity) and its nutritional values. We also highlighted the role of omics and metabolic engineering tools for increasing the content of key saffron bioactive molecules for its mass production. Finally, pre-clinical and clinical studies seem to be necessary to establish its therapeutic potential against human diseases. [ABSTRACT FROM AUTHOR]

Abstract 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.

Crocins are water-soluble apocarotenoids isolated from the flowers of crocus and gardenia. They exhibit various pharmacological effects, including neuroprotection, anti-inflammatory properties, hepatorenal protection, and anticancer activity. They are often used as coloring and seasoning agents. Due to the limited content of crocins in plants and the high cost of chemical synthesis, the supply of crocins is insufficient to meet current demand. The biosynthetic pathways for crocins have been elucidated to date, which allows the heterologous production of these valuable compounds in microorganisms by fermentation. This review article provides a comprehensive overview of the chemistry, pharmacological activity, biosynthetic pathways, and heterologous production of crocins, aiming to lay the foundation for the large-scale production of these valuable natural products by using engineered microbial cell factories.

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. Highlights: • This study uncovers yellow-tepal crocuses as sources of saffron-like compounds, meeting industry demand. Metabolomic and transcriptomic research reveals insights into crocins and flavonoids biosynthesis, offering a promising production avenue. [ABSTRACT FROM AUTHOR]

Abstract Saffron has many pharmacological properties in addition to being a frequently used food seasoning. Crocin and picrocrocin which accumulate in saffron stigma, are responsible for these pharmacological properties. These natural products have health‐promoting effects for the prevention and treatment of numerous diseases, including age‐related cognitive and memory disfunction. Currently, crocin and picrocrocin are obtained from saffron, considered as the spice with the highest price in the market. To develop an efficient and low‐cost approach to producing these compounds with high bioactivity, biosynthetic genes isolated from saffron can be exploited in the metabolic engineering of heterologous hosts and the production of crocins in productive crop plants. Recently, we engineered tomato fruit producing crocins (Tomafran). In this study, we demonstrated that crocin-rich extract, encapsulated in chitosan or in exosomes may function as a neuroprotective strategy. Crocins contained in the Tomafran extracts and much lower doses in chitosan nanoparticles or exosomes were enough to rescue the neuroblastoma cell line SH-SY5Y after damage caused by okadaic acid. Our results confirm the neuroprotective effect of Tomafran and its exosomes that may be useful for the delay or prevention of neurodegenerative disorders such as Alzheimer’s disease. Graphical Abstract

The saffron composition is being widely studied for authenticity and traceability, but very few works have been carried out to investigate the relationship between the chemical and physico-chemical properties of saffron solutes and their technological functionality in colloidal systems. This study aims at evaluating the surface properties of saffron extracts obtained using solvents of different polarities to achieve extracts with different compositions in terms of the pattern and content of polar and medium polarity crocins. The air–water surface was evaluated alone and in the presence of Tween 20 at different surfactant–extract ratios. Saffron extracts were able to decrease the surface tension of the aqueous phase, indicating the presence of surface-active compounds. In the mixed saffron extract–Tween 20 systems, competitive adsorption at the air–water interface occurred when the surfactant was present at a low concentration, while at concentrations higher than the CMC, Tween 20 hindered the adsorption of the extract surface-active compounds. The results highlight the interesting technological functionality of saffron extracts for applications in colloidal systems. To better exploit their use in the design and development of formulated foods, nutraceutics and pharma products, further studies are needed to unravel the relationship between the composition of saffron extracts and corresponding surface activity.

Summary: Gardenia fruit pomace (GFP) is an industrial by‐product containing abundant bioactive components. Incorporating GFP into wheat flour improves the exterior quality of food and enhances healthy functions. This study developed a suitable GFP‐fortified wheat product by applying different cooking methods (steaming, boiling and baking). The fate of bioactive crocin derivatives, physical properties and in vitro digestion of GFP‐fortified wheat products was evaluated. The results showed that the steaming, boiling and baking treatments reduced the trans‐crocin‐4 content by 14.41%, 27.54% and 35.17%, respectively, but had no significant effect on geniposide. Combined with the colour change, the steaming treatment was suitable for developing GFP‐fortified steamed bread. With increasing GFP concentration, the increasing b* value indicated that the steamed bread developed a yellow hue. Meanwhile, GFP enrichment of steamed bread could retain most crocins and slow starch digestion by decreasing rapidly digestible starch content. Furthermore, the steamed bread with 1% GFP showed good overall sensory acceptance (flavour, taste, and mouthfeel). Therefore, GFP is a promising option for improving steamed bread's appearance and nutritional quality. [ABSTRACT FROM AUTHOR]

Most research on saffron has focused on its composition and beneficial effects, while the culinary perspective to enhance its gastronomic potential remains unexplored. This study aims to define the transfer of the main compounds responsible for color, flavor, and aromatic properties, evaluating three critical variables: temperature (60 °C, 80 °C and 100 °C), infusion time (ranging from 10 to 30 min), and the composition of the medium (water, oil, and water/oil). Samples were analyzed using the LC-QTOF MS/MS and ISO 3632-1:2011 methods. The major compounds were crocins, including trans-crocin and picrocrocin. Among the flavonoids, kaempferol 3-O-sophoroside stands out. Regarding extraction conditions, crocins, glycoside flavonoids, and picrocrocin were enhanced in water, the former in 100% water and at low temperatures, while picrocrocin proved to be the most stable compound with extraction favored at high temperatures. The variable with the greatest incidence of picrocrocin isolation seemed to be the concentration of water since water/oil compositions reported higher concentrations. Safranal and kaempferol were enriched in the oil phase and at lower temperatures. This study provides a chemical interpretation for the appropriate gastronomic use of saffron according to its versatility. Finally, the determination of safranal using the ISO method did not correlate with that obtained using chromatography.

