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.     

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.     

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.     

Effect of ultrasonic waves on crocin and safranal content and expression of their controlling genes in suspension culture of saffron (Crocus sativus L.)

The expression of biosynthesis controlling genes of crocin and safranal in saffron (Crocus sativus) can be influenced by ultrasonic waves. Sterilized saffron corms were cultured in a ½-MS medium supplemented by 2-4-D and BAP. Saffron callus cells were treated with ultrasonic waves in a cellular suspension culture under optimal growth conditions. The samples were collected at 24 and 72 hours after treatment in three replications. The secondary metabolites were measured by high-performance liquid chromatography and the gene expression was analysed by the real-time polymerase chain reaction. Results indicate that this elicitor can influence the expressions of genes CsBCH, CsLYC and CsGT-2; the ultrasonic waves acted as an effective mechanical stimulus to the suspension cultures. The analysis of variance of the ultrasonically produced amounts of safranal and crocin indicates that there is a significant difference between once- and twice-treated samples in that the amount of safranal was the highest within the samples taken from the twice-treated suspension culture at 72 h after the ultrasound treatment, and the crocin was maximised after 24 h passed the twice-applied ultrasound treatment.     

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.     

Extraction of crocin from saffron (Crocus sativus) using molecularly imprinted polymer solid‐phase extraction

A new molecularly imprinted polymer for extraction of crocin from saffron stigmas was prepared using gentiobiose (a glycoside moiety in crocin structure) as a template. Crocin binding to gentiobiose imprinted polymer (Gent‐MIP) was studied in comparison with a blank nonimprinted polymer in aqueous media. Affinity of the Gent‐MIP for the crocin was more than the nonimprinted polymer at all concentrations. In Scatchard analysis, the number of binding sites in each gram of polymer (maximum binding sites) and dissociation constant of crocin to binding sites were 18.4 μmol/g polymer and 11.2 μM, respectively. The Gent‐MIP was then used as the sorbent in an SPE method for isolation and purification of crocin from methanolic extract of saffron stigmas. The recovery of crocin, safranal and picrocrocin was determined in washing and elution steps. The Gent‐MIP had significantly higher affinity for crocin than other compounds and enabled selective extraction of crocin with a high recovery (84%) from a complex mixture. The results demonstrated the possibility of using a part of a big molecule in preparing a molecularly imprinted polymer with a good selectivity for the main structure.     

Simultaneous quantification of five major biologically active ingredients of saffron by high-performance liquid chromatography.

A simple, sensitive and specific high-performance liquid chromatography-UV (HPLC-UV) method has been developed for the first time to simultaneously quantify the five major biologically active ingredients of saffron, namely crocin 1, crocin 2, crocin 3, crocin 4 and crocetin. Calibration curves were derived by spiking authentic compounds and internal standard, 13-cis-retinoic acid, into herbal samples prior to extraction. Extraction was conducted simply by stirring dried herb (20 mg) with 80% aqueous methanol (5 ml) at ambient temperature in the dark for 2 h. The HPLC assay was performed on a reversed-phase C18 column with linear gradient elution using methanol and 1% aqueous acetic acid. Calibrations were linear (r2 = 0.999) for all five analytes, with overall intra- and inter-day RSDs of less than 11%. The assay was successfully applied to the determination of four crocins and crocetin in three saffron samples and two Zhizi, another crocin-containing herb. Results indicate that the developed HPLC assay can be readily utilized as a quality control method for crocin-containing medicinal herbs.     

Saffron and Its Major Ingredients’ Effect on Colon Cancer Cells with Mismatch Repair Deficiency and Microsatellite Instability

Background: Colorectal cancer (CRC) is one of the most common cancers worldwide. One of its subtypes is associated with defective mismatch repair (dMMR) genes. Saffron has many potentially protective roles against colon malignancy. However, these roles in the context of dMMR tumors have not been explored. In this study, we aimed to investigate the effects of saffron and its constituents in CRC cell lines with dMMR. Methods: Saffron crude extracts and specific compounds (safranal and crocin) were used in the human colorectal cancer cell lines HCT116, HCT116+3 (inserted MLH1), HCT116+5 (inserted MSH3), and HCT116+3+5 (inserted MLH1 and MSH3). CDC25b, p-H2AX, TPDP1, and GAPDH were analyzed by Western blot. Proliferation and cytotoxicity were analyzed by MTT. The scratch wound assay was also performed. Results: Saffron crude extracts restricted (up to 70%) the proliferation in colon cells with deficient MMR (HCT116) compared to proficient MMR. The wound healing assay indicates that deficient MMR cells are doing better (up to 90%) than proficient MMR cells when treated with saffron. CDC25b and TDP1 downregulated (up to 20-fold) in proficient MMR cells compared to deficient MMR cells, while p.H2AX was significantly upregulated in both cell types, particularly at >10 mg/mL saffron in a concentration-dependent manner. The reduction in cellular proliferation was accompanied with upregulation of caspase 3 and 7. The major active saffron compounds, safranal and crocin reproduced most of the saffron crude extracts’ effects. Conclusions: Saffron’s anti-proliferative effect is significant in cells with deficient MMR. This novel effect may have therapeutic implications and benefits for MSI CRC patients who are generally not recommended for the 5-fluorouracil-based treatment.     

