Metabolites from Marine Macroorganisms of the Red Sea Acting as Promoters or Inhibitors of Amylin Aggregation

Amylin is part of the endocrine pancreatic system that contributes to glycemic control, regulating blood glucose levels. However, human amylin has a high tendency to aggregate, forming isolated amylin deposits that are observed in patients with type 2 diabetes mellitus. In search of new inhibitors of amylin aggregation, we undertook the chemical analyses of five marine macroorganisms encountered in high populations in the Red Sea and selected a panel of 10 metabolites belonging to different chemical classes to evaluate their ability to inhibit the formation of amyloid deposits in the human amylin peptide. The thioflavin T assay was used to examine the kinetics of amyloid aggregation, and atomic force microscopy was employed to conduct a thorough morphological examination of the formed fibrils. The potential ability of these compounds to interact with the backbone of peptides and compete with β-sheet formation was analyzed by quantum calculations, and the interactions with the amylin peptide were computationally examined using molecular docking. Despite their structural similarity, it could be observed that the hydrophobic and hydrogen bond interactions of pyrrolidinones 9 and 10 with the protein sheets result in one case in a stable aggregation, while in the other, they cause distortion from aggregation.This research was partially supported by the Subaward Agreement Ref. OSR-2017-CPF-3627-2 funded by the King Abdullah University of Science and Technology. The authors gratefully acknowledge the financing of the MINECO project (PID2021-127907NB-I00) founded by the Spanish Ministry of Science and Innovation and the financing from the Basque Government (IT1584-22)

Thuwalallenes A–E and Thuwalenynes A–C: New C15 Acetogenins with Anti-Inflammatory Activity from a Saudi Arabian Red Sea Laurencia sp.

Thuwalallenes A–E (1–3, 5 and 8) and thuwalenynes A–C (4, 6, 7), new C15 acetogenins featuring uncommon ring systems, along with cis-maneonene D (9), thyrsiferol (10) and 23-acetyl-thyrsiferol (11) were isolated from the organic extract of a population of the red alga Laurencia sp., collected at Rose Reef off the village of Thuwal in the Red Sea waters of the Kingdom of Saudi Arabia. The structure elucidation of the isolated natural products was based on extensive analysis of their spectroscopic data. Compounds 1–6, 8, 10 and 11 were evaluated for their anti-inflammatory activity by quantifying nitric oxide (NO) release in response to TLR4 stimulation in macrophages. Besides compound 4 that did not exhibit any activity, all other tested metabolites inhibited NO production from activated macrophages. Among them, thyrsiferol (10) and 23-acetylthyrsiferol (11) displayed activity with IC50 values in the low nM scale without cytotoxicity

Comparative study of the essential oil composition of the foliage of 54 Pinus taxa: isolation and structure elucidation of secondary metabolites from the species PINUS HELDREICHII Christ., PINUS PINEA L. and PINUS NIGRA subsp. NIGRA Arn.

