Use of non-aqueous capillary electrophoresis for the quality control of commercial saffron samples

A non-aqueous capillary electrophoresis (NACE) method for quantifying the seven crocin metabolites that are the major biologically active ingredients of saffron was developed. Separation is done by using a fused silica capillary filled with a 12.5 mM H3BO3/37.5 mM sodium tetraborate methanolic solution as background electrolyte. The results obtained were compared with the total index "safranal value", widely used as a quality measure of saffron products. The comparison revealed that the proposed NACE method provides useful information not obtained in the safranal value. Infact, samples with a similar safranal value can contain crocin metabolites in different concentrations and relative proportions. This new method is very useful for quality control in commercial saffron samples.     

藏红花挥发油共有组分的气相色谱-质谱分析

本文采用气相色谱-质谱(GC-MS)法分析不同批次的藏红花挥发性成分,探讨同一产地不同批次藏红花挥发组分的成分与含量,并用峰面积归一化法确定各成分的相对含量,以其中的主要挥发组分作为考察指标,观察不同批次藏红花挥发组分的变化情况.初步鉴定出42种成分,且同一产地不同批次藏红花挥发性成分具有一定相似性.     

LC Determination of Adulterated Saffron Prepared by Adding Styles Colored with Some Natural Colorants

A high-performance liquid chromatographic method previously developed (Lozano et al. in J Chromatogr A 830:477–483, 1999) for simultaneous detection, identification and quantification of the secondary metabolites in commercial saffron was extended for the detection of adulterated saffron prepared by adding styles colored with the natural colorants extracted from saffron petals, safflower, madder and red beet. The chromatograms of the methanol-water (50%, v/v) extracts of pure and adulterated saffron were obtained at the assayed wavelengths, 402 (or 254), 260 and 535 (or 440) nm and then by applying two-way analysis of variance (ANOVA) to the obtained data the presence of the styles colored with the colorant of safflower (>14.3%), styles colored with the colorant of madder (>9.1%) and styles colored with the colorant of red beet (>14.3%) in saffron were significantly detected. But the detection of adulterated saffron prepared with the colorant of saffron petals was not successful.     

Extracted apocarotenoids from saffron stigmas and evaluated the quality of saffron

Saffron is a kind of expensive spice and therapeutic agent. In order to establish a set of methods to evaluate the quality of saffron, High Performance Liquid Chromatography/Mass Spectrometry (HPLC/MS) were used to identify the apocarotenoids extracted from saffron stigmas. TOPSIS method was applied to evaluate the qualities of saffron. The conditions of HPLC/MS and chemical fingerprints of saffron had been established. The 14 chromatographic peaks and 13 main apocarotenoids of saffron with high reproducibility, stability and specificity were confirmed. The qualities of 40 kinds of saffron from different countries and regions were ranked.     

Determination of total safranal by in situ acid hydrolysis in supercritical fluid media: Application to the quality control of commercial saffron

A procedure allowing hydrolysis reactions to be conducted in a dynamic supercritical-CO₂ medium was developed for quantifying total safranal (viz. free safranal present in the sample + safranal resulting from picrocrocin hydrolysis), which are the main component of the essential oil and responsible for the characteristic aroma of saffron. The proposed method allows total safranal amounts over the ranges 0.05-1.5 mg mL⁻¹ to be determined. The standard deviation achieved was 2%. This method was applied to the determination of safranal in natural saffron samples. The results obtained were compared with the “safranal value” total index, which is widely used as a quality measure of saffron products. The comparison revealed that the proposed method provides useful information not contained in the safranal value, based on the fact that, some samples with a high “safranal index” contain low concentrations of safranal. The proposed method is very useful for quality control in commercial saffron samples.     

Effective Isolation of Picrocrocin and Crocins from Saffron: From HPTLC to Working Standard Obtaining

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.     

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

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

1H-NMR Metabolic Profiling and Antioxidant Activity of Saffron (Crocus sativus) Cultivated in Lebanon

Despite the beneficial health properties shown by Lebanese saffron, its qualitative and quantitative composition has never been investigated before. In the present study, NMR spectroscopy, together with antioxidant activity assays, were applied to evaluate the chemical composition of saffron samples of different geographical origins (Lebanon, Italy, Iran, and India) and to categorize the Lebanese saffron for the first time. The distinction between Lebanese saffron and that produced in other countries was attributed to its higher linolenic and linoleic fatty acids, glucose and picrocrocin contents. Moreover, spices produced in three different regions of the Lebanese territory have been clearly differentiated. Saffron cultivated in the Qaa region displayed a high glucose, fatty acids and polyphenols content, whereas Hermel saffron exhibited the largest rate of picrocrocin and glycosylated carotenoids. Finally, samples from Baalbeck showed lower rates for the majority of metabolites. Moreover, Lebanese saffron showed a high antioxidant activity in ABTS and DPPH assays. A low dose of saffron extract (10 µg/mL) inhibited the growth of human lung adenocarcinoma cells, probably due to the high polyphenolic content. This study highlights the quality and peculiarity of Lebanese saffron cultivated in Northern Beqaa district and allows for a good discrimination between spices produced in relatively close territory.     