Considering the interest in the bioactive properties of saffron (Crocus sativus L.), as well as its limited production and high price, saffron-based food supplements (SFS) are highly susceptible to adulteration. However, their complex composition and the wide variety of potential fraudulent practices make the comprehensive assessment of SFS quality a challenging task that has been scarcely addressed. To that aim, a new multianalytical strategy based on gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography with diode array detection coupled to mass spectrometry (HPLC-DAD-MS) was developed and validated in order to detect different frauds affecting SFS. Dried saffron stigmas and a commercial standardized saffron extract (affron®) were selected as reference samples (RS) to obtain an authenticity profile, which was further used to evaluate the quality of 17 SFS. Up to 17 crocins and crocetins, 5 kaempferol glycosides, picrocrocin (determined for the first time by GC-MS), safranal, furanone and isophorone-related compounds were determined in RS. Safranal and crocins were identified in all SFS except for one sample. However, discrepancies with the content declared were detected in 65% of the cases. Moreover, this multianalytical methodology also allowed identifying undeclared additives and the non-declared addition of vegetable sources other than saffron. [ABSTRACT FROM AUTHOR]

Crocins are important natural products predominantly obtained from the stigma of saffron, and that can be utilized as a medicinal compound, spice, and colorant with significant promise in the pharmaceutical, food, and cosmetic industries. Carotenoid cleavage dioxygenase 2 (CsCCD2) is a crucial limiting enzyme that has been reported to be responsible for the cleavage of zeaxanthin in the crocin biosynthetic pathway. However, the catalytic activity of CsCCD2 on β-carotene/lycopene remains elusive, and the soluble expression of CsCCD2 remains a big challenge. In this study, we reported the functional characteristics of CsCCD2, that can catalyze not only zeaxanthin cleavage but also β-carotene and lycopene cleavage. The molecular basis of the divergent functionality of CsCCD2 was elucidated using bioinformatic analysis and truncation studies. The protein expression optimization results demonstrated that the use of a maltose-binding protein (MBP) tag and the optimization of the induction conditions resulted in the production of more soluble protein. Correspondingly, the catalytic efficiency of soluble CsCCD2 was higher than that of the insoluble one, and the results further validated its functional verification. This study not only broadened the substrate profile of CsCCD2, but also achieved the soluble expression of CsCCD2. It provides a firm platform for CsCCD2 crystal structure resolution and facilitates the synthesis of crocetin and crocins. [ABSTRACT FROM AUTHOR]

Crocins are a group of highly valuable water-soluble carotenoids that are reported to have many pharmacological activities, such as anticancer properties, and the potential for treating neurodegenerative diseases including Alzheimer's disease. Crocins are mainly biosynthesized in the stigmas of food–medicine herbs Crocus sativus L. and Gardenia jasminoides fruits. The distribution is narrow in nature and deficient in resources, which are scarce and expensive. Recently, the synthesis of metabolites in the heterologous host has opened up the potential for large-scale and sustainable production of crocins, especially for the main active compounds crocin I and crocin II. In this study, GjCCD4a, GjALDH2C3, GjUGT74F8, and GjUGT94E13 from G. jasminoides fruits were expressed in Nicotiana benthamiana. The highest total content of crocins in T1 generation tobacco can reach 78,362 ng/g FW (fresh weight) and the dry weight is expected to reach 1,058,945 ng/g DW (dry weight). Surprisingly, the primary effective constituents crocin I and crocin II can account for 99% of the total crocins in transgenic plants. The strategy mentioned here provides an alternative platform for the scale-up production of crocin I and crocin II in tobacco. [ABSTRACT FROM AUTHOR]

Plants are an important source of essential bioactive compounds that not only have a beneficial role in human health and nutrition but also act as drivers for shaping gut microbiome. However, the mechanism of their functional attributes is not fully understood despite their significance. One such important plant is Crocus sativus, also known as saffron, which possesses huge medicinal, nutritional, and industrial applications like food and cosmetics. The importance of this plant is grossly attributed to its incredible bioactive constituents such as crocins, crocetin, safranal, picrocrocin, and glycosides. These bioactive compounds possess a wide range of therapeutic activities against multiple human ailments. Since a huge number of studies have revealed negative unwanted side effects of modern-day drugs, the scientific communities at the global level are investigating a large number of medicinal plants to explore natural products as the best alternatives. Taken into consideration, the available research findings indicate that saffron has a huge scope to be further explored to establish alternative natural-product-based drugs for health benefits. In this review, we are providing an update on the role of bioactive compounds of saffron as therapeutic agents (human disorders and antimicrobial activity) and its nutritional values. We also highlighted the role of omics and metabolic engineering tools for increasing the content of key saffron bioactive molecules for its mass production. Finally, pre-clinical and clinical studies seem to be necessary to establish its therapeutic potential against human diseases.

Saffron (Crocus sativus L.), is a spice of vulnerable medical importance. It is often adulterated to lower its quality and efficacy. The present study sought to detect the level of adulteration in five traded saffron collected from different localities using a high-performance liquid chromatography coupled with high resolution tandem mass spectrometry (UPLC-HR-MS/MS) technique. In total, 62 metabolites were identified in the five extracts namely from monoterpene aldehydes, flavonoid derivatives, apocarotenoids and carotenoids classes as well as chemical markers of adulteration. One common adulterant; Gardenia jasminoides Ellis was detected through the simultaneous determination of its specific constituents including geniposide, shanzhiside and major jasminosides. Also, one synthetic dye; auramine-O was detected in one marketed saffron product. The anticholinesterase activity of the five marketed saffron was also evaluated. Furthermore, saffron samples that contain high crocin levels possessed a promising anticholinesterase activity. It was detected for the first time that auramine-O dye is sometimes used as a potential adulterant of saffron. In spite of the grand researches on saffron, it still lacks an accurate evaluation of the commercial saffron products. The results of our study showed that high adulteration rate of trade saffron not only reduces saffron efficacy as a herbal remedy but may also simultaneously represent a health risk.