Inhibitory effect of safranal and crocin,two principle compounds of <Emphasis Type="Italic">Crocus sativus</Emphasis>, on fibrillation of lysozyme

A key feature in more than twenty amyloid-related diseases is the aggregation of intra-and/or extracellular misfolded proteins as amyloid fibrils. Therefore, preventing or reversing amyloid aggregation by using of small molecules is considered as useful approaches to the treatment of these diseases. We have evaluated the ability of safranal and crocin, to inhibit amyloid self-assembly of hen egg white lysozyme (HEWL), as an in vitro model system. Structural properties of HEWL in the presence of these compounds were investigated individually using thioflavin T, anilinonaphthalene-8-sulfonic acid fluorescence assays, far-UV circular dichroism and scanning electron microscopy as well as docking method. Our results showed that incubation of HEWL with either crocin or safranal at various concentrations leads a significant inhibition in the rate of amyloid formation. Docking analysis revealed crocin and safranal interact with the central hydrophobic region of lysozyme through van der Waals interaction. Hydroxyl group in crocin through hydrogen bonds connected to the several hydrophilic amino acids of lysozyme, while in safranal there are just one aldehyde group that through hydrogen bonds connected to aspartic acid in lysozyme. It can be concluded that both hydrophobic and hydrophilic groups contribute to lower lysozyme fibril accumulation.     

Development and validation method for determination of Paroxetine and its metabolites by nonaqueous capillary electrophoresis in human urine. Experimental design for evaluating the ruggedness of the method

A simple, rapid, and sensitive procedure using nonaqueous capillary electrophoresis (NACE) to measure Paroxetine (one of the mostly used antidepressants for mental diseases treatment) and three metabolites has been developed and validated. Optimum separation of paroxetine and metabolites was obtained on a 57 cm x 75 microm capillary using a nonaqueous buffer system of 9:1 methanol-acetonitrile containing 25 mM ammonium acetate and 1% acetic acid, with temperature and voltage of 25 degrees C and 15 kV, respectively, and hydrodynamic injection. Fluoxetine was used as an internal standard. Good results were obtained for different aspects including stability of the solutions, linearity, accuracy, and precision. Detection limits between 9.3 and 23.1 microg.L(-1) were obtained for paroxetine and its metabolites. A ruggedness test of the method was carried out using the Plackett-Burman fractional factorial model with a matrix of 15 experiments. This method has been used to determine paroxetine and its main metabolite B at clinically relevant levels in human urine. Prior to NACE determination, the samples were purified and enriched by means of an extraction-preconcentration step with a preconditioned C18 cartridge and eluting the compounds with methanol.     

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.     

Analysis of Saffron (Crocus sativus L. Stigma) Components by LC–MS–MS

A liquid chromatographic tandem mass spectrometric validated method was developed for the detection of chemicals attributing color, flavor, taste and medicinal properties to saffron (Crocus sativus L. stigma). Ultrasonic extractions of saffron stigmas were followed by LC procedure with Pinnacle II Cyano (5 μm 150 × 2.1 mm) column and acetonitrile: water (70:30, v/v) as mobile phase. Deprotonated ions formed by a turbo ion spray in negative MS mode were used to detect the analytes. MS–MS detection was by monitoring precursors (m/z) fragmentations; of 149 → 113 (safranal), 327 → 283 (crocetin), 329 → 167 (picrocrocin), 355 → 327 (dimethyl crocetin), 489 → 327 (crocin E), 535 → 489 (carotenes), 651 → 327 (crocin C), 813 → 652 (crocin B), 975 → 651 (crocin A) and 1,137 → 813 (crocin F). The method was validated for linearity, precision, repeatability and specificity.     

