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Is galvinoxyl antioxidant assay possible using NMR spectroscopy?

Is galvinoxyl antioxidant assay possible using NMR spectroscopy?



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I would like to perform an antioxidant assay using the galvinoxyl protocol. The protocol states that we need EPR spectroscopy, but only NMR spectroscopy is available at my institution. Is there an alternate way where I perform a galvinoxyl assay using NMR spectroscopy.


Galvinoxyl is a free radical, which means it has an unpaired electron that can be detected with EPR. Antioxidants reduce the radical, which is then not detectable in the EPR anymore as it doesn't have an unpaired electron. What you need the EPR for in the assay is to determine how much of the galvinoxyl is still a radical.

Paramagnetic molecules like galvinoxyl are problematic for NMR, they bleach signals in close proximity to the radical and cause line-broadening. You still might be able to quantify the amount of oxidized and reduced galvinoxyl, but I'm not sure how reliable this would be. The oxidized galvinoxyl would probably be not visible in the NMR, while the reduced version would give a signal. You'd have to measure a few spectra to see if you can determine the percentage of reduced galvinoxyl from the NMR spectra.

EPR is the far easier method here, while I don't think doing this with NMR is impossible, there are quite a few ways it could be inaccurate. I'd strongly recommend to either use an EPR machine somewhere else, or use an entirely different assay that doesn't require such specialized equipment. I have no experience with antioxidation assays, but I'd guess that some exist that don't require EPR.


Is galvinoxyl antioxidant assay possible using NMR spectroscopy? - Biology

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Chapter 13 - Brief review on applications of continuous-wave electron paramagnetic resonance spectroscopy in natural product free radical research

Electron paramagnetic resonance (EPR) spectroscopy, also known as electron spin resonance (ESR) is a technique that detects paramagnetic atoms and molecules containing unpaired electrons. Unpaired electrons occur in free radicals and many transition metals complexes. It is a highly sensitive, versatile and specific technique that enables static and dynamic studies of materials, chemical samples, and biological systems. EPR's specificity to paramagnetic species makes it valuable to research and development in biology and medicine and physical sciences, as well as pharmaceutical and food industries. EPR has many applications in natural product research. Using EPR it is possible to detect antioxidant capacity to quench free radicals. For example, radical scavenging activity of silymarin and its major flavonolignans components as well as white tea extracts and its major catechin components were measured using this technique. EPR allowed the identification of slow-rate, intermediate-rate and fast-rate antioxidants in differently colored lettuce cultivars. In addition, antioxidant capacity of 21 kinds of fruits including strawberry, mulberry, lemon, and banana was measured using EPR and UV–vis spectroscopy and the two techniques were found highly correlated. EPR spectroscopy was used for studying the antioxidant activity of lemon balm, eyebright, St. John's Wort and sage.

EPR spectroscopy has useful applications in pharmaceutical and food industry involving natural products. Heat, light, oxygen, moisture, sterilization processes, impurities, and excipient interactions are some of the factors that can compromise natural products stability. If natural products are involved in manufacturing of pharmaceuticals, these factors may cause degradation of the active pharmaceutical ingredients, excipients, or formulations resulting in loss of product potency or toxic by-product generation. Degradation processes quite often involve free radicals that are responsible for the majority of the damage that occurs in drug products. For example, oxidative stability of olive oil was studied using EPR and correlated to its content of polyphenols and tocopherols. EPR finds application in detecting free radicals in plants induced by irradiation used for decontamination as alternative to heat and fumigation. The versatility of EPR technique opens future perspectives for more applications in elucidating metabolism of secondary metabolites involving paramagnetic species.


Abstract

(+)-Catechin, ethyl gallate, ascorbic acid, and α-tocopherol were reacted with 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the reaction mixtures were subjected to 13 C-nuclear magnetic resonance (NMR) analyses to clarify the molecular mechanisms of the antioxidative and radical-scavenging activities of each antioxidant. When ascorbic acid was reacted with DPPH, it was oxidized to dehydroascorbic acid by DPPH. When a mixture of ascorbic acid and (+)-catechin was reacted with DPPH, ascorbic acid scavenged DPPH radical faster than (+)-catechin. Ascorbic acid also scavenged DPPH radical faster than ethyl gallate and α-tocopherol. When (+)-catechin was reacted with DPPH, the B-ring of (+)-catechin changed to an o-quinone structure. However, it was reduced to (+)-catechin by ethyl gallate or α-tocopherol. α-Tocopherol and ethyl gallate had almost identical antioxidative activities. Therefore, the order of radical-scavenging ability (speed) suggested by our 13 C NMR study was as follows: ascorbic acid > α-tocopherol = ethyl gallate > (+)-catechin.

Keywords: Antioxidation mechanism NMR DPPH catechins antioxidants

Author to whom correspondence should be addressed (telephone +81-547-45-4950 fax +81-547-46-2169 e-mail [email protected]).