In the framework of this study, the chemical composition of the organic extracts of three Pinus species, as well as the needle essential oils of 54 Pinus taxa were investigated. Specifically, cones of Pinus heldreichii, fresh needles of Pinus nigra subsp. nigra and bark of Pinus pinea were exhaustively extracted and the crude extracts were subjected to a series of chromatographic separations. The cones of P. heldreichii yielded 38 metabolites in total. Specifically, twenty abietane diterpenes, eight diterpenes featuring the pimarane skeleton, four labdane diterpenes, two macrocyclic cembrane diterpenes, two podocarpene derivatives, one oxygenated sesquiterpene and one steroid. Among these, three metabolites were new natural products (one abietane and two pimarane diterpenes), one abietane diterpene was isolated for the first time from Gymnospermae, four metabolites were isolated for the first time from the genus Pinus and twenty-seven diterpenes were reported for the first time from P. heldreichii. In addition, the spectroscopic data of 20 metabolites were fully assigned for the first time.In total, 12 metabolites were isolated from the fresh needles of P. nigra subsp. nigra, five of which were identified as abietane diterpenes, four diterpenes possessed a labdane skeleton, one was a steroid and two were phenolic compounds. Among these metabolites, one abietane diterpene and one phenolic compound were new natural products, five metabolites were reported for the first time from this species, while full assignment of the spectroscopic data for six diterpenes was reported.Furthermore, the chromatographic separation of P. pinea bark extract resulted in the isolation of a flavonoid derivative and a mixture of ferulic acid esters.The fresh needles of 46 pine species, including 37 and 17 taxa of the subgenera Pinus and Strobus, respectively, were subjected to hydrodistillation and the essential oils obtained were analyzed by means of GC–FID and GC–MS. Among the analyzed taxa, significant quantitative and qualitative differences were observed. Monoterpenes, sesquiterpenes and diterpenes dominated the pine foliage oils, with most of the oils characterized by the presence of the monoterpene hydrocarbons α- and β-pinene and the sesquiterpene hydrocarbon germacrene D. This is the rst report on the chemical composition of the essential oils of 21 pine taxa, including 15 taxa of the subgenus Pinus and six taxa of the subgenus Strobus.Στα πλαίσια της παρούσας διδακτορικής διατριβής μελετήθηκε η χημική σύσταση των οργανικών εκχυλισμάτων διαφορετικών φυτικών τμημάτων των ειδών Pinus heldreichii Christ., Pinus nigra subsp. nigra Arn. και Pinus pinea L. Επίσης, μελετήθηκε η χημική σύσταση των αιθερίων ελαίων από το φύλλωμα 46 ειδών του γένους Pinus (54 taxa) που συλλέχθηκαν είτε από φυσικές θέσεις είτε από βοτανικούς κήπους.Οι κώνοι του είδους P. heldreichii υποβλήθηκαν σε εξαντλητική εκχύλιση με οργανικούς διαλύτες και το εκχύλισμα που προέκυψε υποβλήθηκε σε σειρά χρωματογραφικών διαχωρισμών μέσω των οποίων απομονώθηκαν και ταυτοποιήθηκαν συνολικά 38 μεταβολίτες, συγκεκριμένα είκοσι διτερπένια αβιετικού τύπου, οκτώ με σκελετό πιμαρανίου, τέσσερα λαβδανικού τύπου, δύο μακροκυκλικά διτερπένια τύπου κεβρανίου, δύο διτερπένια παράγωγα ποδοκαρπενίου, ένα οξυγονωμένο σεσκιτερπένιο και ένα στεροειδές. Μεταξύ αυτών, τρείς διτερπενικοί μεταβολίτες (δύο πιμαρικού και ένας αβιετικού τύπου) ήταν νέα φυσικά προϊόντα, ένας μεταβολίτης σκελετού αβιετανίου αναφέρεται πρώτη φορά σε Γυμνόσπερμα, τέσσερεις μεταβολίτες αναφέρονται πρώτη φορά στο γένος Pinus, ενώ εικοσιεπτά απομονώνονται πρώτη φορά από το είδος P. heldreichii. Επιπλέον, για είκοσι γνωστούς μεταβολίτες πραγματοποιήθηκε πρώτη φορά η πλήρης απόδοση των φασματοσκοπικών τους δεδομένων.Τα βελονοειδή φύλλα του είδους P. nigra subsp. nigra εκχυλίστηκαν εξαντλητικά με οργανικούς διαλύτες και το εκχύλισμα που παραλήφθηκε υποβλήθηκε σε επιμερισμό μεσω υγρής-υγρής εκχύλισης και στη συνέχεια υποβλήθηκε σε σειρά χρωματογραφικών διαχωρισμών. Συνολικά απομονώθηκαν και ταυτοποιήθηκαν 12 μεταβολίτες και συγκεκριμένα τέσσερα διτερπένια λαβδανικού τύπου, πέντε διτερπένια με σκελετό αβιετανίου, ένα στεροειδές και δύο φαινολικά παράγωγα. Μεταξύ αυτών, δύο μεταβολίτες αποτελούν νέα φυσικά προϊόντα, πέντε αναφέρονται πρώτη φορά από το συγκεκριμένο είδος, ενώ για έξι γίνεται η πλήρης απόδοση των φασματοσκοπικών τους δεδομένων για πρώτη φορά.Επίσης, σε εξαντλητική εκχύλιση με οργανικούς διαλύτες υποβλήθηκε ο φλοιός του είδους P. pinea και το εκχύλισμα που παραλήφθηκε αναλύθηκε χρωματογραφικά οδηγώντας στην απομόνωση ενός φλαβονοειδούς και μίγματος λιπαρών εστέρων του φερουλικού οξέος.Νωπά βελονοειδή φύλλα από 37 taxa του υπογένους Pinus και 17 taxa του υπογένους Strobus υποβλήθηκαν σε υδραπόσταξη και τα αιθέρια έλαια που παραλήφθηκαν, αναλύθηκαν μέσω GC-FID και GC-MS. Μεταξύ των δειγμάτων παρατηρήθηκαν σημαντικές ποσοτικές, αλλά και ποιοτικές διαφορές. Μονοτερπένια, σεσκιτερπένια και διτερπένια κυριαρχούσαν στις αναλύσεις, με τους μονοτερπενικούς υδρογονάνθρακες α- και β-πινένιο και το σεσκιτερπένιο γερμακρένιο D να χαρακτηρίζουν την πλειοψηφία των δειγμάτων. Στην παρούσα μελέτη αναλύθηκε για πρώτη φορά το αιθέριο έλαιο των βελόνων εικοσιενός taxa, συμπεριλαμβανομένων δεκαπέντε του υπογένους Pinus και έξι του υπογένους Strobus

