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15 pages, 2140 KiB

Open AccessFeature PaperArticle

DFT Calculations of ¹H- and ¹³C-NMR Chemical Shifts of Geometric Isomers of Conjugated Linoleic Acid (18:2 ω-7) and Model Compounds in Solution

by Themistoklis Venianakis, Christina Oikonomaki, Michael G. Siskos, Panayiotis C. Varras, Alexandra Primikyri, Eleni Alexandri and Ioannis P. Gerothanassis

Molecules 2020, 25(16), 3660; https://doi.org/10.3390/molecules25163660 - 11 Aug 2020

Cited by 12 | Viewed by 4321

Abstract

A density functional theory (DFT) study of the ¹H- and ¹³C-NMR chemical shifts of the geometric isomers of 18:2 ω-7 conjugated linoleic acid (CLA) and nine model compounds is presented, using five functionals and two basis sets. The results are compared [...] Read more.

A density functional theory (DFT) study of the ¹H- and ¹³C-NMR chemical shifts of the geometric isomers of 18:2 ω-7 conjugated linoleic acid (CLA) and nine model compounds is presented, using five functionals and two basis sets. The results are compared with available experimental data from solution high resolution nuclear magnetic resonance (NMR). The experimental ¹H chemical shifts exhibit highly diagnostic resonances due to the olefinic protons of the conjugated double bonds. The “inside” olefinic protons of the conjugated double bonds are deshielded than those of the “outside” protons. Furthermore, in the cis/trans isomers, the signals of the cis bonds are more deshielded than those of the trans bonds. These regularities of the experimental ¹H chemical shifts of the olefinic protons of the conjugated double bonds are reproduced very accurately for the lowest energy DFT optimized single conformer, for all functionals and basis sets used. The other low energy conformers have negligible effects on the computational ¹H-NMR chemical shifts. We conclude that proton NMR chemical shifts are more discriminating than carbon, and DFT calculations can provide a valuable tool for (i) the accurate prediction of ¹H-NMR chemical shifts even with less demanding functionals and basis sets; (ii) the unequivocal identification of geometric isomerism of CLAs that occur in nature, and (iii) to derive high resolution structures in solution. Full article

(This article belongs to the Special Issue NMR Spectroscopy as a Structural and Analytical Probe of Natural Products: Emerging Advancements and Future Insights)

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11 pages, 2563 KiB

Open AccessFeature PaperArticle

NMR-Based Chemical Profiling, Isolation and Evaluation of the Cytotoxic Potential of the Diterpenoid Siderol from Cultivated Sideritis euboea Heldr.

by Ekaterina-Michaela Tomou, Maria V. Chatziathanasiadou, Paschalina Chatzopoulou, Andreas G. Tzakos and Helen Skaltsa

Molecules 2020, 25(10), 2382; https://doi.org/10.3390/molecules25102382 - 20 May 2020

Cited by 20 | Viewed by 6613

Abstract

Diterpenes are characteristic compounds from the genus Sideritis L., possessing an array of biological activities. Siderol is the main constituent of the ent-kaurene diterpenes in Sideritis species. In order to isolate the specific compound and evaluate for the first time its cytotoxic [...] Read more.

Diterpenes are characteristic compounds from the genus Sideritis L., possessing an array of biological activities. Siderol is the main constituent of the ent-kaurene diterpenes in Sideritis species. In order to isolate the specific compound and evaluate for the first time its cytotoxic activity, we explored the dichloromethane extract of cultivated Sideritis euboea Heldr. To track the specific natural bioactive agent, we applied NMR spectroscopy to the crude plant extract, since NMR can serve as a powerful and rapid tool both to navigate the targeted isolation process of bioactive constituents, and to also reveal the identity of bioactive components. Along these lines, from the rapid 1D ¹H NMR spectrum of the total crude plant extract, we were able to determine the characteristic proton NMR signals of siderol. Furthermore, with the same NMR spectrum, we were able to categorize several secondary metabolites into chemical groups as a control of the isolation process. Therefore, this non-polar extract was explored, for the first time, revealing eleven compounds—one fatty acid ester; 2-(p-hydroxyphenyl)ethylstearate (1), three phytosterols; β-sitosterol (2), stigmasterol (3), and campesterol (4); one triterpenoid; ursolic acid (5), four diterpenoids; siderol (6), eubol (7), eubotriol (8), 7-epicandicandiol (9) and two flavonoids; xanthomicrol (10) and penduletin (11). The main isolated constituent was siderol. The antiproliferative potential of siderol was evaluated, using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay, on three human cancer cell lines DLD1, HeLa, and A549, where the IC₅₀ values were estimated at 26.4 ± 3.7, 44.7 ± 7.2, and 46.0 ± 4.9 μΜ, respectively. The most potent activity was recorded in the human colon cancer cell line DLD1, where siderol exhibited the lowest IC₅₀. Our study unveiled the beneficial potential of siderol as a remarkable cytotoxic agent and the significant contribution of NMR spectroscopy towards the isolation and identification of this potent anticancer agent. Full article