Materials and Methods

Plant Material and Preparation of Extracts

The plant material for extraction was collected in the area of Turkey during summer 2019 (H. neurocalycinum: Hadim village, Dedemli Valley, 3,140 m, Konya H. triquetrifolium: Anamur village, the ancient city of Anemurium, 5 m, Mersin). Taxonomic identification was performed by the botanist Dr. Evren Yıldıztugay (Selcuk University, Department of Biotechnology, Konya, Turkey) and one voucher specimen for each species (voucher ID numbers: EY-3110 for H. neurocalycinum and EY-3072 for H. triquetrifolium) was deposited at the herbarium of Selcuk University. The aerial parts (flowers, leaves and stem as mix) and roots were carefully separated. Then, plant materials were dried in a shaded and well-ventilated environment. After drying (about 10ꃚys), plant materials were powdered using a laboratory mill. Powdered plant materials were stored in a dark and cool place and they were kept away from sunlight.

In the study, maceration was preferred to obtain methanol extract. Maceration could be useful to extract thermolabile compounds and this method could be easily performed in further applications. Briefly, powdered plant samples (5 g) were stirred with 100 ml of methanol for 24 h at room temperature. Afterwards, the mixture was filtered and the solvent was evaporated by using rotary-evaporator. Infusion was selected for water extracts. Briefly, the material (5 g) was kept in boiled water (100 ml) for 15 min, then the extract was filtered and lyophilized. Obtained dry extracts were stored at 4ଌ (Etienne et al., 2021 Sinan et al., 2021).

Determination of Total Phenolic and Total Flavonoid Contents

Spectrophotometric methods were used to determine total phenolic and flavonoid contents, as already reported in earlier papers. Standard equivalents (gallic acid equivalent: GAE, for phenolics rutin equivalent: RE, for flavonoids) were used to explain the contents in the plant extracts (Slinkard and Singleton, 1977 Zengin et al., 2016).

Phytochemical Investigations

For the preliminary NMR analyses, a sample of H. triquetifolium methanol extract was dissolved in methanol/water (50%) mixture (22.5 mg/ml) and the solution was loaded on a Bondelute C-18 solid phase extraction (SPE) cartridge (3 ml). Cartridge was washed with water (2 column volumes), then compounds were eluted using methanol/water (2 volumes) and methanol (2 volumes).

NMR Spectroscopy

NMR spectra were recorded at 600 MHz on Bruker Avance NEO spectrometer equipped with a Cryo probe Prodigy TCI 5 mmm. All experiments were performed at 298 K. COSY, TOCSY, edited-HSQC, HMBC spectra were obtained using gradient selected pulse sequences. The spectral widths were 7,000 and 25,000 Hz for the 1 H- and 13 C-dimensions, respectively. The number of collected complex points was 1,024 for 1 H-dimension with a recycle delay of 1.5 s. TOCSY experiments were acquired with 16 transients, 512 increments in second dimension and a 70 ms of spin lock period. Heteronuclear spectra were acquired with 64� transients, and 140� time increments in 13 C-dimension. HSQC experiments used a one-bond carbon-proton coupling constant of 145 Hz, HMBC experiments used a long-range carbon-proton coupling constant of 8 Hz. 2D spectra were processed (software Topspin 4.0.6, Bruker BioSpin) using zero filling to 1,024 in F1 dimension, squared sine-bell apodization in both dimensions, prior to Fourier transformations.

LC-DAD-MS n (Ion Trap) and UPLC-QTOF Analyses

LC-DAD-MS n analyses were obtained using an Agilent LC system (Series 1260) equipped with DAD, autosampler and column oven. After the chromatographic column, a “T” connection splitted the flow equally to DAD and MS. As mass spectrometer, a Varian MS 500 Ion trap equipped with Electrospray Ion Source (ESI) was used, working in negative ion mode and acquiring the data in the m/z range 100𠄲,000. Fragmentation of most intense ion species was obtained using the turbo data depending scanning (tdds ® ) function of the instrument. Parameters were as follows: spray shield, 600 V nebulizer pressure, 25 psi drying gas pressure, 15 psi capillary voltage, 80 V RF loading, 80% needle voltage, 4,500 V. An Agilent XDB C-18 column (3.0 × 150 mm, 3.5 µm) was used as stationary phase. Solvents were: 1% formic acid in water A), acetonitrile B) and methanol C). Gradient was as follows: 0 min, 98% A and 2% B isocratic up to 5 min 25 min, 80% A, 10% B, and 10% C 40 min, 60% A, 30% B, 10% C 45 min, 20% A, 70% B, and 10% C isocratic up to 60 min. The flow rate was 400 ml/min.

As reference compounds for quantitative analyses, chlorogenic acid, gallic acid, epicatechin, quercetin-3-glucoside, quercetin, hyperoside, rutin, hypericin and hyperforin were used, and calibration curves were built. Chlorogenic acid solutions were used for quantification of hydoxycinnamic derivatives at 330 nm, and the calibration curve was y = 165.6x − 382.1 (R = 0.99991). For the quantification of small phenolics, catechin and procyanidin derivatives, gallic acid and epicatechin solutions were used and analyzed at 280 nm. Calibration curves were y = 122.2x + 16.0 (R = 1) and y = 27.8x + 111.6 (R = 0.9908), respectively. Quercetin, quercetin-3-glucoside, hyperoside and rutin solutions were used for the quantification of flavonoid and flavonoid glycosides, and they were analyzed at 280 nm. Calibration curves were y = 80.9x – 74.4 (R = 0.9999), y = 39.3x + 227.1 (R = 0.9889), y = 89.9x + 417.9 (R = 0.9964) and y = 39.2x + 19.6 (R = 0.9996), respectively. Naphthodianthrone derivatives were quantified with hypericin solutions at 590 nm, and the calibration curve was y = 266x + 8,199 (R = 0.9988). Quantification of phloroglucinols was obtained using MS. Hyperforin solutions were used, and the calibration curve was y = 5.58e ʵ x + 1.14e ʷ (R = 0.9999). Solutions were prepared in the range of 100𠄰.1 μg/ml.

