Isolation of bioactive compounds from cruciferous vegetables: identification and quantification by synthetic and spectroscopic techniques

Vegetables of the Cruciferae family (order Brassicales) contain a number of organic compounds with biological activity. Among these are glucosinolates, which are found only in the order Brassicales. Upon enzymatic hydrolysis of glucosinolates, aliphatic isothiocyanates, aromatic isothiocyanates and indole derivatives, are released. The isothiocyanate sulforaphane and the indole derivatives, indole-3-carbinol, indole-3-acetonitrile, indole-3-carbaldehyde and ascorbigen were synthesized and used as standards for the developing of new qualitative and quantitative methods applied in cruciferous vegetables. Indole derivatives such as indole-3-acetamide and amide conjugates of indole-3-acetic acid with 10 amino acids were also synthesized. All synthesized compounds were studied by NMR and FT-IR spectroscopy and HRMS. A new method for the direct quantification of total isothiocyanate content of cruciferous vegetables by Fourier Transform Infrared Spectroscopy, using the Attenuated Total Reflection (ATR) technique, in combination with the PLS algorithm, was developed. The assay was based on the absorbance of the 2150-2120 cm-1 region, where the asymmetric stretching of N=C=S appears. The new method proved equivalent to the UV-Vis literature method, based on the F and t statistical tests and the % recovery, which were within the acceptable limits. Subsequently, a new method for the simultaneous determination of sulforaphane and indole-3-carbinol was developed, using a high-resolution mass spectrometer with a Q-TOF mass analyzer. The new UPLC-HRMS/MS method was validated and characterized by excellent linearity and low detection limits. The new UPLC-HRMS/MS method is the first that utilizes high resolution mass spectrometry for the simultaneous determination of sulforaphane and indole-3-carbinol in cruciferous vegetables. Indole-3-carbaldehyde, indole-3-carbinol, indole-3-acetonitrile and ascorbigen, were determined by UHPLC-HRMS/MS. The indole derivatives were studied in extracts of 7 cruciferous vegetables (red and white cabbage, green and purple broccoli, turnip, radish, white cauliflower) through a new UHPLC-HRMS/MS method which was developed for the study. This method is the first application of UHPLC-HRMS/MS for the simultaneous determination of glucobrassicin hydrolysis products in cruciferous vegetables. In the final stage of dissertation, a determination of phytohormones in eight cruciferous vegetables was carried out. The compounds included in the study (27 compounds) were indole-3-acetic acid, 4-chloroindole-3-acetic acid, jasmonic acid and salicylic acid. Indole-3-acetonitrile and indole-3-acetamide and indole-3-carbaldehyde were also studied. Finally, the amide conjugates of indole-3-acetic acid with the amino acids alanine, glycine, tryptophan, serine, methionine, tyrosine, phenylalanine, valine, aspartic acid and glutamic acid were studied. The new UHPLC-HRMS/MS method enabled the identification and quantification of new compounds with unknown role in plants, providing accuracy and reliability of results and can be applied for the study of phytohormones in other plant species.