Antimicrobial Activity of the Essential Oil of Greek Endemic Stachys spruneri and its Main Component, Isoabienol

The essential oils of Stachys spruneri Boiss. (sample A and sample B) were analyzed by GC and GC-MS. (+)-Isoabienol was the dominant component (49.5 and 48.2%, respectively of the total oils) among seventy-two identified constituents. Isoabienol was separated, purified by preparative thin-slayer chromatography, and further identified by means of physicochemical and spectrometric analysis. The microbial growth inhibitory properties of the essential oil and its main metabolite, the labdane diterpene isoabienol, were determined using the broth microdilution method against eight laboratory strains of bacteria (Gram- positive: Staphylococcus aureus, S. epidermidis, Micrococcus luteus, Enterococcus faecalis, Bacillus subtilis, and Gram- negative: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and two strains of the yeast Candida albicans. Both essential oil and isoabienol showed considerable activity against all the microorganisms tested, with the isolated compound being most active

Composition and Antimicrobial Activity of the Essential Oils from Different Parts of Cachrys cristata DC. from Greece

Fresh leaves, stems and roots of Cachrys cristata, were subjected to hydrodistillation and the oils obtained were analyzed by means of GC and GC-MS. Sixty eight constituents were in total identified, with (Z)-beta-ocimene (44.2% and 30.5%) dominating in leaves and stems oils, while the major metabolite of the roots was found to be p-tolualdehyde (39.6%), followed by (Z)-beta-ocimene (15.2%). The microbial growth inhibitory properties of the essential oils were determined using the broth microdilution method against seven laboratory strains of bacteria - Gram positive: Staphylococcus aureus, S. epidermidis, Micrococcus luteus, Enterococcus faecalis and Gram negative: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and two strains of the yeast Candida albicans. The essential oils exhibited considerable activity against certain strains of the microorganisms tested, with the leaves oil demonstrating higher antimicrobial activity among the tested samples

Antimicrobial Activity and Essential Oil Composition of Five Sideritis taxa of Empedoclia and Hesiodia Sect. from Greece

Dried aerial parts of five taxa of Greek Sideritis were subjected to hydrodistillation and the oils obtained were analyzed by using GC and GC-MS. A total of 82 compounds were identified and the analysis showed important differences between the samples not only quantitatively but also qualitatively. The microbial growth inhibitory properties of the essential oils were determined using the broth microdilution method against eight laboratory strains of bacteria - Gram positive: Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, Enterococcus faecalis, Bacillus subtilis and Gram negative: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and two strains of the yeast Candida albicans. The tested essential oils exhibited considerable activity against certain strains of the microorganisms tested, with S. lanata oil presenting MIC values to S. aureus and M. luteus comparable to those of the reference antibiotics

Conium divaricatum Boiss. & Orph.