Identification of compounds was obtained comparing the MS fragmentation spectra with the literature, and MS and retention times (R.T.) with those of available standard compounds.

Accurate m/z values were obtained using a Waters Acquity UPLC system coupled to a Waters Xevo G2 QTOF MS detector, operating in ESI (-) mode. For chromatographic separation, an Agilent Eclipse plus C18 column (2.1 × 50 mm, 1.8 µm) was used as stationary phase, and a gradient mixture of methanol 1) and 0.1% formic acid in water 2) as mobile phase. The gradient was: 0 min, 2% A 0.75 min, 2% A 11 min, 100% A 13.5 min, 100% A 14 min, 2% A and isocratic up to 15 min. Flow rate was 0.4 ml/min. MS parameters were as follows: sampling cone voltage, 40 V source offset, 80 V capillary voltage, 3,500 V nebulizer gas (N2) flow rate, 800 L/h desolvation temperature, 450ଌ. The mass accuracy and reproducibility were maintained by infusing lockmass (leucine-enkephalin [M–H] − = 554.2620 m/z) thorough Lockspray at a flow rate of 20 μl/min. Centroided data were collected in the m/z range 50𠄱,200, and the m/z values were automatically corrected during acquisition using lockmass.

Determination of Antioxidant and Enzyme Inhibitory Effects

Different protocols were performed to explain the antioxidant properties of Hypericum extracts. The protocols included reducing power (CUPric Reducing Antioxidant Capacity assay: CUPRAC and Ferric Antioxidant Power assay: FRAP), metal chelating, phosphomolybenum and free radical scavenging (2,2-diphenyl-1-picryl-hydrazyl-hydrate assay: DPPH and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay: ABTS). Experimental details were given in our previous paper (Grochowski et al., 2017). Inhibitory effects of Hypericum extracts were tested against different enzymes (tyrosinase, α-amylase, α-glucosidase and cholinesterase). Both antioxidant and enzyme inhibition assays were explained by standard equivalents (trolox and EDTA for antioxidant galantamine for cholinesterase kojic acid for tyrosinase acarbose for amylase and glucosidase) (Grochowski et al., 2019 Stojković et al., 2020).

Data Analysis

One-way ANOVA followed by Turkey post-hoc test was performed to assess the difference between the averages of the samples. The analysis was performed using XLSTAT software v. 2018. The p-value for each parameter was evaluated, and a p < 0.05 was considered as statistically significant. After the univariate analysis, a supervised PLS-DA analysis was carried out through the R package mixOmics to discriminate the two studied species. The variable importance on projection (VIP) score of each bioactivity was calculated to reveal the most discriminant, and Student’s t-test was performed to compare the species considering those discriminant bioactivities.

Correlation Analysis

Correlation analysis between phenolic compounds identified in Hypericum methanol extracts and biological activities was performed using the Spearman rank correlation test. Data pre-processing included the removal of variables with more than 80% missing values, the imputation of the remaining missing values using the K-nearest neighbors (KNN) algorithm, and finally data normalization by means of log transformation and Pareto scaling. Analysis was performed using the Metaboanalyst v. 4.0 platform (Chong et al., 2019).


Footnotes

† Present address: Structural Biology Department, St Jude Children's Research Hospital, Memphis, TN 38105, USA.

Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

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Abstract

Species of Eugenia have been used as an antidiabetic natural source. Chemical, antioxidant and antiglycant screening of extracts from pedra-ume caá (Eugenia punicifolia) fruits were performed. 1 H NMR assisted by non-supervised chemometric methods were employed for the evaluation of the chemical profiles which were distinguished according to the color of fruit maturation stages, as well as for pulp and seed fruit. Furthermore, 1 H NMR fingerprint analysis of the crude extract allowed the identification of quercitrin and myricitrin, beside other nine compounds. The extracts of the yellow (YP) and green (GP) pulps presented higher antiglycant and antioxidant activities. Fresh juice from E. punicifolia was encapsulated in microcapsules produced with dextrose equivalent (DE) of 10, 20 or 30 as wall materials for the maintainment of their antioxidant and antiglycant properties. The more efficient retention of the bioactive compounds was found using the DE30. The Encapsulation Efficiency (EE) and the Retention Efficiency (RE) of this system was found around 89.7% and 97.6%, respectively. In addition, NMR spectra revealed the presence of flavonoids O-glycosylated (quercitrin and myricitrin) which might be related to the antiglycant and antioxidant activities. The YP presented larger content of quercitrin (117.6 ± 0.4 mg per each 100 g of fresh fruit). Therefore, pedra-ume caá should be employed as an alternative nutraceutical source, as well as intherapeutic pourposes.