Characterization of volatile components of Iranian saffron using factorial-based response surface modeling of ultrasonic extraction combined with gas chromatography–mass spectrometry analysis

The volatile components of Iranian saffron were extracted using ultrasonic solvent extraction (USE) technique and then were separated and detected by gas chromatography–mass spectrometry (GC–MS). Variables affecting the extraction procedure were screened by using a 2⁵⁻¹ fractional factorial design and among them; sample amount, solvent volume, solvent ratio and extraction time were optimized by applying a rotatable central composite design (CCD). The optimum values of factors were: 2.38 g sample, 29.04 mL solvent, 69.23% MeOH solvent ratio and 71.8 min for the extraction time. Forty constituents were identified for Iranian saffron by GC–MS representing 90% of the total peak area. The major components were 2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde, namely safranal (26.29%), bicyclo[3,2,0]hept-2-ene-,4-ethoxy-,endo (5.69%), linoleic acid (4.77%), 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde, namely HTCC (4.44%), and nonadecanol (3.32%). Some new compounds were identified for the first time in saffron. In addition, the results of this study were compared with those of Greek saffron.     

Saffron Characterization by a Multidisciplinary Approach

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.     

Authentication of Iranian Saffron (Crocus sativus) Using Stable Isotopes δ13C and δ2H and Metabolites Quantification

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.     

Phytochemical Differentiation of Saffron (Crocus sativus L.) by High Resolution Mass Spectrometry Metabolomic Studies

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

HPLC/PDA/ESI-MS evaluation of saffron (Crocus sativus L.) adulteration

The present study evaluated the reliability of the ISO/TS 3632-2 UV-Vis spectrometric method for saffron classification, making experiments on saffron samples to which were added increasing concentrations of common saffron spice adulterants (safflower, marigold and turmeric). The results showed that the ISO/TS 3632-2 method is not able to detect addition of up to 10-20%, w/w, of saffron adulterants. For additions from 20 to 50%, w/w, of the three adulterants, saffron was classified in a wrong category; addition of higher than 50%, w/w, determined variations in the investigated parameters that did not allow identification of the product as "saffron". In all cases, the method did not permit the recognition of the nature of the adulterant. On the contrary, the specificity of the HPLC/PDA/MS technique allowed the unequivocal identification of adulterant characteristic marker molecules that could be recognized by the values of absorbance and mass. The selection of characteristic ions of each marker molecule has revealed concentrations of up to 5%, w/w, for safflower and marigold and up to 2% for turmeric. In addition, the high dyeing power of turmeric allowed the determination of 2%, w/w, addition using exclusively the HPLC/PDA technique.     

A rapid site‐specific PCR based one‐tube authentication method for saffron in crude drugs and processed herbal medicines

Saffron is one of the world's most precious medicinal herbs and often found to be adulterated with other cheaper materials. The chemical compounds in Crocus sativus L. such as crocin, picrocrocin also exist in other plants, which makes the chemotype‐driven analysis not ideal for the quality control of saffron. Herein, we developed a rapid authentication method for saffron in crude drugs by the site‐specific PCR. In order to realize fast high‐throughput analysis, a one‐tube identification approach was further established by using a universal fluorescent dye to detect the PCR products. In addition, this method was also applied to the authentication of saffron in a processed herbal medicine “Er shi wu wei shan hu wan” which consists of twenty‐five kinds of medicinal materials including plants, minerals and animals. Additionally, this method was also proved to be with high specificity and repeatability. The flexibility of choosing different primers also made this method versatile for other medicinal materials.     