Saffron is a spice that is obtained by dehydrating the stigmas of the Crocus sativus flower. Iran is the country that produces the largest amount of saffron, exceeding 90% of world production. Currently, there is a growing medicinal use which implies that there is more demand than supply worldwide, in turn, a large amount of labor is required to obtain it; for these two reasons, it reaches a high price in the international market. This demand is due to the high concentration of apocarotenoid metabolites that it biosynthesizes. In this work, the content of these metabolites of saffron from six production areas of Iran and neighbouring countries infected with saffron latent virus (SaLV) and dehydrated at two temperatures is compared. The corms of the six provenances were planted in a homogeneous plot and the stigmas analyzed were those of the second year after planting. The analysis showed that corms do not completely retain the memory of their original origin. In general, the ratio of the sum of mmol/kg of HTCC derivatives to the sum of the mmol of crocins is greater than two. This implies that the biosynthesis of saffron apocarotenoids due to the degradation of β-carotene towards HTCC is more important than that of zeaxanthin formation, which later gives HTCC and crocetin dialdehyde. [ABSTRACT FROM AUTHOR]

Crocins are glycosylated apocarotenoids with strong coloring power and anti-oxidant, anticancer, and neuro-protective properties. We previously dissected the saffron crocin biosynthesis pathway, and demonstrated that the CsCCD2 enzyme, catalyzing the carotenoid cleavage step, shows a strong preference for the xanthophyll zeaxanthin in vitro and in bacterio. In order to investigate substrate specificity in planta and to establish a plant-based bio-factory system for crocin production, we compared wild-type Nicotiana benthamiana plants, accumulating various xanthophylls together with α- and β-carotene, with genome-edited lines, in which all the xanthophylls normally accumulated in leaves were replaced by a single xanthophyll, zeaxanthin. These plants were used as chassis for the production in leaves of saffron apocarotenoids (crocins, picrocrocin) using two transient expression methods to overexpress CsCCD2: agroinfiltration and inoculation with a viral vector derived from tobacco etch virus (TEV). The results indicated the superior performance of the zeaxanthin-accumulating line and of the use of the viral vector to express CsCCD2. The results also suggested a relaxed substrate specificity of CsCCD2 in planta, cleaving additional carotenoid substrates. [ABSTRACT FROM AUTHOR]

In order to valorize the species Crocus sativus from Morocco and to prepare new products with high added value that can be used in the food and pharmaceutical industry, our interest was focused on the phytochemical characterization and the biological and pharmacological properties of the stigmas of this plant. For this purpose, the essential oil of this species, extracted by hydrodistillation and then analyzed by GC-MS, revealed a predominance of phorone (12.90%); (R)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol (11.65%); isopropyl palmitate (9.68%); dihydro-β-ionone (8.62%); safranal (6.39%); trans-β-ionone (4.81%); 4-keto-isophorone (4.72%); and 1-eicosanol (4.55%) as the major compounds. The extraction of phenolic compounds was performed by decoction and Soxhlet extraction. The results of the determination of flavonoids, total polyphenols, condensed tannins, and hydrolyzable tannins determined by spectrophotometric methods on aqueous and organic extracts have proved the richness of Crocus sativus in phenolic compounds. Chromatographic analysis by HPLC/UV-ESI-MS of Crocus sativus extracts revealed the presence of crocin, picrocrocin, crocetin, and safranal molecules specific to this species. The study of antioxidant activity by three methods (DPPH, FRAP, and total antioxidant capacity) has proved that C. sativus is a potential source of natural antioxidants. Antimicrobial activity of the aqueous extract (E0) was investigated by microdilution on a microplate. The results have revealed the efficacy of the aqueous extract against Acinetobacter baumannii and Shigella sp. with MIC ≤ 600 µg/mL and against Aspergillus niger, Candida kyfer, and Candida parapsilosis with MIC = 2500 µg/mL. Measurements of pro-thrombin time (PT) and activated partial thromboplastin time (aPTT) in citrated plasma obtained from routine healthy blood donors were used to determine the anticoagulant activity of aqueous extract (E0). The anticoagulant activity of the extract (E0) studied showed that this extract can significantly prolong the partial thromboplastin time (p < 0.001) with a 359 µg/mL concentration. The antihyperglycemic effect of aqueous extract was studied in albino Wistar rats. The aqueous extract (E0) showed strong in vitro inhibitory activity of α-amylase and α-glucosidase compared with acarbose. Thus, it very significantly inhibited postprandial hyperglycemia in albino Wistar rats. According to the demonstrated results, we can affirm the richness of Crocus sativus stigmas in bioactive molecules and its use in traditional medicine. [ABSTRACT FROM AUTHOR]

Saffron is a spice derived from the flower of Crocus sativus used as a flavoring and coloring agent in the food industry which also possesses medicinal properties. In the current study, the optimum Solid Phase Extraction (SPE) conditions for separating picrocrocin and crocins from aqueous saffron extracts were investigated, using Rotatable-Central Composite Design (RCCD) in combination with Response Surface Methodology (RSM). The optimized factors were volume of saffron extract (3 mL), elution solvent (15% v/v ACN/water for picrocrocin; and 50% v/v ACN/water for crocins), and volume of elution solvent (ACN/water) (10 mL). The response factor measured was the UV-Vis absorbance. The presence of picrocrocin and crocins in the solutions obtained from SPE was confirmed using Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (UPLC-QToF-MS). The above optimized SPE procedure provides economy in reagents and consumables. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Contrary to the biosynthetic pathways of many terpenoids, which are well characterized and elucidated, their transport inside subcellular compartments and the secretion of reaction intermediates and final products at the short- (cell-to-cell), medium- (tissue-to-tissue), and long-distance (organ-to-organ) levels are still poorly understood, with some limited exceptions. In this review, we aim to describe the state of the art of the transport of several terpene classes that have important physiological and ecological roles or that represent high-value bioactive molecules. Among the tens of thousands of terpenoids identified in the plant kingdom, only less than 20 have been characterized from the point of view of their transport and localization. Most terpenoids are secreted in the apoplast or stored in the vacuoles by the action of ATP-binding cassette (ABC) transporters. However, little information is available regarding the movement of terpenoid biosynthetic intermediates from plastids and the endoplasmic reticulum to the cytosol. Through a description of the transport mechanisms of cytosol- or plastid-synthesized terpenes, we attempt to provide some hypotheses, suggestions, and general schemes about the trafficking of different substrates, intermediates, and final products, which might help develop novel strategies and approaches to allow for the future identification of terpenoid transporters that are still uncharacterized. [ABSTRACT FROM AUTHOR]