<i>2.1. Chemical composition of essential oils of C. divaricatum and</i> <i>C. maculatum</i> <p> The chemical composition of twenty-five <i>C. divaricatum</i> and ten <i>C. maculatum</i> essential oils obtained after hydrodistillation of separate plant tissues (infructescences, inflorescences, leaves, stems, roots) collected from either natural populations or cultivated plants was investigated using gas chromatography with flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) (Table 1).</p> <p> In total 68 components were identified in the <i>C. divaricatum</i> essential oils (Table 2). The chemical group that dominated the essential oils of <i>C</i>. <i>divaricatum</i> was that of oxygenated hydrocarbons, mainly represented by esters. Among them, one of the major constituents (<b>1</b> with RRI 1980; subsequently identified as 4 ′ -oxodecyl hexanoate) of the <i>C. divaricatum</i> essential oils (2.8%–88.4%), consistently abundant in stems’ essential oils, regardless of the collection date or site, could not be readily identified on the basis of its mass spectral data. Nonetheless, the highest percentage of compound <b>1</b> was detected in the essential oil of the infructescences collected from Mt Pateras in 2010. Furthermore, two more metabolites (<b>2</b> and <b>3</b> with RRI 2153 and 1767, respectively, subsequently identified as 4 ′ -oxododecyl hexanoate and 4 ′ -oxooctyl hexanoate) were also abundant in the majority of the investigated <i>C. divaricatum</i> samples. Moreover, sesquiterpenes and non-oxygenated hydrocarbons (alkanes and alkenes) were constantly present, but in lower ratios. The roots’ essential oils exhibited a different chemical profile compared to the aerial parts, as they were rich in sesquiterpenes, with <i>cis -α</i> -bergamotene and <i>β</i> -bisabolene being the major components. Additionally, elemicin and osthol were compounds detected exclusively in the essential oils of roots in significant percentages.</p> <p> According to the chemical analyses of the essential oils of <i>C. divaricatum</i> collected from Mt Imittos, hydrocarbon esters dominated the aerial parts’ essential oils, followed by sesquiterpenes, alkanes and alkenes, exhibiting a chemical profile that characterizes <i>C. divaricatum.</i> Hydrocarbon esters were also abundant in the essential oils of <i>C. divaricatum</i> aerial parts collected from Mt Pateras and Mt Parnon. However, the percentage of fatty acids was strikingly higher in the samples collected from Mt Pateras. In contrast to the consistently high content in hydrocarbon esters detected in the samples of <i>C. divaricatum</i> from Mt Imittos, Mt Pateras and Mt Parnon, the analysis of the essential oils from Erythres revealed a different chemical profile, as hydrocarbons were also in this case the most abundant chemical group, represented however mainly by alkanes, alkenes and ketones, whereas esters were detected in lower amounts. This variation in the chemical profile within the same species could be attributed to the different collection period and different vegetative stage, namely to a seasonal variation, since the samples from Erythres were collected in a more advanced fruiting stage, however lacking ripe fruits. Therefore, it can be concluded that hydrocarbon esters are either important for the plant defense or play a key role in reproduction, as they are found in abundance during the most important vegetative stage, the flowering stage. To the best of our knowledge this is the first report on the essential oil composition of <i>C. divaricatum.</i></p> <p> In the essential oils of <i>C. maculatum</i>, 67 compounds were identified in total (Table 3). The two investigated taxa showed significant differences in their chemical profile, since in the case of <i>C. maculatum</i> hydrocarbons were detected in lower amounts, and most importantly hydrocarbon esters that characterized <i>C. divaricatum</i> samples were either absent or detected only in traces.</p> <p> Many quantitative differences were also observed among the essential oils of <i>C. maculatum</i>, with the percentages of the main chemical groups showing a rather significant variation. (<i>Z</i>)-Falcarinol was the main constituent in the infructescences’ essential oils collected from the areas of Thiva and Avlona, while the inflorescences’ oil was characterized by the abundance of alkanes and alkenes, followed by sesquiterpenes. Sesquiterpene derivatives were the main chemical group in the leaves’ essential oil, with germacrene D being the most abundant representative. (<i>Z</i>)-Falcarinol, a natural pesticide, was also the most abundant metabolite in the essential oils of the stems and the roots. Furthermore, the chemical profile of cultivated samples showed some differences in comparison to wild grown plant specimens. In particular, the essential oil of the cultivated plant was characterized by an almost equal ratio of monoterpenes (21.9%) and sesquiterpenes (21.5%). The sample also presented a strikingly lower percentage of (<i>Z</i>)-falcarinol (3.4%), when compared to the essential oils obtained from wild grown plants.</p> <p> <b>Table 3</b></p> <p> Chemical composition of the essential oils a of <i>Conium maculatum</i>.