Τα λαχανικά της οικογένειας των σταυρανθών (τάξη Brassicales) περιέχουν πλήθος βιοδραστικών οργανικών ενώσεων. Μεταξύ αυτών ξεχωρίζουν οι γλυκοζινολίτες οι οποίοι συναντώνται στην τάξη Brassicales. Κατά την ενζυμική υδρόλυση των γλυκοζινολιτών, απελευθερώνονται αλειφατικές ισοθειοκυανικές ενώσεις, αρωματικές ισοθειοκυανικές ενώσεις και παράγωγα του ινδολίου. Στα πλαίσια της διατριβής, συντέθηκαν, η ισοθειοκυανική ένωση σουλφοραφάνη, οι ινδολικές ενώσεις ινδολο-3-καρβινόλη, ινδολο-3-ακετονιτρίλιο, ινδολο-3-καρβαλδεΰδη και ασκορβιγόνο. Επίσης, συντέθηκαν τα ινδολικά παράγωγα, ινδολο-3-ακεταμίδιο και τα αμιδικά συζεύγματα του ινδολο-3-οξικού οξέος με 10 αμινοξέα. Όλες οι ενώσεις που συντέθηκαν, μελετήθηκαν με φασματοσκοπία NMR και FT-IR και φασματομετρία μάζας HRMS. Αναπτύχθηκε μια νέα μέθοδος άμεσου ποσοτικού προσδιορισμού του συνόλου των ισοθειοκυανικών ενώσεων στα σταυρανθή λαχανικά, με φασματοσκοπία υπερύθρου με μετασχηματισμό Fourier, αξιοποιώντας την τεχνική της εξασθενημένης ολικής ανάκλασης (ATR FT-IR), σε συνδυασμό με τον αλγόριθμο PLS. Ο ποσοτικός προσδιορισμός βασίστηκε στις απορροφήσεις της περιοχής 2150-2120 cm-1 όπου εμφανίζεται η ασύμμετρη τάση N=C=S. Η νέα μέθοδος αποδείχθηκε ισοδύναμη με την αντίστοιχη μέθοδο UV-Vis της βιβλιογραφίας, βάση των στατιστικών ελέγχων F και t και της % ανάκτησης, που ήταν εντός των αποδεκτών ορίων. Στη συνέχεια της διατριβής, αναπτύχθηκε μια νέα μέθοδος ποιοτικού και ποσοτικού προσδιορισμού της σουλφοραφάνης και της ινδολο-3-καρβινόλης, χρησιμοποιώντας φασματογράφο μάζας υψηλής διακριτικής ικανότητας με αναλυτή μαζών Q-TOF. Η νέα μέθοδος UPLC-HRMS/MS επικυρώθηκε και χαρακτηρίστηκε από εξαιρετική γραμμικότητα και χαμηλά όρια ανίχνευσης. Επιπλέον, προσδιορίστηκαν με UΗPLC-HRMS/MS οι ενώσεις, ινδολο-3-καρβαλδεΰδη, ινδολο-3-καρβινόλη, ινδολο-3-ακετονιτρίλιο και ασκορβιγόνο. Η συγκριμένη μέθοδος αποτελεί την πρώτη εφαρμογή του UHPLC-HRMS/MS για τον ταυτόχρονο προσδιορισμό των προϊόντων ενζυμικής υδρόλυσης της γλυκομπρασικίνης σε σταυρανθή λαχανικά. Στο τελευταίο στάδιο της διατριβής πραγματοποιήθηκε προσδιορισμός των φυτορμονών με UΗPLC-HRMS/MS σε οχτώ σταυρανθή λαχανικά. Οι ενώσεις που συμπεριλήφθηκαν στη μελέτη (27 ενώσεις) ήταν οι φυτορμόνες, ινδολο-3-οξικό οξύ, 4-χλωροϊνδολο-3-οξικό οξύ, ιασμονικό οξύ και σαλικυλικό οξύ. Επίσης προσδιορίστηκαν, το ινδολο-3-ακετονιτρίλιο, το ινδολο-3-ακεταμίδιο και η ινδολο-3-καρβαλδεΰδη. Τέλος, προσδιορίστηκαν στοχευμένα τα αμιδικά συζεύγματα του ινδολο-3-οξικού οξέος με τα αμινοξέα αλανίνη, γλυκίνη, θρυπτοφάνη, σερίνη, μεθειονίνη, τυροσίνη, φαινυλαλανίνη, βαλίνη, ασπαρτικό οξύ και γλουταμινικό οξύ. Η νέα μέθοδος UΗPLC-HRMS/MS που αναπτύχθηκε, επέτρεψε την ταυτοποίηση και ποσοτικοποίηση νέων ενώσεων, που δεν είναι γνωστός ο ρόλος τους στα φυτά, παρέχοντας ακρίβεια και αξιοπιστία των αποτελεσμάτων και μπορεί να εφαρμοστεί για την μελέτη των φυτορμονών και σε άλλα είδη φυτών