Saffron is known as the most expensive spice in the world. It is comprised of the dried stigmas of the pistil of the Crocus sativus L., which is a cultivated, sterile crocus plant. This plant material is now recognized as the unique edible source of certain bioactive apocarotenoids for which in-vivo antioxidant properties have been reported. Among the latter, crocins, red-orange natural colorants, and their parent molecule crocetin prevail in bioactivity significance. This review is focused on the strategies developed so far for their nanoencapsulation in relation to the characteristics of the starting material, extraction procedures of the bioactive antioxidants and analytical methods applied for their characterization and quantification throughout the process. The literature so far points out gaps that lead to publishable data, on one hand, but not necessarily to repeatable and meaningful processes due to incomplete characterization of the starting and the released material in efficiency and stability studies of the nanoencapsulates. Accurate terminology and quantitative chromatographic or spectrophotometric procedures for the determination of the core compounds are needed. Authenticity control and quality of saffron samples, and the verification of the concentrations of compounds in commercial preparations labeled as 'crocin,' are prerequisites in any experimental design setup. [ABSTRACT FROM AUTHOR]

Saffron is a spice obtained from the drying process of the stigmas of the flower Crocus sativus Linnaeus. It is well known that the organoleptic characteristics of this spice are closely linked to the production area and harvesting year. The present work aims to evaluate whether saffron samples produced in different years and origins present sensibly different crocin profiles. To achieve this goal, 120 saffron samples were harvested between 2016 and 2020 in four different Italian areas. The crocins were analysed, identified, and quantified by high-performance liquid chromatography–electrospray–tandem mass spectrometry (HPLC–ESI–MS/MS) in multiple reaction monitoring mode (MRM). Subsequently, ANOVA–simultaneous component analysis (ASCA) was used to evaluate whether the origin and annuity significantly affected the composition of the crocins. ASCA confirmed the relevance of these effects. Eventually, soft independent modelling by class analogy (SIMCA) models were created for each of the four different origins. Mixtures of saffron from different areas were also prepared to test the robustness of the models. SIMCA provided satisfying results; in fact, models provided 100% sensitivity for three origins (Cascia, Sardinia, and Città della Pieve) on the external test set (48 samples) and 88% (sensitivity on the external test set) for the Spoleto class. [ABSTRACT FROM AUTHOR]

Considering the interest in the bioactive properties of saffron (Crocus sativus L.), as well as its limited production and high price, saffron-based food supplements (SFS) are highly susceptible to adulteration. However, their complex composition and the wide variety of potential fraudulent practices make the comprehensive assessment of SFS quality a challenging task that has been scarcely addressed. To that aim, a new multianalytical strategy based on gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography with diode array detection coupled to mass spectrometry (HPLC-DAD-MS) was developed and validated in order to detect different frauds affecting SFS. Dried saffron stigmas and a commercial standardized saffron extract (affron®) were selected as reference samples (RS) to obtain an authenticity profile, which was further used to evaluate the quality of 17 SFS. Up to 17 crocins and crocetins, 5 kaempferol glycosides, picrocrocin (determined for the first time by GC-MS), safranal, furanone and isophorone-related compounds were determined in RS. Safranal and crocins were identified in all SFS except for one sample. However, discrepancies with the content declared were detected in 65% of the cases. Moreover, this multianalytical methodology also allowed identifying undeclared additives and the non-declared addition of vegetable sources other than saffron.

Crocins are important natural products predominantly obtained from the stigma of saffron, and that can be utilized as a medicinal compound, spice, and colorant with significant promise in the pharmaceutical, food, and cosmetic industries. Carotenoid cleavage dioxygenase 2 (CsCCD2) is a crucial limiting enzyme that has been reported to be responsible for the cleavage of zeaxanthin in the crocin biosynthetic pathway. However, the catalytic activity of CsCCD2 on β-carotene/lycopene remains elusive, and the soluble expression of CsCCD2 remains a big challenge. In this study, we reported the functional characteristics of CsCCD2, that can catalyze not only zeaxanthin cleavage but also β-carotene and lycopene cleavage. The molecular basis of the divergent functionality of CsCCD2 was elucidated using bioinformatic analysis and truncation studies. The protein expression optimization results demonstrated that the use of a maltose-binding protein (MBP) tag and the optimization of the induction conditions resulted in the production of more soluble protein. Correspondingly, the catalytic efficiency of soluble CsCCD2 was higher than that of the insoluble one, and the results further validated its functional verification. This study not only broadened the substrate profile of CsCCD2, but also achieved the soluble expression of CsCCD2. It provides a firm platform for CsCCD2 crystal structure resolution and facilitates the synthesis of crocetin and crocins.

Crocins are a group of highly valuable water-soluble carotenoids that are reported to have many pharmacological activities, such as anticancer properties, and the potential for treating neurodegenerative diseases including Alzheimer’s disease. Crocins are mainly biosynthesized in the stigmas of food–medicine herbs Crocus sativus L. and Gardenia jasminoides fruits. The distribution is narrow in nature and deficient in resources, which are scarce and expensive. Recently, the synthesis of metabolites in the heterologous host has opened up the potential for large-scale and sustainable production of crocins, especially for the main active compounds crocin I and crocin II. In this study, GjCCD4a, GjALDH2C3, GjUGT74F8, and GjUGT94E13 from G. jasminoides fruits were expressed in Nicotiana benthamiana. The highest total content of crocins in T1 generation tobacco can reach 78,362 ng/g FW (fresh weight) and the dry weight is expected to reach 1,058,945 ng/g DW (dry weight). Surprisingly, the primary effective constituents crocin I and crocin II can account for 99% of the total crocins in transgenic plants. The strategy mentioned here provides an alternative platform for the scale-up production of crocin I and crocin II in tobacco.

Crocins are high-value water soluble pigments that have long been recognized for their medicinal properties, and whose demand has increased worldwide in recent years. In the present study transgenic tomato fruits engineered for the production of crocins and hp3/Bsh tomato mutants with alterations in the carotenoid metabolism, have been combined to increase the levels of crocins in tomato fruits. Tomato fruits from F4 plants showed high antioxidant capacity and the crocin-rich juice of the produced tomato fruit was protected neuroblastoma cells against oxidative insult, through its ability to activate factor 2 related to nuclear erythroid factor 2 (Nrf2). The bio-accessibility of crocins in the juice showed values similar to the ones observed with saffron. Overall, our results support that crocin-fortified tomatoes could result in higher crocin availability and have additional health-promoting effects and could provide better protection against oxidative stress related chronic diseases in humans.