</p> <p> (<i>continued on next page</i>)</p> <p> tr: detected in traces (<i><</i> 0.1%), -: not detected.</p> <p> a Numbering of samples according to Table 1.</p> <p> b Compounds listed in the order of elution on a HP-5MS column under the specified chromatographic conditions.</p> <p> c Relative retention indices calculated from the retention times of the compounds in relation to those of a series of <i>n</i> -alkanes (C –C) analyzed under the same 8 30 chromatographic conditions.</p> <p> d The base peak and molecular ion peak (when observed) in the mass spectrum of the unidentified compound are included in parentheses.</p> <p> e Including only the identified compounds.</p> <p> Our results are generally in accordance with the literature data reporting the chemical analysis of essential oils from Iranian and Serbian hemlock (Masoudi et al., 2006; Radulovic et al., 2008). According to Radulovic et al. (2008), the leaves’ and the inflorescences’ essential oils from Serbia are rich in sesquiterpenes (54.4% and 46.3%, respectively), with germacrene D being the most abundant metabolite. Sesquiterpenes are also the main chemical group in the essential oils of <i>C. maculatum</i> aerial parts, wild grown in Iran, with germacrene D being the most abundant representative (Masoudi et al., 2006). On the contrary, the essential oils of hemlock from Sicily showed different chemical profiles. In particular, the essential oil of the inflorescences was characterized by the presence of 1-butylpiperidine and myrcene in high abundance (26.4% and 24.0%, respectively), while (<i>E</i>)-caryophyllene dominated (54.8%) the essential oil of the leaves (Di Napoli et al., 2019).</p> <p> Comparison of the chemical profiles of the essential oils obtained from the different plant parts of <i>C. divaricatum</i> and <i>C. maculatum</i> revealed significant qualitative differences between the two taxa. In particular, (<i>Z</i>)-falcarinol was present in high abundance (18.1–81.1%) in all samples of <i>C. maculatum</i>, with the exception of the cultivated population M1, whereas it was completely absent from specimens of <i>C. divaricatum</i>. In contrast, all samples of <i>C. divaricatum</i> aerial parts, with the exception of samples D6 and D16 which were collected from Erythres in a more advanced fruiting stage, were characterized by the presence of compounds <b>1–3</b> in high abundance (50.5–95.0%).</p> <i>2.2. Isolation and structure elucidation of metabolites 1–3 from C.</i> <i>divaricatum</i> <p> Since the major constituents of the <i>C. divaricatum</i> essential oils could not be readily identified on the basis of their mass spectral data, chromatographic separations were undertaken in order to isolate and elucidate the chemical structures of compounds <b>1–3</b> (Fig. 1). Towards this, <i>C. divaricatum</i> stems collected from Mt Imittos were subjected to biphasic alkaline extraction and the organic phase was repeatedly fractionated to yield compounds <b>1–3</b> and 12-nonacosanone in pure form.</p> <p> Compound <b>1</b> was isolated as colourless oil. The molecular formula C 16 H 30 O 3 was deduced by EIMS data ([M] + observed at <i>m/z</i> 270). The 13 C NMR and HSQC-DEPT spectra revealed 16 carbon signals, including an ester and a ketone carbonyl at <i>δ</i> 173.9 and 211.1, respectively, one oxygenated sp 3 carbon (<i>δ</i> 64.1), and thirteen non-functionalized sp 3 carbons (<i>δ</i> 13.7–44.6), among which eleven methylenes and two methyls (Table 4). The 1 H NMR spectrum revealed two triplets ascribed to two aliphatic methyls at <i>δ</i> 0.83 (t, <i>J</i> = 6.9 Hz, H 3 -6) and 0.85 (t, <i>J</i> = 7.1 Hz, H 3 -1 ′), as well as two multiplets at <i>δ</i> 1.48–1.60 and <i>δ</i> 1.22–1.34, each assigned to four aliphatic methylenes. Three triplets were also observed at <i>δ</i> 2.23 (t, <i>J</i> =7.6 Hz, H 2 -2), 2.31 (t, <i>J</i> =7.1 Hz, H 2 -3 ′), and 2.33 (t, <i>J</i> = 7.0 Hz, H 2 -5 ′) attributed to three relatively deshielded methylenes, as well as a triplet at 3.99 ppm (t, <i>J</i> = 6.7 Hz, H 2 -10 ′) assigned to an oxygenated methylene (Table 4). Analysis of the HSQC-DEPT, HMBC and COSY spectra revealed the planar structure of compound 1 (Fig. 2). Specifically, the COSY correlations of H 3 -1’/H 2 -2 ′, H 2 - 2’/H 2 -3 ′, H 2 -5’/H 2 -6 ′, H 2 -6’/H 2 -7 ′, H 2 -7’/H 2 -8 ′, H 2 -8’/H 2 -9 ′, H 2 -9’/H 2 - 10 ′, H 2 -2/H 2 -3, H 2 -3/H 2 -4, H 2 -4/H 2 -5, and H 2 -5/H 3 -6 revealed three separate spin systems. Additionally, HMBC correlations of C-4 ′ with H 2 - 2 ′, H 2 -3 ′, H 2 -5 ′ and H 2 -6 ′ were observed, connecting the two separate spin systems. Moreover, HMBC correlations of C-1 with H 2 -2, H 2 -3 and H 2 -10 ′ were observed, concluding the structure of compound <b>1</b>, which was identified as 4 ′ -oxodecyl hexanoate, representing an undescribed natural product.</p> <p>nd: not detected.</p> <p> a Recorded at 400 MHz.</p> <p> b Recorded at 50.3 MHz.</p> <p> c Determined through HSQC-DEPT correlations.