Development of a UPLC-Q-ToF-MS Method for the Determination of Sulforaphane and Iberin in Cruciferous Vegetables

Sulforaphane (1-isothiocyanato-4-(methylsulfinyl)-butane) and iberin (1-isothiocyanato-3-methylsulfinylpropane) have attracted widespread attention due to their anti-inflammatory and cancer-preventive properties. These isothiocyanates are products of the enzymatic hydrolysis of the glucosinolates glucoraphanin and glucoiberin, which are found only in the plants of the order Brassicales. Cruciferous vegetables, such as broccoli, cabbage and cauliflower, belong to the order Brassicales, specifically, in the Brassicaceae family. Our aim was to develop an efficient and accurate method for the simultaneous determination of sulforaphane and iberin in cruciferous vegetables using Ultra-high Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UPLC-Q-ToF-MS). The method was applied for the quantitative determination of these compounds in a variety of cruciferous vegetables (green and purple broccoli, white and purple cabbage, radish, turnip, arugula, watercress and cauliflower). The results showed that green and purple broccoli contained the highest levels of sulforaphane (660.14 ± 34.29 to 210.11 ± 9.76 μg g−1 dry weight), while the highest concentration of iberin was detected in purple broccoli (144.98 ± 3.56 μg g−1 dry weight). The lowest concentrations of sulforaphane and iberin were measured in watercress and radish. The differences in the content of these compounds can be attributed to the variability among Brassicaceae species, geography, season and various environmental factors

Greek Honey Authentication: Botanical Approach

Honey is a functional, honeybee product with a useful role in human nutrition and several health benefits. Greece is a Mediterranean region with several types of monofloral honey. Today, Greek honey has acquired an important position in national and international markets. Due to this increased industrialization and globalization, quality control is a necessity. Mislabeling constitutes one of the most notable types of fraudulence, while most consumers are looking for authentic honey. Moreover, producers and suppliers are searching for rapid and analytical methodologies to secure Greek honey in a competitive environment. In this context, we aimed to describe the classical (melissopalynological, physicochemical) and analytical (chromatographic, spectrometric, and spectroscopic) methods for the standardization of the botanical origin of Greek honey

Greek Honey Authentication: Botanical Approach

Honey is a functional, honeybee product with a useful role in human nutrition and several health benefits. Greece is a Mediterranean region with several types of monofloral honey. Today, Greek honey has acquired an important position in national and international markets. Due to this increased industrialization and globalization, quality control is a necessity. Mislabeling constitutes one of the most notable types of fraudulence, while most consumers are looking for authentic honey. Moreover, producers and suppliers are searching for rapid and analytical methodologies to secure Greek honey in a competitive environment. In this context, we aimed to describe the classical (melissopalynological, physicochemical) and analytical (chromatographic, spectrometric, and spectroscopic) methods for the standardization of the botanical origin of Greek honey

Unifloral Autumn Heather Honey from Indigenous Greek Erica manipuliflora Salisb.: SPME/GC-MS Characterization of the Volatile Fraction and Optimization of the Isolation Parameters

For long heather honey has been a special variety due to its unique organoleptic characteristics. This study aimed to characterize and optimize the isolation of the dominant volatile fraction of Greek autumn heather honey using solid-phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS). The described approach pointed out 13 main volatile components more closely related to honey botanical origin, in terms of occurrence and relative abundance. These volatiles include phenolic compounds and norisoprenoids, with benzaldehyde, safranal and p-anisaldehyde present in higher amounts, while ethyl 4-methoxybenzoate is reported for the first time in honey. Then, an experimental design was developed based on five numeric factors and one categorical factor and evaluated the optimum conditions (temperature: 60 °C, equilibration time: 30 min extraction time: 15 min magnetic stirrer velocity: 100 rpm sample volume: 6 mL water: honey ratio: 1:3 (v/w)). Additionally, a validation test set reinforces the above methodology investigation. Honey is very complex and variable with respect to its volatile components given the high diversity of the floral source. As a result, customizing the isolation parameters for each honey is a good approach for streamlining the isolation volatile compounds. This study could provide a good basis for future recognition of monofloral autumn heather honey