Abstract

Previous research has demonstrated that certain combinations of compounds result in a decrease in toxic or pro-oxidative effects, previously noted when compounds were administered singly. Thus, there is a need to study many complex interactions further. Two in vitro techniques [electron paramagnetic resonance (EPR) and oxygen radical absorbance capacity (ORAC) assays] were used in this study to assess pro- and antioxidant capacity and synergistic potential of various compounds. Rutin, p-coumaric acid, abscisic acid, ascorbic acid, and a sugar solution were evaluated individually at various concentrations and in all 26 possible combinations at concentrations found in certain foods (honey or papaya), both before and after simulated digestion. EPR results indicated sugar-containing combinations provided significantly higher antioxidant capacity those combinations containing sugars and ascorbic acid demonstrated synergistic potential. The ORAC assay suggested additive effects, with some combinations having synergistic potential, although fewer combinations were significantly synergistic after digestion. Finally, ascorbic acid, caffeic acid, quercetin, and urate were evaluated at serum-achievable levels. EPR analysis did not demonstrate additive or synergistic potential, although ORAC analysis did, principally in combinations containing ascorbic acid.


Abstract

Certain meroterpenoids isolated from brown alga of the genus Sargassum are known to be antioxidant agents. Herein, density functional theory has been performed to analyze the preferred antioxidant mechanism of the two reactive antioxidant compounds derived from the Sargassum genus, that is, Sargahydroquinoic acid and Sargachromanol and some of their derivatives. Their global reactivity descriptors have been calculated to reveal their reactivity as an antioxidant. Molecule 1 is the most reactive antioxidant according to calculated descriptors. The results of molecule 1 are comparable to that of Trolox, suggesting their similar activity. The calculated descriptors are closely matched with experimental pieces of evidence. It has been found that hydrogen atom transfer (HAT) is more favored in gas media. Also, the effect of solvent polarity on the antioxidant activity has been explored for molecule 1. The results disclose that the polarity of the solvent increases the contribution of two other mechanisms, that is, single-electron transfer, followed by proton transfer and sequential proton loss electron transfer.


Results

Selective cytotoxicity of Urtica dioica extract in human lung cancer cells

The cytotoxic activity of the UD extract was assessed on human NSCLC H460, H1299, A549 and H322 cell lines, which were previously selected by our group as EGFR wild-type cell models with a low sensitivity to cisplatin-based therapies 4 . Plant extract cytotoxicity was evaluated through MTT assay exploring a broad spectrum of doses in two time periods (48 and 72 h). Results from these experiments proved that UD decreased NSCLC cell proliferation in a time and dose-dependent manner (Fig.  1A ). The studied cell lines showed a diverse sensitivity to the treatment, since the plant extract exerted a two-fold higher activity in H1299 and A549 (IC50 values: 52.333 ±𠂐.003 and 47.466 ±𠂐.003 µg/mL, for H1299 and A549 respectively) compared with H460 and H322 (IC50 values: 84.333 ±𠂐.002 and 78.333 ±𠂐.002 µg/mL for H460 and H322 respectively). H1299 and A549 are particularly refractory to cisplatin treatment 4 , thus these were selected for further investigations aimed at figuring out the underlying mechanism(s) by which nettle induces cell death. In addition to this, it was investigated whether the UD extract extended its cytotoxic effect on normal lung cells and, for this purpose, plant treatment was also performed on Beas2B and Wi38, normal bronchial epithelial and lung fibroblast cells, respectively. Interestingly, cytotoxicity was only appreciable at higher doses, suggesting that UD preferentially inhibited the growth of malignant lung cancer cells A549 and H1299 (Fig.  1A ).

Effects of Urtica dioica extract on NSCLC (H1299, A549, H460 and H322) cell lines, normal bronchial epithelial (Beas2b) cells and human fibroblasts (Wi38). (A) Urtica dioica extract treatment was performed at the indicated doses for 48 and 72 h. Cell proliferation was measured with the MTT assay, as described in Materials and Methods, in human NSCLC cell lines and in normal bronchial epithelial cells and human fibroblasts. The results are the average ± sd of three independent experiments, each done in triplicate. All the used doses are statistically significant as determined by the Student-t test (**P ≤𠂐.01). For sake of simplicity asterisks (**) are not reported in the graph. (B) Cell cycle distribution of H1299, A549, Beas2B and Wi38 cells after treatment with Urtica dioica extract for 72 h. Each experiment was done in duplicate ± sd. Statistically significant data are evidenced with asterisks (**P ≤𠂐.01).

Apoptosis induction by Urtica dioica extract in human lung cancer cells

In an attempt to understand how nettle impairs cell growth, firstly, the effect of the plant extract on the cell cycle by using a flow cytometer was determined. After treating cells with 60 μg/mL of UD for 3 days, H1299 and A549 accumulated in the G2/M phase conversely, no detectable changes were noted in Beas2B and Wi38 (Fig.  1B ).

Subsequently, the induction of apoptosis in H1299 and A549 tumour cell lines in comparison with the non-tumour cell lines Wi38 and Beas2b was analysed, after the treatment with (60 μg/mL for 72 h) and without (CTR-) UD. As a result, approximately 25.3% for H1299 and 30% for A549 experienced apoptosis, meanwhile Beas2b and Wi38 did not show any post-treatment effects. Histogram data are expressed as a percentage of both early and late apoptotic cells, moreover representative dot plots diagrams of flow cytometric analysis of H1299 and Wi38 cell apoptosis are shown in the Fig.  2A .