Summary: Crocins are beneficial antioxidants and potential chemotherapeutics that give raise, together with picrocrocin, to the colour and taste of saffron, the most expensive spice, respectively. Crocins are formed from crocetin dialdehyde that is produced in Crocus sativus from zeaxanthin by the carotenoid cleavage dioxygenase 2L (CsCCD2L), while GjCCD4a from Gardenia jasminoides, another major source of crocins, converted different carotenoids, including zeaxanthin, into crocetin dialdehyde in bacterio. To establish a biotechnological platform for sustainable production of crocins, we investigated the enzymatic activity of GjCCD4a, in comparison with CsCCD2L, in citrus callus engineered by Agrobacterium‐mediated supertransformation of multi genes and in transiently transformed Nicotiana benthamiana leaves. We demonstrate that co‐expression of GjCCD4a with phytoene synthase and β‐carotene hydroxylase genes is an optimal combination for heterologous production of crocetin, crocins and picrocrocin in citrus callus. By profiling apocarotenoids and using in vitro assays, we show that GjCCD4a cleaved β‐carotene, in planta, and produced crocetin dialdehyde via C30 β‐apocarotenoid intermediate. GjCCD4a also cleaved C27 β‐apocarotenoids, providing a new route for C17‐dialdehyde biosynthesis. Callus lines overexpressing GjCCD4a contained higher number of plastoglobuli in chromoplast‐like plastids and increased contents in phytoene, C17:0 fatty acid (FA), and C18:1 cis‐9 and C22:0 FA esters. GjCCD4a showed a wider substrate specificity and higher efficiency in Nicotiana leaves, leading to the accumulation of up to 1.6 mg/g dry weight crocins. In summary, we established a system for investigating CCD enzymatic activity in planta and an efficient biotechnological platform for crocins production in green and non‐green crop tissues/organs. [ABSTRACT FROM AUTHOR]

Saffron is a very high value-added ingredient used in the food supplement market and contains a high level of safranal. Adding synthetic safranal to saffron, which is significantly cheaper, and falsifying the origin of saffron may represent recurrent fraud. Saffron from different countries was analyzed to determine the stable isotope ratios δ13C and δ2H from safranal by gas chromatography coupled with isotope-ratio mass spectrometry (GC-C/P-IRMS) and the concentration of saffron metabolites with ultra-high performance liquid chromatography coupled with diode array detector (UHPLC-DAD). The isotopic analysis highlighted a higher ratio of δ2H in synthetic safranal than in natural safranal; the mean values were 36‰ (+/− 40) and −210‰ (+/− 35), respectively. The δ13C between Iranian, Spanish and other saffron was significantly different and represents median values of −28.62‰, −30.12‰ and −30.70‰, respectively. Moreover, linear and quadratic discriminant analyses (LDA and QDA) were computed using the two isotope ratios of safranal and the saffron metabolites. A first QDA showed that trans-crocetin and the δ13C of safranal, picrocrocin, and crocin C3 concentrations clearly differentiated Iranian saffron from other origins. A second model identified δ13C, trans-crocetin, crocin C2, crocin C3, and picrocrocin as good predictors to discriminate saffron samples from Iran, Spain, or other origins, with a total ability score classification matrix of 100% and a prediction matrix of 82.5%. This combined approach may be a useful tool to authenticate the origin of unknown saffron. [ABSTRACT FROM AUTHOR]

The preparation process of yellow pigment (YP) from gardenia (Gardenia jasminoides) fruit was investigated, and the main components of YP were characterized by liquid chromatography-time of flight-mass spectrometer/mass spectrometer (LC-TOF–MS/MS). Furthermore, cytotoxic activity in HepG2 cells by induction of apoptosis was also evaluated. The preparation results indicated that the color value of YP was 498.34, which was 8.6 times higher than crude YP. Fifteen compounds in YP were identified, and crocins were the predominant compounds. The cell experiment results showed that YP inhibited the proliferation of HepG2 cells in a time- and dose-dependent manner. Moreover, YP also inhibited HepG2 cells in G2/M stage, increased the level of intracellular reactive oxygen species (ROS), and enhanced cell apoptosis. Real-time quantitative polymerase chain reaction (RT-PCR) analysis revealed the up-regulation of caspase-3, 8, 9, and bax and down-regulation of bcl-2 in HepG2 cells. Overall, these findings suggested that YP had potential cytotoxic activity in HepG2 cells by induction of apoptosis, which might be beneficial to human health. [ABSTRACT FROM AUTHOR]

The fruits of Gardenia jasminoides Ellis are an important herb medicine in Traditional Chinese Medicine (TCM) and have been used for thousands of years for clearing away heat and toxic materials. It mainly contains iridoids, pigments, organic acids, and flavonoids. Although belonging to one species, it has two kinds of cultivars and one variety widely distributed and sold. This study aims to develop an integrated and efficient analytical strategy for comprehensive profiling of phytochemicals and clarify the differences in all three populations. Based on reversed-phase ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC/ESI-QTOFMS), an optimized analytical approach for comprehensive profiling of phytochemicals in the fruits of G. jasminoides was established in negative ionization mode. The holistic metabolites profiling was carried out on UHPLC/ESI-QTOFMS and data analysis program Progenesis QI, and a total of 80 metabolites were obtained and interpreted by chromatographic and tandem mass spectral data. The interpretation of metabolites comprises iridoids, pigments, organic acids, and flavonoids. Principal component analysis and partial least square-discriminant analysis were performed, and 19 main different components could be obtained to distinguish the three populations. Combined with non-targeted and targeted data analysis, the integrated strategy developed in this study was feasibly applied to discern differences in the profiles of the phytochemicals accumulating in the fruits of three populations of G. jasminoides. [ABSTRACT FROM AUTHOR]