Authentication of the Botanical and Geographical Origin and Detection of Adulteration of Olive Oil Using Gas Chromatography, Infrared and Raman Spectroscopy Techniques: A Review

Olive oil is among the most popular supplements of the Mediterranean diet due to its high nutritional value. However, at the same time, because of economical purposes, it is also one of the products most subjected to adulteration. As a result, authenticity is an important issue of concern among authorities. Many analytical techniques, able to detect adulteration of olive oil, to identify its geographical and botanical origin and consequently guarantee its quality and authenticity, have been developed. This review paper discusses the use of infrared and Raman spectroscopy as candidate tools to examine the authenticity of olive oils. It also considers the volatile fraction as a marker to distinguish between different varieties and adulterated olive oils, using SPME combined with gas chromatography technique

The Use of SPME-GC-MS IR and Raman Techniques for Botanical and Geographical Authentication and Detection of Adulteration of Honey

The aim of this review is to describe the chromatographic, spectrometric, and spectroscopic techniques applied to honey for the determination of botanical and geographical origin and detection of adulteration. Based on the volatile profile of honey and using Solid Phase microextraction-Gas chromatography-Mass spectrometry (SPME-GC-MS) analytical technique, botanical and geographical characterization of honey can be successfully determined. In addition, the use of vibrational spectroscopic techniques, in particular, infrared (IR) and Raman spectroscopy, are discussed as a tool for the detection of honey adulteration and verification of its botanical and geographical origin. Manipulation of the obtained data regarding all the above-mentioned techniques was performed using chemometric analysis. This article reviews the literature between 2007 and 2020

Optimized Isolation of Safranal from Saffron by Solid-Phase Microextraction (SPME) and Rotatable Central Composite Design-Response Surface Methodology (RCCD-RSM)

Safranal is the main aroma component of saffron stigmas. It is also a great antioxidant with known pharmacological properties and is a potent indicator for the grading and authentication of saffron. In this study, the optimum extraction conditions of safranal from saffron stigmas were investigated using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) and response surface methodology (RSM). A rotatable-central composite design was applied, and a linear regression model has been used for the model building. The optimized factors were as follows: sample weight (15 mg), water volume (4 mL), exposure time in the headspace (20 min), and extraction temperature (45 °C). All factors were found significant; however, extraction temperature and exposure time were the most important for the isolation of safranal. The obtained model was successfully validated with a test set of saffron samples analyzed under the optimum extraction conditions. The optimized SPME extraction conditions of safranal found in this study contribute to the efforts towards the detection of saffron authentication and adulteration

Optimized Isolation of Safranal from Saffron by Solid-Phase Microextraction (SPME) and Rotatable Central Composite Design-Response Surface Methodology (RCCD-RSM)

Safranal is the main aroma component of saffron stigmas. It is also a great antioxidant with known pharmacological properties and is a potent indicator for the grading and authentication of saffron. In this study, the optimum extraction conditions of safranal from saffron stigmas were investigated using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) and response surface methodology (RSM). A rotatable-central composite design was applied, and a linear regression model has been used for the model building. The optimized factors were as follows: sample weight (15 mg), water volume (4 mL), exposure time in the headspace (20 min), and extraction temperature (45 °C). All factors were found significant; however, extraction temperature and exposure time were the most important for the isolation of safranal. The obtained model was successfully validated with a test set of saffron samples analyzed under the optimum extraction conditions. The optimized SPME extraction conditions of safranal found in this study contribute to the efforts towards the detection of saffron authentication and adulteration