Determination of flurbiprofen enantiomers in plasma using a single-isomer amino cyclodextrin derivative in nonaqueous capillary electrophoresis

A nonaqueous capillary electrophoresis (NACE) assay was developed for the separation and determination of flurbiprofen enantiomers in plasma samples using 6-monodeoxy-6-mono(3-hydroxy)propylamino-beta-cyclodextrin as chiral selector. The nonaqueous background electrolyte was made up of 40 mM ammonium acetate in methanol (MeOH), and flufenamic acid was employed as internal standard. Solid-phase extraction was used for sample cleanup prior to the NACE separation. The NACE method reproducibility was optimized by evaluating different capillary washing sequences between runs. After having tested various conditions, trifluoroacetic acid (1 M) in MeOH was finally selected. Concerning the solid-phase extraction procedure, good and reproducible analyte recoveries were obtained using MeOH for protein denaturation and a polymeric phase combining hydrophobic interactions with anion exchange properties (Oasis) MAX) was selected as extraction sorbent. The method selectivity was not only demonstrated toward a blank plasma sample but also toward other non-steroidal anti-inflammatory drugs. The method was then successfully validated with respect to response function, trueness, precision, accuracy, linearity and limit of quantification.     

Non-aqueous capillary zone electrophoresis method for the analysis of paroxetine, tamoxifen, and their main metabolites in urine

The potential of non-aqueous capillary electrophoresis (NACE) was investigated for the simultaneous separation of paroxetine, tamoxifen, and their main metabolites. Baseline separation of the studied solutes was obtained on a  μm capillary using a non-aqueous buffer composed of 18 mM ammonium acetate and 1.1% acetic acid in 80:20 (v/v) methanol-acetonitrile, with a temperature and voltage of 22 °C and 15 kV, respectively. Clomipramine was used as internal standard. Aspects such as stability of the solutions, linearity, accuracy, precision and ruggedness were examined in order to validate the proposed method. Detection limits obtained for all the studied compounds ranged between 3.0 and 7.1 μg l⁻¹. The developed method is sensitive and robust and was used to determine paroxetine, tamoxifen, and their metabolites at clinically relevant levels in human urine. Before NACE determination, the samples were purified and enriched by means of an extraction-pre-concentration step with a pre-conditioned C₁₈ cartridge. Determination of these analytes in the urine of four females urines was demonstrated.     

Cow and Ewe Cheeses Made with Saffron: Characterization of Bioactive Compounds and Their Antiproliferative Effect in Cervical Adenocarcinoma (HeLa) and Breast Cancer (MDA-MB-231) Cells

Saffron is a widespread consumed spice containing many phytochemicals. It is often used in dairy technologies to enhance color and flavor of cheeses, but it is also known for its several therapeutic effects, as well as its antiproliferative and anticancer properties. In this study High Performance Liquid Chromatography was used to characterize saffron bioactive compounds in cow and ewe cheeses made with saffron, and the antiproliferative effect of the crocin-rich extracts from cheeses was investigated on different cellular lines (CaCo2, MDA-MB-231 and HeLa) by MTT assay. Crocins were observed in all cheese samples, with the total content ranging between 0.54 and 30.57 mg trans-4-GG/100 g cheese, according to the different cheese making process. Picrocrocin was detected in no cheese (probably due to its degradation during cheese making), while safranal was detected only in one ewe cheese (mainly due to its high volatility). HeLa and MDA-MB-231 cells were sensitive to treatment with crocin-rich extracts from cheeses, while no effect was observed on CaCo2 cells. The chemical environment of the food matrix seems to have a great influence on the crocin antiproliferative effect: the crocin-rich extracts from cheese with both high residual N/protein and fat contents showed increased antiproliferative effect compared to pure crocin (trans-4-GG), but cheeses from different milk species (type of fats and proteins) could also play an important role in modulating crocin’s antiproliferative effects.     

Nonaqueous capillary electrophoresis method for the analysis of tamoxifen, imipramine and their main metabolites in urine

The viability of nonaqueous capillary electrophoresis (NACE) was investigated for the simultaneous determination of tamoxifen, imipramine and their main metabolites (4-hydroxytamoxifen and desipramine, respectively). Baseline separation of the studied solutes was obtained on a 57 cm × 75 μm capillary using a nonaqueous solution composed of 17 mM ammonium acetate and 1.25% acetic acid in 80:20 (v:v) methanol-acetonitrile, temperature and voltage 22 °C and 15 kV, respectively, and hydrodynamic injection. Paroxetine was used as internal standard. Different aspects including linearity, accuracy, ruggedness and precision was studied. Detection limits between 9.0 and 15.0 μg L⁻¹ were obtained for all the studied compounds. The developed method is simple, rapid and sensitive and has been used to determine tamoxifen, imipramine and their metabolites at clinically relevant levels in human urine. Before NACE determination, a solid phase extraction (SPE) procedure with a C₁₈ cartridge was necessary. Real determination of these analytes in three females urines were done.     