</p> <p> Compound <b>2</b>, isolated as colourless oil, displayed a molecular ion peak at <i>m</i> / <i>z</i> 298 (EIMS), corresponding to C 18 H 34 O 3. The spectroscopic characteristics of compound <b>2</b> (Table 4) revealed high similarity to those of <b>1</b>. Specifically, in the 1 H NMR spectrum two triplets were observed at <i>δ</i> 0.86 (t, <i>J</i> = 7.0 Hz, H 3 -6) and 0.89 (t, <i>J</i> = 7.2 Hz, H 3 -1 ′), assigned to two aliphatic methyls, and two multiplets observed at <i>δ</i> 1.50–1.62 and 1.23–1.37 were ascribed to four and six aliphatic methylenes, respectively. Moreover, three triplets resonating at <i>δ</i> 2.26 (t, <i>J</i> =7.5 Hz, H 2 -2), 2.35 (t, <i>J</i> =7.2 Hz, H 2 -3 ′), and 2.37 (t, <i>J</i> =7.2 Hz, H 2 -5 ′) were assigned to three relatively deshielded methylenes, while a triplet at 4.02 ppm (t, <i>J</i> = 6.7 Hz, H 2 -12 ′) was attributed to an oxygenated methylene. In a similar manner to <b>1</b>, analysis of the 2D NMR spectra led to the identification of compound <b>2</b> as 4 ′ -oxododecyl hexanoate, an undescribed natural product.</p> <p> Compound <b>3</b>, obtained as colourless oil, had the molecular formula C 14 H 26 O 3, as determined by EIMS data ([M] + observed at <i>m/z</i> 242). The spectroscopic data of compound <b>3</b> (Table 4) were rather similar to those of compounds <b>1</b> and <b>2</b>. Direct comparison of the 1 H NMR spectra of <b>1–3</b>, revealed that <b>3</b> contained only four methylenes in the middle spin system, in contrast to 1 and 2 containing six and eight methylenes, respectively. Thus, compound <b>3</b> was identified as an undescribed natural product, namely 4 ′ -oxooctyl hexanoate.</p> <i>2.3. Detection of alkaloids and determination of the total alkaloids</i> <i>content</i> <p> The genus <i>Conium</i> is characterized by the abundance of alkaloids. In order to confirm their presence in the investigated taxa, two extracts of different polarity, namely an organic extract resulting from maceration of the air-dried aerial parts in CH 2 Cl 2 /MeOH (1:1) containing less polar constituents and a hydromethanolic extract resulting from maceration of the air-dried aerial parts in MeOH/H 2 O (4:1) containing more polar constituents, were prepared from samples of both taxa and the presence of alkaloids was investigated employing specific reagents (Dragendorff, Wagner and Mayer) for the detection of alkaloids. All extracts upon addition of the reagents resulted in the formation of precipitate, confirming the presence of alkaloids in both taxa (Table 5).</p> <p> The total alkaloids content of both taxa was determined in triplicate in their hydroethanolic extracts, resulting from maceration of the air-dried samples in EtOH/H 2 O (3: 1), following a standard acid-base titrimetric procedure described in the literature (Mellon and Tigelaar, 1932). The total alkaloids content of <i>C. maculatum</i> was determined as 0.203 ± 0.096%, while for <i>C. divaricatum</i> it was measured at 0.060 ± 0.026%, expressed in both cases as coniine (Paech and Tracey, 1955). Regarding the total alkaloid content of <i>C. maculatum</i>, our results are in agreement with literature data (0.01–0.9% w/w for various extracts) (L´opez et al., 1999), while there are no previous reports for <i>C. divaricatum</i>.</p> <i>2.4. Evaluation of toxicity</i> <p> In this work, the brine shrimp lethality assay was used to assess the toxicity of the organic extracts and essential oils of both taxa, as well as of 4 ′ -oxodecyl hexanoate (<b>1</b>), the main component of the essential oil of <i>C. divaricatum</i>. The lethality exerted on the nauplii of the brine shrimp <i>Artemia salina</i> L. (Artemiidae) has been widely used in laboratory bioassays as a predictor of chemical toxicity on organisms in aquatic environments through the estimation of the medium lethal concentration (LC 50) (Meyer et al., 1982). The results of the evaluation of the toxicity on the brine shrimp after 24 h of incubation are presented in Table 6. The toxicity assessment against the crustacean <i>A. salina</i> did not reveal significant differences for the two investigated taxa, displaying moderate toxicity with LC 50 values between 0.1 and 0.5 mg /mL (Mitic´et al., 2021).</p> 3. Concluding remarks <p> The distinctive differences observed between the chemical profiles of the essential oils obtained from various plant parts of <i>C. divaricatum</i> and of <i>C. maculatum</i>, in conjunction with the morphological differences observed for the two taxa, strongly support that <i>C. divaricatum</i> should be classified as a separate species within the genus, as it was originally proposed by Boissier and Orphanides in 1856, and not as a synonym of</p> <p> +: moderate formation of precipitate; ++: intense formation of precipitate. <i>C. maculatum</i>, as it is found in several botanical sources. Since both species, forming rich populations in Attica and around Athens, exhibit similar levels of toxicity, Socrates may have been poisoned by either of the two.</p>Published as part of <i>Vlassi, Anthi, Koutsaviti, Aikaterini, Constantinidis, Theophanis, Ioannou, Efstathia & Tzakou, Olga, 2022, What Socrates drank? Comparative chemical investigation of two Greek Conium taxa exhibiting diverse chemical profiles, pp. 1-10 in Phytochemistry (113060) (113060) 195</i> on pages 2-7, DOI: 10.1016/j.phytochem.2021.113060, <a href="http://zenodo.org/record/8234268">http://zenodo.org/record/8234268</a&gt