Carotenoids are C40 isoprenoids with well-established roles in photosynthesis, pollination, photoprotection, and hormone biosynthesis. The enzymatic or ROS-induced cleavage of carotenoids generates a group of compounds named apocarotenoids, with an increasing interest by virtue of their metabolic, physiological, and ecological activities. Both classes are used industrially in a variety of fields as colorants, supplements, and bio-actives. Crocins and picrocrocin, two saffron apocarotenoids, are examples of high-value pigments utilized in the food, feed, and pharmaceutical industries. In this study, a unique construct was achieved, namely O6, which contains CsCCD2L, UGT74AD1, and UGT709G1 genes responsible for the biosynthesis of saffron apocarotenoids driven by a patatin promoter for the generation of potato tubers producing crocins and picrocrocin. Different tuber potatoes accumulated crocins and picrocrocin ranging from 19.41–360 to 105–800 μg/g DW, respectively, with crocetin, crocin 1 [(crocetin-(β-D-glucosyl)-ester)] and crocin 2 [(crocetin)-(β-D-glucosyl)-(β-D-glucosyl)-ester)] being the main compounds detected. The pattern of carotenoids and apocarotenoids were distinct between wild type and transgenic tubers and were related to changes in the expression of the pathway genes, especially from PSY2, CCD1, and CCD4. In addition, the engineered tubers showed higher antioxidant capacity, up to almost 4-fold more than the wild type, which is a promising sign for the potential health advantages of these lines. In order to better investigate these aspects, different cooking methods were applied, and each process displayed a significant impact on the retention of apocarotenoids. More in detail, the in vitro bioaccessibility of these metabolites was found to be higher in boiled potatoes (97.23%) compared to raw, baked, and fried ones (80.97, 78.96, and 76.18%, respectively). Overall, this work shows that potatoes can be engineered to accumulate saffron apocarotenoids that, when consumed, can potentially offer better health benefits. Moreover, the high bioaccessibility of these compounds revealed that potato is an excellent way to deliver crocins and picrocrocin, while also helping to improve its nutritional value.

Saffron is a spice that is obtained by dehydrating the stigmas of the Crocus sativus flower. Iran is the country that produces the largest amount of saffron, exceeding 90% of world production. Currently, there is a growing medicinal use which implies that there is more demand than supply worldwide, in turn, a large amount of labor is required to obtain it; for these two reasons, it reaches a high price in the international market. This demand is due to the high concentration of apocarotenoid metabolites that it biosynthesizes. In this work, the content of these metabolites of saffron from six production areas of Iran and neighbouring countries infected with saffron latent virus (SaLV) and dehydrated at two temperatures is compared. The corms of the six provenances were planted in a homogeneous plot and the stigmas analyzed were those of the second year after planting. The analysis showed that corms do not completely retain the memory of their original origin. In general, the ratio of the sum of mmol/kg of HTCC derivatives to the sum of the mmol of crocins is greater than two. This implies that the biosynthesis of saffron apocarotenoids due to the degradation of β-carotene towards HTCC is more important than that of zeaxanthin formation, which later gives HTCC and crocetin dialdehyde.

Crocins are glycosylated apocarotenoids with strong coloring power and anti-oxidant, anticancer, and neuro-protective properties. We previously dissected the saffron crocin biosynthesis pathway, and demonstrated that the CsCCD2 enzyme, catalyzing the carotenoid cleavage step, shows a strong preference for the xanthophyll zeaxanthin in vitro and in bacterio. In order to investigate substrate specificity in planta and to establish a plant-based bio-factory system for crocin production, we compared wild-type Nicotiana benthamiana plants, accumulating various xanthophylls together with α- and β-carotene, with genome-edited lines, in which all the xanthophylls normally accumulated in leaves were replaced by a single xanthophyll, zeaxanthin. These plants were used as chassis for the production in leaves of saffron apocarotenoids (crocins, picrocrocin) using two transient expression methods to overexpress CsCCD2: agroinfiltration and inoculation with a viral vector derived from tobacco etch virus (TEV). The results indicated the superior performance of the zeaxanthin-accumulating line and of the use of the viral vector to express CsCCD2. The results also suggested a relaxed substrate specificity of CsCCD2 in planta, cleaving additional carotenoid substrates.

Crocins (CRs) and the related active constituents derived from Crocus sativus L. (Saffron) have demonstrated protective effects against cerebral ischemia and ischemic stroke, with various bioactivities including neuroprotection, antineuroinflammation, antioxidant, and cardiovascular protection. Among CRs, crocin (CR) has been shown to act on multiple mechanisms and signaling pathways involved in ischemic stroke, including mitochondrial apoptosis, nuclear factor kappa light chain enhancer of B cells pathway, S100 calcium-binding protein B, interleukin-6 and vascular endothelial growth factor-A. CR is generally safe and well-tolerated. Pharmacokinetic studies indicate that CR has poor bioavailability and needs to convert to crocetin (CC) in order to cross the blood-brain barrier. Clinical studies have shown the efficacy of saffron and CR in treating various conditions, including metabolic syndrome, depression, Alzheimer's disease, and coronary artery disease. There is evidence supporting CR as a treatment for ischemic stroke, although further studies are needed to confirm their efficacy and safety in clinical settings. [ABSTRACT FROM AUTHOR]

Saffron is widely cultivated and used as a spice. Recently published data on the chemical composition and pharmacological potential of saffron determine its use in pharmacy and medicine. The proposed high-performance thin-layer chromatography (HPTLC) method allows good separation of 11 analytes. The saffron quality (Iran, Ukraine, Spain, Morocco samples) assessment was based on the European Pharmacopoeia monograph and ISO 3632. The HPTLC method for the safranal, crocin, and picrocrocin quantification was proposed and validated. The crocins content in Ukrainian saffron was from 17.80% to 33.25%. Based on qualitative and quantitative assessment results, the saffron sample from Zaporizhzhia (Ukraine) had the highest compounds content and was chosen to obtain the working standards of picrocrocin and crocins (trans-4GG, trans-2G, trans-3Gg) by preparative chromatography. The compounds were isolated from lyophilized extract of saffron using a Symmetry Prep C18 column (300 × 19 mm × 7 µm), and identified by spectroscopic techniques (HPLC-DAD, UPLC-ESI-MS/MS). The purity of crocins and picrocrocin was more than 97%. A novel method proposed to obtain working standards is simple and reproducible for the routine analysis of saffron quality control. [ABSTRACT FROM AUTHOR]