(This article belongs to the Special Issue NMR Spectroscopy as a Structural and Analytical Probe of Natural Products: Emerging Advancements and Future Insights)

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15 pages, 1776 KiB

Open AccessArticle

NMR Relaxation Measurements on Complex Samples Based on Real-Time Pure Shift Techniques

by Xiaoqing Lin, Haolin Zhan, Hong Li, Yuqing Huang and Zhong Chen

Molecules 2020, 25(3), 473; https://doi.org/10.3390/molecules25030473 - 22 Jan 2020

Cited by 5 | Viewed by 4709

Abstract

Longitudinal spin-lattice relaxation (T₁) and transverse spin-spin relaxation (T₂) reveal valuable information for studying molecular dynamics in NMR applications. Accurate relaxation measurements from conventional 1D proton spectra are generally subject to challenges of spectral congestion caused by [...] Read more.

Longitudinal spin-lattice relaxation (T₁) and transverse spin-spin relaxation (T₂) reveal valuable information for studying molecular dynamics in NMR applications. Accurate relaxation measurements from conventional 1D proton spectra are generally subject to challenges of spectral congestion caused by J coupling splittings and spectral line broadenings due to magnetic field inhomogeneity. Here, we present an NMR relaxation method based on real-time pure shift techniques to overcome these two challenges and achieve accurate measurements of T₁ and T₂ relaxation times from complex samples that contain crowded NMR resonances even under inhomogeneous magnetic fields. Both theoretical analyses and detailed experiments are performed to demonstrate the effectiveness and ability of the proposed method for accurate relaxation measurements on complex samples and its practicability to non-ideal magnetic field conditions. Full article

(This article belongs to the Special Issue NMR Spectroscopy as a Structural and Analytical Probe of Natural Products: Emerging Advancements and Future Insights)

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14 pages, 1284 KiB

Open AccessArticle

Identification and Quantitation of Ursolic and Oleanolic Acids in Ilex aquifolium L. Leaf Extracts Using ¹³C and ¹H-NMR Spectroscopy

by Doreen Palu, Ange Bighelli, Joseph Casanova and Mathieu Paoli

Molecules 2019, 24(23), 4413; https://doi.org/10.3390/molecules24234413 - 03 Dec 2019

Cited by 15 | Viewed by 4970

Abstract

Leaves of Ilex aquifolium L. have been used for their therapeutic properties. In previous studies, components contained in the leaves were first isolated by various chromatographic techniques. Then, quantitation of oleanolic and ursolic acids, which are responsible for the biological and therapeutic activities [...] Read more.

Leaves of Ilex aquifolium L. have been used for their therapeutic properties. In previous studies, components contained in the leaves were first isolated by various chromatographic techniques. Then, quantitation of oleanolic and ursolic acids, which are responsible for the biological and therapeutic activities of the plant, was performed by HPLC, HPTLC, and somewhat by GC-MS. Our objective was to develop a simple method that allows the identification of compounds contained in the leaves of Corsican I. aquifolium and to quantify ursolic and oleanolic acids. Leaves were successively extracted with hexane and dichloromethane. The extracts were chromatographed on silica gel and the fractions of column chromatography submitted to ¹³C-NMR analysis, following a computerized method developed in the laboratory. ¹³C-NMR allowed the identification of various triterpenes including ursolic acid and oleanolic acid. Quantitation of both acids was achieved, for the first time, by ¹H-NMR after validation of the method (accuracy, precision, linearity, limit of detection and limit of quantitation). Ursolic and oleanolic acids accounted for 55.3% and 20.8% of the dichloromethane extract, respectively. This represents 1.3% and 0.5% of the mass of dried leaves. ¹H-NMR spectroscopy appeared as a powerful tool for a rapid quantitation of biologically active compounds from I. aquifolium. Full article

(This article belongs to the Special Issue NMR Spectroscopy as a Structural and Analytical Probe of Natural Products: Emerging Advancements and Future Insights)

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19 pages, 4797 KiB

Open AccessArticle

Solvent-Dependent Structures of Natural Products Based on the Combined Use of DFT Calculations and ¹H-NMR Chemical Shifts

by Saima H. Mari, Panayiotis C. Varras, Atia-tul-Wahab, Iqbal M. Choudhary, Michael G. Siskos and Ioannis P. Gerothanassis

Molecules 2019, 24(12), 2290; https://doi.org/10.3390/molecules24122290 - 20 Jun 2019

Cited by 24 | Viewed by 3791

Abstract

Detailed solvent and temperature effects on the experimental ¹H-NMR chemical shifts of the natural products chrysophanol (1), emodin (2), and physcion (3) are reported for the investigation of hydrogen bonding, solvation and conformation effects in solution. [...] Read more.