Recent Advances on the Anticancer Properties of Saffron (Crocus sativus L.) and Its Major Constituents

Cancer is the second leading cause of death globally with an estimated 9.6 million deaths in 2018 and a sustained rise in its incidence in both developing and developed countries. According to the WHO, about 1 in 6 deaths is due to cancer. Despite the emergence of many pioneer therapeutic options for patients with cancer, their efficacy is still time-limited and noncurative. Thus, continuous intensive screening for superior and safer drugs is still ongoing and has resulted in the detection of the anticancer properties of several phytochemicals. Among the spices, Crocus sativus L. (saffron) and its main constituents, crocin, crocetin, and safranal, have attracted the interest of the scientific community. Pharmacological experiments have established numerous beneficial properties for this brilliant reddish-orange dye derived from the flowers of a humble crocus family species. Studies in cultured human malignant cell lines and animal models have demonstrated the cancer prevention and antitumor activities of saffron and its main ingredients. This review provides an insight into the advances in research on the anticancer properties of saffron and its components, discussing preclinical data, clinical trials, and patents aiming to improve the pharmacological properties of saffron and its major ingredients.     

Nonaqueous capillary electrophoresis method for the analysis of gleevec and its main metabolite in human urine

The viability of nonaqueous capillary electrophoresis (NACE) was investigated for determination of gleevec and its main metabolite in human urine using a fused-silica capillary. Baseline separation of the studied solutes was obtained using a nonaqueous solution composed of 12 mM ammonium acetate and 87.6 mM acetic acid in methanol-acetonitrile (ACN) (80:20, v:v) providing analysis time shorter than 3 min. Different aspects including stability of the solutions, linearity, accuracy and precision were studied in order to validate the method in the urine matrix. Detection limits of 24 microg L(-1) for gleevec and its metabolite were obtained. A robustness test of the method was carried out using the Plackett-Burman fractional factorial model with a matrix of 15 experiments. The developed method is simple, rapid and sensitive and has been used to determine gleveec and its metabolite at clinically relevant levels in human urine. Before NACE determination, a solid-phase extraction (SPE) procedure with a C18 cartridge was necessary. Real determination of these analytes in two patient urines were done.     

Nonaqueous capillary electrophoresis method for the enantiomeric purity determination of S-timolol using heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-cyclodextrin: validation using the accuracy profile strategy and estimation of uncertainty

Nonaqueous capillary electrophoresis (NACE) was successfully applied to the enantiomeric purity determination of S-timolol maleate using heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-cyclodextrin (HDMS-beta-CD) as chiral selector. With a background electrolyte made up of a methanolic solution of 0.75 M formic acid, 30 mM potassium camphorsulfonate and containing 30 mM HDMS-beta-CD, the determination of 0.1% of R-timolol in S-timolol could be performed with an enantiomeric resolution of 8.5. Pyridoxine was selected as internal standard. The NACE method was then fully validated by applying a novel strategy using accuracy profiles. It is based on beta-expectation tolerance intervals for the total measurement error which includes trueness and intermediate precision. The uncertainty of measurements derived from beta-expectation tolerance intervals was estimated at each concentration level of the validation standards. To confirm the suitability of the developed and validated method, several real samples of S-timolol maleate containing R-timolol maleate at different concentrations were analysed and the results were compared to those obtained by liquid chromatography.     

Development and validation method for determination of fluoxetine and its main metabolite norfluoxetine by nonaqueous capillary electrophoresis in human urine

A simple, rapid and sensitive procedure using nonaqueous capillary electrophoresis (NACE) to measure fluoxetine and its main metabolite norfluoxetine has been developed and validated. Optimum separation of fluoxetine and norfluoxetine, by measuring at 230 nm, was obtained on a 60 cm × 75 μm capillary using a nonaqueous solution system of 7:3 methanol-acetonitrile containing 15 mM ammonium acetate, capillary temperature and voltage 25 °C and 25 kV, respectively and hydrodynamic injection. Paroxetine was used as internal standard. Good results were obtained for different aspects including stability of the solutions, linearity, and precision. Detection limits of 10 μg L⁻¹ were obtained for fluoxetine and its metabolite. This method has been used to determine fluoxetine and it main metabolite at clinically relevant levels in human urine. Before NACE determination, the samples were purified and enriched by means of extraction-preconcentration step with a preconditioned C₁₈ cartridge and eluting the compounds with methanol.