Crocins are high-value compounds with industrial and food applications. Saffron is currently the main source of these soluble pigments, but its high market price hinders its use by sectors, such as pharmaceutics. Enzymes involved in the production of these compounds have been identified in saffron, Buddleja, and gardenia. In this study, the enzyme from Buddleja, BdCCD4.1, was constitutively expressed in Nicotiana glauca, a tobacco species with carotenoid-pigmented petals. The transgenic lines produced significant levels of crocins in their leaves and petals. However, the accumulation of crocins was, in general, higher in the leaves than in the petals, reaching almost 302 µg/g DW. The production of crocins was associated with decreased levels of endogenous carotenoids, mainly β-carotene. The stability of crocins in leaf and petal tissues was evaluated after three years of storage, showing an average reduction of 58.06 ± 2.20% in the petals, and 78.37 ± 5.08% in the leaves. This study illustrates the use of BdCCD4.1 as an effective tool for crocin production in N. glauca and how the tissue has an important impact on the stability of produced high-value metabolites during storage. [ABSTRACT FROM AUTHOR]

Crocins (CRs) and the related active constituents derived from Crocus sativus L. (Saffron) have demonstrated protective effects against cerebral ischemia and ischemic stroke, with various bioactivities including neuroprotection, anti-neuroinflammation, antioxidant, and cardiovascular protection. Among CRs, crocin (CR) has been shown to act on multiple mechanisms and signaling pathways involved in ischemic stroke, including mitochondrial apoptosis, nuclear factor kappa light chain enhancer of B cells pathway, S100 calcium-binding protein B, interleukin-6 and vascular endothelial growth factor-A. CR is generally safe and well-tolerated. Pharmacokinetic studies indicate that CR has poor bioavailability and needs to convert to crocetin (CC) in order to cross the blood-brain barrier. Clinical studies have shown the efficacy of saffron and CR in treating various conditions, including metabolic syndrome, depression, Alzheimer’s disease, and coronary artery disease. There is evidence supporting CR as a treatment for ischemic stroke, although further studies are needed to confirm their efficacy and safety in clinical settings.

In order to valorize the species Crocus sativus from Morocco and to prepare new products with high added value that can be used in the food and pharmaceutical industry, our interest was focused on the phytochemical characterization and the biological and pharmacological properties of the stigmas of this plant. For this purpose, the essential oil of this species, extracted by hydrodistillation and then analyzed by GC-MS, revealed a predominance of phorone (12.90%); (R)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol (11.65%); isopropyl palmitate (9.68%); dihydro-β-ionone (8.62%); safranal (6.39%); trans-β-ionone (4.81%); 4-keto-isophorone (4.72%); and 1-eicosanol (4.55%) as the major compounds. The extraction of phenolic compounds was performed by decoction and Soxhlet extraction. The results of the determination of flavonoids, total polyphenols, condensed tannins, and hydrolyzable tannins determined by spectrophotometric methods on aqueous and organic extracts have proved the richness of Crocus sativus in phenolic compounds. Chromatographic analysis by HPLC/UV-ESI-MS of Crocus sativus extracts revealed the presence of crocin, picrocrocin, crocetin, and safranal molecules specific to this species. The study of antioxidant activity by three methods (DPPH, FRAP, and total antioxidant capacity) has proved that C. sativus is a potential source of natural antioxidants. Antimicrobial activity of the aqueous extract (E0) was investigated by microdilution on a microplate. The results have revealed the efficacy of the aqueous extract against Acinetobacter baumannii and Shigella sp. with MIC ≤ 600 µg/mL and against Aspergillus niger, Candida kyfer, and Candida parapsilosis with MIC = 2500 µg/mL. Measurements of pro-thrombin time (PT) and activated partial thromboplastin time (aPTT) in citrated plasma obtained from routine healthy blood donors were used to determine the anticoagulant activity of aqueous extract (E0). The anticoagulant activity of the extract (E0) studied showed that this extract can significantly prolong the partial thromboplastin time (p < 0.001) with a 359 µg/mL concentration. The antihyperglycemic effect of aqueous extract was studied in albino Wistar rats. The aqueous extract (E0) showed strong in vitro inhibitory activity of α-amylase and α-glucosidase compared with acarbose. Thus, it very significantly inhibited postprandial hyperglycemia in albino Wistar rats. According to the demonstrated results, we can affirm the richness of Crocus sativus stigmas in bioactive molecules and its use in traditional medicine.

Crocins are high-value soluble pigments that are used as colorants and supplements, their presence in nature is extremely limited and, consequently, the high cost of these metabolites hinders their use by other sectors, such as the pharmaceutical and cosmetic industries. The carotenoid cleavage dioxygenase 2L (CsCCD2L) is the key enzyme in the biosynthetic pathway of crocins in Crocus sativus. In this study, CsCCD2L was introduced into Nicotiana tabacum and Nicotiana glauca for the production of crocins. In addition, a chimeric construct containing the Brevundimonas sp. β-carotene hydroxylase (BrCrtZ), the Arabidopsis thaliana ORANGE mutant gene (AtOrMut), and CsCCD2L was also introduced into N. tabacum. Quantitative and qualitative studies on carotenoids and apocarotenoids in the transgenic plants expressing CsCCD2L alone showed higher crocin level accumulation in N. glauca transgenic plants, reaching almost 400 μg/g DW in leaves, while in N. tabacum 36 μg/g DW was obtained. In contrast, N. tabacum plants coexpressing CsCCD2L , BrCrtZ , and AtOrMut accumulated, 3.5-fold compared to N. tabacum plants only expressing CsCCD2L. Crocins with three and four sugar molecules were the main molecular species in both host systems. Our results demonstrate that the production of saffron apocarotenoids is feasible in engineered Nicotiana species and establishes a basis for the development of strategies that may ultimately lead to the commercial exploitation of these valuable pigments for multiple applications. [ABSTRACT FROM AUTHOR]