Induction of apoptosis in NSCLC (H1299 and A549) cell lines, normal bronchial epithelial (Beas2b) cells and human fibroblasts (Wi38). (A) Apoptosis was evaluated as described in Materials and Methods with Annexin V staining in H1299, A549, Beas2B and Wi38 cells after treatment with (60 μg/mL for 72 h) and without (CTR-) Urtica dioica extract. Representative dot plot diagrams of flow cytometric analysis of H1299 and Wi38 cell apoptosis are shown. Dot plot diagrams show the different stages of apoptosis. % indicated in the UL (Upper Left) quadrant represent cells positive for Annexin V and negative for 7AAD, considered as early apoptotic cells % in UR (Upper Right) quadrant indicate cells positive for both Annexin V and 7AAD, showing the late apoptotic or necrotic cells population % in LL (Lower Left) quadrant are negative for both markers and represent viable cells. Histogram of data expressed as percentage of both early and late apoptotic cells. Bars represent mean values obtained from three separate experiments. P values <𠂐.05 were considered as statistically significant (**) at Student-t test. (B) The Western Blot analyses, which were carried out using antibodies against PARP, (89)-cleaved-PARP fragment, pro-caspase3, pro-caspase 8 and cleaved BID, were performed on protein lysates from cell after the indicated treatment with (+) and without (−) Urtica dioica extract.

Following this, the expression of the main apoptosis-related enzymes 20 by using western blotting analysis was evaluated (see Fig.  2B and S1 in the Supplementary Information File). Decreased levels of the pro-caspase-3 and -8 indicated the activation of the proteolytic enzymes caspase-3 and -8, respectively. Furthermore, a concomitant increase of cleaved poly (ADP) ribose polymerase (c-PARP) (89 KDa) and truncated Bid (tBid) (15 KDa), that represented the correspondent substrates of caspase-3 and -8, undoubtedly confirmed this data.

To identify the cellular signalling responsible for caspase activation, the MAPK and PI3K/Akt pathways were initially investigated, as they both play a pivotal role in regulating cell proliferation, apoptosis 20 , 21 and cisplatin cytotoxicity 3 . The dysregulation of MAPK and PI3K/Akt pathways were found to impair cisplatin sensitiveness 22 , 23 and this led to suppose that UD treatment could restore the activation of MAPK and PI3K/Akt pathways finally fostering apoptosis. In order to validate this hypothesis, the levels of phosphorylated MAPK (p44/42 MAPK) and Akt were analysed, since the activities of these proteins are modulated by phosphorylation. Nevertheless, the unphosphorylated/phosphorylated levels of MAPK and Akt had proven similar both before and after the treatment, suggesting that UD did not affect these proteins (See Fig.  3 and S2 in the Supplementary Information File).

The expression levels of ER-stress related proteins were investigated on H1299 and A549 cancer cells following treatment with (+) and without (−) Urtica dioica extract (60 μg/mL) for 72 h. The Western Blot analyses were carried out using antibodies against MAPK, Phosho MAPK, AKT, Phosho AKT, GADD 153, DR5. β-actin was used as the loading control.

Thus, other molecular and cellular determinants known to be responsible for caspase activation were studied. Previous works sustained that caspase-8 activity is likely to be externally stimulated by surface death receptors 24 , and therefore, these proteins were analysed, indicating the expression of the death receptor DR5 remarkably increased after UD treatment in both H1299 and A549 cells (Fig.  3 ). These findings revealed that UD promoted the extrinsic apoptotic pathway through DR5, which upon activation, triggered the caspase cascade along with the cleavage of cytosolic BID in tBID 20 . Moreover, DR5 up-regulation was known to be directly linked to the activation of the growth arrest and DNA damage-inducible gene 153 (GADD153), also known as the C/EBP homologous transcription factor (CHOP), which was considered a marker of endoplasmic reticulum (ER) stress 25 , 26 . Therefore, GADD153 levels were examined, displaying that this protein was evidently up-regulated in H1299 and A549 cells after 72 h of incubation with UD (Fig.  3 ).

Taken together, the findings supported a mechanistic scenario in which UD treatment induced ER-stress by up-regulating GADD153. In turn, this event resulted in the overexpression of DR5, which directly promoted the extrinsic apoptotic pathway and indirectly stimulated the mitochondria apoptotic machinery via BID activation.

The synergistic anti-proliferative effect of Urtica dioica extract and cisplatin against human lung cancer cells

To extend the pre-clinical observations, it was evaluated whether UD was able to improve the sensitivity of H1299 and A549 cell lines to cisplatin. For this purpose, cells were treated with cisplatin and/or UD (Fig.  4A ). As expected, treatment with cisplatin alone, weakly inhibited cell proliferation (IC50 =�.012 ±𠂐.004 and 22.156 ±𠂐.003 µg/mL, for A549 and H1299, respectively), whereas, the co-treatment of cisplatin and UD exhibited a notable anti-proliferative synergistic effect in both H1299 and A549 cells (Fig.  4A ). The administration of cisplatin at 2.5 μg/mL or UD at 20 μg/mL caused a comparable effect that was approximatively accountable for 20% apoptotic rate conversely, the combination of these dramatically raised the apoptotic percentage reaching approximately 65% (Fig.  4B ). These results indicated that UD extract synergised with cisplatin, radically improving the sensitivity of H1299 and A549 cells to cisplatin treatment.