Diversity of Volatile Compounds in the <i>Inula candida</i> / <i>I. verbascifolia</i> Group (Asteraceae-Inuleae) and Its Impact on Species Delimitation

The members of the Inula candida / I. verbascifolia group are perennial and chasmophytic plants attributed to four species and eleven entities at subspecific or varietal level. They are mostly confined to Greece. Volatile compounds of above-ground flowering parts of twenty-three populations covering ten taxonomic entities were obtained after hydrodistillation and analyzed by gas chromatography and mass spectrometry (GC-MS). In most cases, the total percentage of identified constituents reached 89% or above. Seventy-two components were identified. Oxygenated sesquiterpenes prevailed in the I. candida subgroup (47.3–71.5%), with epi-α-cadinol present in all members (13.4–42.7%) but rarely found in the I. verbascifolia subgroup. Considerable amounts of the hydrocarbon aldehydes decanal, undecanal and particularly tridecanal (0.5–35.0%, rarely absent) were predominately found in the I. verbascifolia members but were mostly absent in the I. candida subgroup. Isoalantolactone (12.4–49.5%), identified only in I. subfloccosa, and γ-(Z)-curcumen-12-ol, found only in I. candida subsp. limonella (22.6–42.1%), may serve as chemotaxonomic markers. Two different chemotypes can be distinguished within I. verbascifolia subsp. aschersoniana: a trans-muurola-4(14),5-diene-, γ-cadinene-rich chemotype from north-eastern Greece and a 1-epi-cubenol-, tridecanal-rich chemotype from central Greece. Different statistical algorithms were used to interpret the chemical diversity and identify the most appropriate number of clusters for the taxa. Cluster analyses indicated that the optimum number of clusters that best explain the metabolomic variability of the taxa is two. The degree of membership for each population based on the fuzzy k-means algorithm supported the I. verbascifolia subsp. aschersoniana samples within the I. candida subgroup, whereas I. subfloccosa may belong to any of the two clusters formed, although it also appears to have some unique characteristics