Detailed solvent and temperature effects on the experimental ¹H-NMR chemical shifts of the natural products chrysophanol (1), emodin (2), and physcion (3) are reported for the investigation of hydrogen bonding, solvation and conformation effects in solution. Very small chemical shift of │Δδ│ < 0.3 ppm and temperature coefficients │Δδ/ΔΤ│ ≤ 2.1 ppb/K were observed in DMSO-d₆, acetone-d₆ and CDCl₃ for the C(1)–OH and C(8)–OH groups which demonstrate that they are involved in a strong intramolecular hydrogen bond. On the contrary, large chemical shift differences of 5.23 ppm at 298 K and Δδ/ΔΤ values in the range of −5.3 to −19.1 ppb/K between DMSO-d₆ and CDCl₃ were observed for the C(3)–OH group which demonstrate that the solvation state of the hydroxyl proton is a key factor in determining the value of the chemical shift. DFT calculated ¹H-NMR chemical shifts, using various functionals and basis sets, the conductor-like polarizable continuum model, and discrete solute-solvent hydrogen bond interactions, were found to be in very good agreement with the experimental ¹H-NMR chemical shifts even with computationally less demanding level of theory. The ¹H-NMR chemical shifts of the OH groups which participate in intramolecular hydrogen bond are dependent on the conformational state of substituents and, thus, can be used as molecular sensors in conformational analysis. When the X-ray structures of chrysophanol (1), emodin (2), and physcion (3) were used as input geometries, the DFT-calculated ¹H-NMR chemical shifts were shown to strongly deviate from the experimental chemical shifts and no functional dependence could be obtained. Comparison of the most important intramolecular data of the DFT calculated and the X-ray structures demonstrate significant differences for distances involving hydrogen atoms, most notably the intramolecular hydrogen bond O–H and C–H bond lengths which deviate by 0.152 tο 0.132 Å and 0.133 to 0.100 Å, respectively, in the two structural methods. Further differences were observed in the conformation of –OH, –CH₃, and –OCH₃ substituents. Full article

(This article belongs to the Special Issue NMR Spectroscopy as a Structural and Analytical Probe of Natural Products: Emerging Advancements and Future Insights)

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12 pages, 1225 KiB

Open AccessFeature PaperArticle

Use of ¹³C-qNMR Spectroscopy for the Analysis of Non-Psychoactive Cannabinoids in Fibre-Type Cannabis sativa L. (Hemp)

by Lucia Marchetti, Virginia Brighenti, Maria Cecilia Rossi, Johanna Sperlea, Federica Pellati and Davide Bertelli

Molecules 2019, 24(6), 1138; https://doi.org/10.3390/molecules24061138 - 22 Mar 2019

Cited by 20 | Viewed by 6617

Abstract

Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The discovery of the presence of many biologically-active metabolites (cannabinoids) in fibre-type Cannabis (hemp) has recently given rise to the valorisation of this variety. In this context, the present study was [...] Read more.

Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The discovery of the presence of many biologically-active metabolites (cannabinoids) in fibre-type Cannabis (hemp) has recently given rise to the valorisation of this variety. In this context, the present study was aimed at the multi-component analysis and determination of the main non-psychoactive cannabinoids (cannabidiol, cannabidiolic acid, cannabigerol and cannabigerolic acid) in female inflorescences of different hemp varieties by means of ¹³C quantitative nuclear magnetic resonance spectroscopy (qNMR). The method proposed here for the first time for the determination of cannabinoids provided reliable results in a competitive time with respect to the more consolidated HPLC technique. In fact, it gave sufficiently precise and sensitive results, with LOQ values lower than 750 μg/mL, which is easily achievable with concentrated extracts, without affecting the quality of ¹³C-qNMR spectra. In conclusion, this method can be considered as a promising and appropriate tool for the comprehensive chemical analysis of bioactive cannabinoids in hemp and other derived products in order to ensure their quality, efficacy and safety. Full article

(This article belongs to the Special Issue NMR Spectroscopy as a Structural and Analytical Probe of Natural Products: Emerging Advancements and Future Insights)