Crocins are high-value soluble pigments that are used as colorants and supplements, their presence in nature is extremely limited and, consequently, the high cost of these metabolites hinders their use by other sectors, such as the pharmaceutical and cosmetic industries. The carotenoid cleavage dioxygenase 2L (CsCCD2L) is the key enzyme in the biosynthetic pathway of crocins in Crocus sativus. In this study, CsCCD2L was introduced into Nicotiana tabacum and Nicotiana glauca for the production of crocins. In addition, a chimeric construct containing the Brevundimonas sp. β-carotene hydroxylase (BrCrtZ), the Arabidopsis thaliana ORANGE mutant gene (AtOrMut), and CsCCD2L was also introduced into N. tabacum. Quantitative and qualitative studies on carotenoids and apocarotenoids in the transgenic plants expressing CsCCD2L alone showed higher crocin level accumulation in N. glauca transgenic plants, reaching almost 400 μg/g DW in leaves, while in N. tabacum 36 μg/g DW was obtained. In contrast, N. tabacum plants coexpressing CsCCD2L, BrCrtZ, and AtOrMut accumulated, 3.5-fold compared to N. tabacum plants only expressing CsCCD2L. Crocins with three and four sugar molecules were the main molecular species in both host systems. Our results demonstrate that the production of saffron apocarotenoids is feasible in engineered Nicotiana species and establishes a basis for the development of strategies that may ultimately lead to the commercial exploitation of these valuable pigments for multiple applications.

Saffron (Crocus sativus L.) spice making requires time-spending manual operations: stigmas are separated from the flower picked in bud early in the morning, and once dried they are preserved protected from light. This study verified how the correct pursuing of these good practices affects saffron quality. Few hours of exposure of the flower to the sun determined a significant decrease in the colouring strength (239.66 ± 10.33 versus 255.35 ± 11.87). The correct cleaning of stigmas determined a very significant increase of colouring strength (247.12 ± 13.32 instead of 224.35 ± 14.88) and a significant increase of flavour strength (99.72 ±7.48 against 90.31 ± 6.32, p < 0.05). In 24 months, all the samples kept in the dark were still of first category of quality while the ones kept in the light dropped in second category. For all samples there was an increase of aroma strength and a decrease of the flavour and colouring strength, but only the colouring strength loss followed a significantly more sloping trendline. A detectable difference in the content of trans-crocin 4 correlated to the ageing or the way of conservation was not found, nevertheless, it was confirmed that the isomers tend towards a photostationary state where the trans isomer is more present. [ABSTRACT FROM AUTHOR]

Crocins are a group of highly valuable apocarotenoid-derived pigments mainly produced in Crocus sativus stigmas and Gardenia jasminoides fruits, which display great pharmacological activities for human health, such as anticancer, reducing the risk of atherosclerosis, and preventing Alzheimer’s disease. However, traditional sources of crocins are no longer sufficient to meet current demands. The recent clarification of the crocin biosynthetic pathway opens up the possibility of large-scale production of crocins by synthetic metabolic engineering methods. In this review, we mainly introduce the crocin biosynthetic pathway, subcellular route, related key enzymes, and its synthetic metabolic engineering, as well as its challenges and prospects, with a view to providing useful references for further studies on the synthetic metabolic engineering of crocins.

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.

Saffron is known as the most expensive spice in the world. It is comprised of the dried stigmas of the pistil of the Crocus sativus L., which is a cultivated, sterile crocus plant. This plant material is now recognized as the unique edible source of certain bioactive apocarotenoids for which in-vivo antioxidant properties have been reported. Among the latter, crocins, red-orange natural colorants, and their parent molecule crocetin prevail in bioactivity significance. This review is focused on the strategies developed so far for their nanoencapsulation in relation to the characteristics of the starting material, extraction procedures of the bioactive antioxidants and analytical methods applied for their characterization and quantification throughout the process. The literature so far points out gaps that lead to publishable data, on one hand, but not necessarily to repeatable and meaningful processes due to incomplete characterization of the starting and the released material in efficiency and stability studies of the nanoencapsulates. Accurate terminology and quantitative chromatographic or spectrophotometric procedures for the determination of the core compounds are needed. Authenticity control and quality of saffron samples, and the verification of the concentrations of compounds in commercial preparations labeled as ‘crocin,’ are prerequisites in any experimental design setup.

Contrary to the biosynthetic pathways of many terpenoids, which are well characterized and elucidated, their transport inside subcellular compartments and the secretion of reaction intermediates and final products at the short- (cell-to-cell), medium- (tissue-to-tissue), and long-distance (organ-to-organ) levels are still poorly understood, with some limited exceptions. In this review, we aim to describe the state of the art of the transport of several terpene classes that have important physiological and ecological roles or that represent high-value bioactive molecules. Among the tens of thousands of terpenoids identified in the plant kingdom, only less than 20 have been characterized from the point of view of their transport and localization. Most terpenoids are secreted in the apoplast or stored in the vacuoles by the action of ATP-binding cassette (ABC) transporters. However, little information is available regarding the movement of terpenoid biosynthetic intermediates from plastids and the endoplasmic reticulum to the cytosol. Through a description of the transport mechanisms of cytosol- or plastid-synthesized terpenes, we attempt to provide some hypotheses, suggestions, and general schemes about the trafficking of different substrates, intermediates, and final products, which might help develop novel strategies and approaches to allow for the future identification of terpenoid transporters that are still uncharacterized.

Saffron is a spice obtained from the drying process of the stigmas of the flower Crocus sativus Linnaeus. It is well known that the organoleptic characteristics of this spice are closely linked to the production area and harvesting year. The present work aims to evaluate whether saffron samples produced in different years and origins present sensibly different crocin profiles. To achieve this goal, 120 saffron samples were harvested between 2016 and 2020 in four different Italian areas. The crocins were analysed, identified, and quantified by high-performance liquid chromatography–electrospray–tandem mass spectrometry (HPLC–ESI–MS/MS) in multiple reaction monitoring mode (MRM). Subsequently, ANOVA–simultaneous component analysis (ASCA) was used to evaluate whether the origin and annuity significantly affected the composition of the crocins. ASCA confirmed the relevance of these effects. Eventually, soft independent modelling by class analogy (SIMCA) models were created for each of the four different origins. Mixtures of saffron from different areas were also prepared to test the robustness of the models. SIMCA provided satisfying results; in fact, models provided 100% sensitivity for three origins (Cascia, Sardinia, and Città della Pieve) on the external test set (48 samples) and 88% (sensitivity on the external test set) for the Spoleto class.