Diversity of Volatile Compounds in the Inula candida / I. verbascifolia Group (Asteraceae-Inuleae) and Its Impact on Species Delimitation

The members of the Inula candida / I. verbascifolia group are perennial and chasmophytic plants attributed to four species and eleven entities at subspecific or varietal level. They are mostly confined to Greece. Volatile compounds of above-ground flowering parts of twenty-three populations covering ten taxonomic entities were obtained after hydrodistillation and analyzed by gas chromatography and mass spectrometry (GC-MS). In most cases, the total percentage of identified constituents reached 89% or above. Seventy-two components were identified. Oxygenated sesquiterpenes prevailed in the I. candida subgroup (47.3&ndash;71.5%), with epi-&alpha;-cadinol present in all members (13.4&ndash;42.7%) but rarely found in the I. verbascifolia subgroup. Considerable amounts of the hydrocarbon aldehydes decanal, undecanal and particularly tridecanal (0.5&ndash;35.0%, rarely absent) were predominately found in the I. verbascifolia members but were mostly absent in the I. candida subgroup. Isoalantolactone (12.4&ndash;49.5%), identified only in I. subfloccosa, and &gamma;-(Z)-curcumen-12-ol, found only in I. candida subsp. limonella (22.6&ndash;42.1%), may serve as chemotaxonomic markers. Two different chemotypes can be distinguished within I. verbascifolia subsp. aschersoniana: a trans-muurola-4(14),5-diene-, &gamma;-cadinene-rich chemotype from north-eastern Greece and a 1-epi-cubenol-, tridecanal-rich chemotype from central Greece. Different statistical algorithms were used to interpret the chemical diversity and identify the most appropriate number of clusters for the taxa. Cluster analyses indicated that the optimum number of clusters that best explain the metabolomic variability of the taxa is two. The degree of membership for each population based on the fuzzy k-means algorithm supported the I. verbascifolia subsp. aschersoniana samples within the I. candida subgroup, whereas I. subfloccosa may belong to any of the two clusters formed, although it also appears to have some unique characteristics