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18 pages, 2713 KiB

Open AccessFeature PaperArticle

NMR-Based Μetabolomics of the Lipid Fraction of Organic and Conventional Bovine Milk

by Constantinos G. Tsiafoulis, Christina Papaemmanouil, Dimitrios Alivertis, Ouranios Tzamaloukas, Despoina Miltiadou, Stéphane Balayssac, Myriam Malet-Martino and Ioannis P. Gerothanassis

Molecules 2019, 24(6), 1067; https://doi.org/10.3390/molecules24061067 - 18 Mar 2019

Cited by 35 | Viewed by 4388

Abstract

Origin and quality identification in dairy products is an important issue and also an extremely challenging and complex experimental procedure. The objective of the present work was to compare the metabolite profile of the lipid fraction of organic and conventional bovine milk using [...] Read more.

Origin and quality identification in dairy products is an important issue and also an extremely challenging and complex experimental procedure. The objective of the present work was to compare the metabolite profile of the lipid fraction of organic and conventional bovine milk using NMR metabolomics analysis. ¹H-NMR and 1D TOCSY NMR methods of analysis were performed on extracted lipid fraction of lyophilized milk. For this purpose, 14 organic and 16 conventional retail milk samples were collected monthly, and 64 bulk-tank (58 conventional and 6 organics) milk samples were collected over a 14-month longitudinal study in Cyprus. Data were treated with multivariate methods (PCA, PLS-DA). Minor components were identified and quantified, and modification of the currently used equations is proposed. A significantly increased % content of conjugated (9-cis, 11-trans)18:2 linoleic acid (CLA), α-linolenic acid, linoleic acid, allylic protons and total unsaturated fatty acids (UFA) and decreased % content for caproleic acid were observed in the organic samples compared to the conventional ones. The present work confirms that lipid profile is affected by contrasting management system (organic vs. conventional), and supports the potential of NMR-based metabolomics for the rapid analysis and authentication of the milk from its lipid profile. Full article

(This article belongs to the Special Issue NMR Spectroscopy as a Structural and Analytical Probe of Natural Products: Emerging Advancements and Future Insights)

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16 pages, 3088 KiB

Open AccessArticle

Response of Bioactive Metabolite and Biosynthesis Related Genes to Methyl Jasmonate Elicitation in Codonopsis pilosula

by Jiao-jiao Ji, Qi Feng, Hai-feng Sun, Xue-jun Zhang, Xiao-xiao Li, Jian-kuan Li and Jian-ping Gao

Molecules 2019, 24(3), 533; https://doi.org/10.3390/molecules24030533 - 01 Feb 2019

Cited by 32 | Viewed by 3701

Abstract

Bioactive metabolites in Codonopsis pilosula are of particular interest as an immunostimulant. Methyl jasmonate (MeJA) plays an important role in the elicitation of metabolite biosynthesis. Here, we explored the response of metabolites to MeJA elicitation in C. pilosula adventitious roots and multiple shoots. [...] Read more.

Bioactive metabolites in Codonopsis pilosula are of particular interest as an immunostimulant. Methyl jasmonate (MeJA) plays an important role in the elicitation of metabolite biosynthesis. Here, we explored the response of metabolites to MeJA elicitation in C. pilosula adventitious roots and multiple shoots. The results showed that the biomass, polysaccharide, and lobetyolin content of adventitious roots exhibited the highest increases with 100 µmol·L⁻¹ MeJA at the 16th day of subculture, whereas the atractylenolide III (a terpenoid) content increased extremely with 50 µmol·L⁻¹ MeJA treatment at the 7th day of subculture. In addition, the biomass and lobetyolin content significantly increased at the 4th day after treatment. Similarly, the polysaccharide and lobetyolin content increased in multiple shoots. Further identification of different metabolites responding to MeJA by ¹H-NMR showed an extremely significant increase of the lobetyolinin level, which coincided with lobetyolin. Accordingly, the precursor, fatty acids, showed a highly significant decrease in their levels. Furthermore, a significant increase in β-d-fructose-butanol glycoside was detected, which was accompanied by a decrease in the sucrose level. Accordingly, the enzyme genes responsible for terpenoid and carbohydrate biosynthesis, CpUGPase, and CpPMK, were up regulated. In conclusion, MeJA promoted culture growth and accelerated bioactive metabolite accumulation by regulating the expression of the metabolite biosynthesis related genes, CpUGPase and CpPMK in C. pilosula. Full article

(This article belongs to the Special Issue NMR Spectroscopy as a Structural and Analytical Probe of Natural Products: Emerging Advancements and Future Insights)

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