Effects of the combination of Urtica dioica extract and cisplatin on NSCLC (H1299 and A549) cell lines. (A) Cell proliferation analysis performed by MTT assay in H1299 and A549 after treatment with the indicated doses of Urtica dioica and cisplatin. The results are the average ± sd of three independent experiments, each done in triplicate. All the used doses are statistically significant as determined by the Student-t test (**P ≤𠂐.01). For sake of simplicity asterisks (**) are not reported in the graph. (B) Apoptosis evaluated in H1299 and A549 after treatment with the indicated doses of nettle and cisplatin. Histogram of data expressed as percentage of both early and late apoptotic cells. Bars represent mean values obtained from three separate experiments. Statistically significant data are evidenced with asterisks (**P ≤𠂐.01).

1D and 2D-NMR spectroscopy investigation of Urtica dioica extract

The encouraging biological results resulted in the performance of a comprehensive NMR analysis with the aim of unveiling the main secondary metabolites present in the plant extract.

Metabolites were identified by comparing peak chemical shifts to those found in literature and in Human Metabolome Database (HMDB). Furthermore, as spectral overlaps of the 1 H resonances in 1D spectra often seriously limited the unambiguous identification of certain metabolites, we also carried out an extensive 2D NMR analysis of the plant extract.

The 1 H NMR spectrum of UD (Fig.  5 ) displayed peculiar chemical shift values of flavonoids evident in the aromatic region of the spectrum in particular, two meta-coupled doublets at δH 6.39 (δC 93.5) and δH 6.20 (δC 98.7) were clearly detectable. These signals, thanks to the CIGAR-HMBC correlations (see Figure  S3 in the Supplementary Information File) along with available literature data 27 , were assigned to quercetin. Moreover, two signals at δH 5.01 and 4.53 also correlated in CIGAR-HMBC experiment with the C-3 carbon (δC 135.2) and the methylene carbon of glucose (δC 68.1), respectively. This data proved to be in line with the presence of quercetin-3-O-rutinoside, a flavonol glycoside known as rutin, which has already been reported as a representative constituent of U. dioica flowers 28 .

1 H NMR spectrum of Urtica dioica crude extract recorded in methanol-d4/phosphate buffer (1:1). R = rutin, Oxy = oxylipins, sugar = glycosidic moiety of rutin.

In the region of the 1 H-NMR spectrum included between 6.00 and 5.00 ppm, several overlapped protons were evident. Furthermore, two methylene triplets (Figs  5 and ​ and6) 6 ) at δH 2.80 (δC 25.2) and 2.23 (δC 35.0) along with overlapped methylene protons at 2.06 (δC 26.8 and 20.3), 1.59 (δC 25.1), 1.32 (δC 29.0) and the methyl triplet at 0.95 (δC 13.1) supported the presence of omega-3 fatty acids. These data were in agreement with previous works, which identified α-linoleic acid and its derivatives as the pre-dominant fatty acids of U.dioica leaf extract 29 . The combination of 2D-NMR techniques, especially DQF-COSY, H2BC and HSQCTOCSY (see Figure  S4 in the Supplementary Information File), allowed the assignment of almost all overlapped H-atom and C-atom signals. Specifically, DQF-COSY homocorrelations (see Figure  S5 in the Supplementary Information File) led to the identification of two types of spin system: CH3-CH2-CH=CH-CH2-CH=CH and CH=CH=CH-CH(O) as shown in Fig.  7 . The former spin system confirmed the presence of an omega-3 fatty acid, while the latter suggested that this compound included a site of hydroxylation. This was further supported by several works that previously demonstrated the presence of oxylipins (polyunsaturated oxidised fatty acid) in U. dioica 30 , 31 . Moreover, in the CIGAR-HMBC experiment, the methine proton at δH 5.70 was heterocorrelated with carbon at δC 128.5 that bonded to the proton at δH 5.94. This latter signal was heterocorrelated with the olefinic carbon at δC 136.7 as well as with an oxygenated C-atom at δC 77.0 (see Figure  S3 in the Supplementary Information File). In turn, this was heterocorrelated with the protons at δH 2.68, 2.26, 1.36. Specifically, the carboxyl carbon at δC 180.1 displayed cross peaks with the methylene protons at δH 2.32 (δC 34.1), which, in turn, heterocorrelated with carbons at δC 29.1 (δH 1.29) and 24.6 (δH 1.64) (see Figure  S3 in the Supplementary Information File). Altogether, the data suggested the presence of hydroxyl polyunsaturated fatty acids. However, due to the lipid nature of these metabolites, it was not possible to define the exact structures of the above-mentioned oxylipins in the crude extract.

(A) Expanded olefinic and (B) aliphatic region of HSQC experiment of Urtica dioica extract. R = rutin. LG = lignan. OXY = oxylipins

Selected CIGAR-HMBC, DQF-COSY and H2BC correlations of rutin and oxylipin derivatives detected in Urtica dioica crude extract.