A Novel FTIR-Based Chemometric Solution for the Assessment of Saffron Adulteration with Non-Fresh Stigmas

The development of fast, non-destructive, and green methods with adequate sensitivity for saffron authentication has important implications in the quality control of the entire production chain of this precious spice. In this context, the highly suitable sensitivity of a spectroscopic method coupled with chemometrics was verified. A total number of 334 samples were analyzed using attenuated-total-reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy; the collected spectra were processed by partial-least-squares discriminant analysis (PLS-DA) to evaluate the feasibility of this study for the discrimination between compliant saffron (fresh samples produced in 2020) and saffron samples adulterated with non-fresh stigmas produced in 2018 and 2016. PLS-DA was able to classify the saffron samples in accordance with the aging time and to discriminate fresh samples from the samples adulterated with non-fresh (legally expired) stigmas, achieving 100% of both sensitivity and specificity in external prediction. Moreover, PLS regression was able to predict the adulteration level with sufficient accuracy (the root-mean-square error of prediction was approximately 3–5%). In summary, ATR-FTIR and chemometrics can be employed to highlight the illegal blending of fresh saffron with unsold stocks of expired saffron, which may be a common fraudulent practice not yet considered in the scientific literature.     

Hormone and microorganism treatments in the cultivation of saffron (Crocus sativus L.) plants

The difficult cultivation of the saffron plant (Crocus Sativus L.) make the spice of the same name made from its dried stigmas very valuable. It is estimated that some 75,000 blossoms or 225,000 hand-picked stigmas are required to make a single pound of saffron, which explains why it is the world's most expensive spice. The aim of this study was to identify ways of increasing the fertility and production of saffron. For this purpose, the treatment of saffron bulbs with a synthetic growth hormone--a mixture of Polystimulins A6 and K--and two different microorganism based materials--biohumus or vermicompost and Effective Microorganisms (EM)--in four different ways (hormone alone, biohumus alone, EM alone and EM+biohumus) was investigated to determine whether these treatments have any statistically meaningful effects on corms and stigmas. It has been shown that EM + biohumus was the most effective choice for improved saffron cultivation.     

Multi-residue contaminants and pollutants analysis in saffron spice by stir bar sorptive extraction and gas chromatography-ion trap tandem mass spectrometry

A method for the simultaneous determination of 46 semi-volatile organic contaminants and pollutants in saffron has been developed for the first time using a stir bar sorptive extraction technique and thermal desorption in combination with gas chromatography-ion trap tandem mass spectrometry. The analytical method proposed was easy, rapid and sensitive and showed good linearity, accuracy, repeatability and reproducibility over the concentration range tested. Moreover, the correlation coefficients were higher than 0.98 for all target compounds and detection limits were lower than 1 microg/kg except for simazine. The present method was also applied for the analysis of trace contaminants in saffron samples.     

Saffron: Chemical Composition and Neuroprotective Activity

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

Identification and Isotopic Analysis of Safranal from Supercritical Fluid Extraction and Alcoholic Extracts of Saffron

Abstract

Saffron is one of the most expensive spices. Consequently, it is not so difficult to understand that fraudulent saffron exists. Thus, it was interesting to study the most important flavouring component, in terms of aroma, volatile compound of saffron - safranal - by ¹³C isotopic analysis. Five saffron samples from different countries have been analysed. Safranal has been extracted by methanol or by Supercritical Fluid Extraction (SFE). The results indicate that there is a significant difference between the synthetic safranal and the natural one. On the contrary, it is difficult to conclude on the difference between the various geographical origins, as the isotopic variations are small. Moreover, it has been found that Supercritical Fluid Extraction allowed the selective extraction of volatile compounds from saffron under optimised conditions. It is a cleaner and faster method of extraction compared to the extraction using organic solvent. Nevertheless, an isotopic fractionation occurs in relation to the extraction yield of safranal.     

Hydrogen and Atom Transfer Activity of Saffron Extracts by Square Wave Voltammetry

Saffron is an edible spice with highly appreciated sensory and antioxidant properties. One of the most representative redox species found in saffron extracts is crocin, whose content is used to evaluate the quality and value of the resulting spice. In this study, a voltammetry method based on the direct detection of crocin at a bare glassy carbon electrode is presented. The principle of the method is based on the monitoring of the anodic wave exhibited by crocin (0.1–1.0 mM) after its mixing with the azo radical initiator AAPH (20 mM) in ethanol:acetonitrile (1 : 1) solution. The decay rate of the anodic peak (E=+434 mV vs. Ag/Ag⁺), as a result of the consumption of crocin by AAPH, was used as index of the hydrogen transfer capacity and, thus, of its antioxidant activity. With a decay rate of k=0.02 h⁻¹, crocin exhibits only a weak antioxidant activity in comparison with tocopherols (k=0.13 h⁻¹), but still sufficient to protect against the oxidation of safranal, a further redox species found in saffron extracts and mainly responsible for its flavor. The proposed approach was finally applied to discriminate saffron extract samples from different geographical origins. The proposed approach is suitable to characterize the quality of saffron extracts and estimate its antioxidant properties.