Besides flavonols and oxylipins, less intense signals, overlapped in the 1 H-NMR spectrum, were detectable in the 2D NMR experiments and ascribable to less abundant UD metabolites. Most of these signals were attributed to lignan-type molecules. Specifically, thanks to the HSQC experiment (Fig.  6 ), six aromatic hydrogen signals, which belonged to two 1,2,4-trisubstituted phenyl groups were detected. Moreover, key CIGAR-HMBC correlations indicated the presence of two guaiacyl groups linked to tetrahydrofuran moiety through an oxymethine (δH 4.73/δC87.1) and an oxymethylene 32 . This data supported the presence of olivil derivatives, tetrahydrofuranic lignans that had already been identified as a constituent of Urtica triangularis 33 .

Identification and isolation of the major components of Urtica dioica that contribute to the cytotoxicity of the active plant extract

Subsequently, it was investigated whether the major components of the UD contributed to its selective cytotoxic effect on NSCL cells. For this purpose, a targeted fractionation of UD was performed using different chromatographic techniques. Initially, the plant extract was portioned between ethyl acetate (UD1) and water (UD2): rutin was isolated from both UD1 and UD2, while an oxylipins’ enriched fraction was obtained exclusively from UD1. The pure compound rutin was analysed trough 1D and 2D NMR (see Fig.  7 and S6 in the Supplementary Information File) thus, its structure was confirmed by comparing NMR data with those available in literature 34 and with the in-house NMR library. Likewise, the oxylipins’ enriched fraction was firstly analysed trough 1D and 2D NMR. In the 1 H-spectrum, overlapped signals resonating in the range 0.88𠄱.00 ppm and 4.40𠄳.80 ppm along with two triplets at δH 5.96 and 5.95 and two double doublets at δH 5.64 and 5.63. These signals were in agreement with those detected in the plant crude extract and supported the presence of omega 3-oxylipins, whose basic skeleton was further confirmed by 2D NMR correlations (see Figures  S7 and S8 in the Supplementary Information File).

Subsequently, the cytotoxicity of these compounds on A549 cell line were assessed through the MTT assay testing four different doses (25, 50, 75 and 100 µg/mL) at 72 hours. These experiments demonstrated that rutin did not show any cytotoxic effect, while the oxylipins’ enriched fraction recapitulated the effect of the crude plant extract (Fig.  8 ).

Cytotoxic effect of rutin, oxylipins and UD extract on A549 cell line. Cell proliferation was evaluated 72 h after treatment by MTT assay results are reported as mean ± sd of 3 independent experiments, each done in triplicate. Inhibition of cell growth exerted by Oxylipins and UD extract vs control was statistically significant at Student- t test (**P ≤𠂐.01).

Due to the interesting biological results, the main aim was to elucidate the structure of the main oxylipin present in the above-mentioned mixture. However, the severe overlaps in the NMR spectra made again not feasible the unambiguous characterisation of these metabolites. Therefore, an HPLC analysis was carried out by identifying and isolating the principal oxylipin of the fraction. Its structure was then elucidated thanks to a combination of ESI Q-TOF HRMS and NMR techniques. The ESI Q-TOF HRMS spectrum in the negative mode showed a pseudomolecular ion [M-H] − at m/z 365.2706, giving the molecular formula C22H38O4 for this oxylipin. Meanwhile, the positive mode of the ESI Q-TOF HRMS spectrum displayed a sodium adduct at m/z at 389.2649 along with two fragments at m/z 349.2723 and 331.2623. The two last peaks suggested the consecutive loss of two molecules of H2O (see Figure  S9 in the Supplementary Information File). These data were in good agreement with the HSQC spectrum (see Figure  S10 and Table  S1 in the Supplementary Information File), in which the presence of two carbinolic methins at δC 69.2 (δH 4.01) and δC 72.9 (δH 4.09) was evident. This last proton was heterocorrelated with the C10 (δC 69.2), two methylene carbons C11 (δC 28.3) and C12 (δC 36.1), and two methin carbons C14 (δC 137.8) and C15 (δC 126.1). This latter in the H2BC experiment showed a 2 J correlation with the triplet at δH 5.99, which correlated with the olephinic carbon C17 at δC 131.5 (Fig.  7 ). In turn, this carbon displayed a cross peak with the double allylic protons H18 at δH 2.95 (δC 26.6). Moreover, the HMBC experiment evidenced key correlations among a series of methylene protons (resonating at δH 2.24, 2.05, 1.6 and 1.34) and the carbonyl carbon at δC 182.2. This data was further confirmed by the tandem MS analysis of the pseudomolecular ion [M - H] − , which showed two fragment ions at m/z 347.1905 and 303.2093, indicating the loss of H2O and H2O along with CO2, respectively. Altogether, these data were in agreement with the presence of 10,13-dihydroxydocosa-12,16,19-trienoic acid.


Results and discussion

I. Synthesis and properties of CT51

As illustrated in Fig 1 , CT51 was synthesized from resorcinol, condensing it with citric acid (Pechmann) to obtain 2-(7-hydroxy-2-oxo-2H-chromen-4-yl) acetic acid (compound 1). To synthesize CT51, compound 1 was esterified under Fischer-Speier conditions, giving ethyl 2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetate (compound 2), which was reacted with 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS). The resulting CT51 product was characterized by 1 H-NMR spectroscopy (S1A Fig), 13 C-NMR spectroscopy (S1B Fig), electrospray ionization mass spectrum (S1C Fig) and X-ray crystallography (S1D Fig and S1 Table). The presence of only 12 carbons instead of 13 atoms in the NMR spectrum stems from the fact that the signal originated by the missing carbon is hidden by the DMSO signal. This carbon atom was assigned to the methylene group, which acts as a bridge between the coumarin ring and the amide group.

Reagents and conditions: a) Citric acid, H2SO4, room temperature for 48 h b) H2SO4, ethanol, reflux 6 h c) 2-amine-2-hydroxymethyl-propane-1,3-diol, ethanol, reflux 48 h.

The CT51 molecule is water-soluble, most probably due to the three hydroxyl groups originating from the TRIS moiety, which also comprises a putative iron coordination site. A fourth hydroxyl group is present in the phenol moiety attached to C7 in the benzopyrone ring, which acts as a strong electron donor [33, 34], leading to polarity-dependent fluorescence intensity that increases with increasing polarity of the medium with little spectral shift [35]. The absorption spectrum of CT51 displayed a maximum at 330 nm and a shoulder near 370 nm (S2A Fig). This result agrees with other absorption spectroscopic studies of 7-hydroxycoumarins, which exhibit absorption maxima in the range 320� nm, depending on the nature of the substituent groups at C3 or C4. Regularly, these compounds have been reported to have band groups of UV absorptions at 270� nm due to π–π* transitions [36]. At 330 nm, CT51 displayed a molar extinction coefficient (ε) of 17,460 M -1 cm -1 . Fluorescence studies revealed that CT51 had an emission band with a maximum at 460 nm (S2B Fig). The emission quantum yield (Φ) of CT51 was Φ = 0.34, determined by using quinine sulfate as standard the lifetime (τ) was 5.95 ns, and the Stokes shift was 130 nm.

The cation selectivity of CT51 was detected by the quenching of fluorescence caused by the following cations: Hg 2+ , Fe 3+ , Fe 2+ , Co 2+ , Cu 2+ , Ca 2+ , Zn 2+ , Mn 2+ , Mg 2+ , Ni 2+ , Pb 2+ or Cd 2+ . Only Fe 2+ , and to a lesser extent Fe 3+ , produced a significant quench of CT51 fluorescence ( Fig 2A ). Increasing the concentration of Fe 2+ up to the 200 μM range partially reduced the fluorescence of 5 μM CT51 ( Fig 2B ). The selectivity of CT51 for Fe 2+ and Fe 3+ makes it a possible candidate for iron chelation in living cells. A similar coumarin-TRIS compound (DAT-1) was recently characterized as a Fe 3+ chelator [37]. At difference with CT51, DAT-1 did not demonstrate selectivity for Fe 2+ . We don’t know the causes for this discrepancy, but the use of 0.2 M citrate, a mild Fe 2+ chelator, in the solutions used to test DAT-1 [37] possibly hindered the interaction of Fe 2+ with DAT-1.

(A) Fluorescence emission spectra of CT51 (20 μM) recorded before and after addition of several different chloride salts of Hg 2+ , Fe 3+ , Fe 2+ , Co 2+ , Cu 2+ , Ca 2+ , Zn 2+ , Mn 2+ , Mg 2+ , Ni 2+ , Pb 2+ or Cd 2+ all salt solutions (200 μM) were prepared in 20 mM HEPES buffer, pH 7.4. (B) Emission spectra of CT51 (20 μM) were recorded (λex = 330 nm) upon addition of increasing concentrations (up to 200 μM) of Fe(NH4)2(SO4)2୶H2O prepared in 20 mM HEPES buffer, pH 7.4. (C) Cyclic voltammetric analysis. The voltammograms represent the behavior of solutions containing 0.58 mM Fe 2+ without CT51 (black line), or in the presence of 0.96 mM CT51 (red line). The voltammograms were performed at a scan rate of 100 mV s -1 , using a potentiostat DropSens μStat 400.

Cyclic voltammetry analysis represents a useful tool to study redox processes. Hence, to confirm iron chelation, we performed voltammetric assays using a Screen Printed Carbon Electrode (SPCE) and a solution of 0.58 mM Fe 2+ in the absence (black line, pH 3.0) and in the presence of 0.96 mM CT51 (red line, pH 6.8) ( Fig 2C ). These assays showed an oxidation current for the reaction Fe 2+ ↔ Fe 3+ + e- at 0.18 V (reaction a), and a cathodic current for the reaction Fe 3+ + e- ↔ Fe 2+ at 0.034 V (reaction b), with ΔV = 0.146 V, indicating a quasi-reversible process. When an optimal concentration of CT51 was added to the Fe 2+ -containing solution, a decrease in the Fe 2+ anodic current was noted. Also, a change in the potential at 0.94 V emerged (reaction c), which may be due to CT51 oxidation in the complex with Fe 3+ . Importantly, we did not observe a cathodic signal for the reaction Fe 3+ + e- ↔ Fe 2+ . Probably, Fe 3+ generated from the oxidation of Fe 2+ forms a stable complex with CT51 and therefore it is not reduced back to Fe 2+ , so that in the reaction Fe 2+ → Fe 3+ + e-, Fe 3+ becomes irreversibly oxidized. This is a relevant finding, since stopping Fe 2+ /Fe 3+ redox cycling in cells should prevent the hydroxyl radical production resulting from the Fenton reaction [8]. Notwithstanding, undetermined cellular reactions could decompose the CT51-Fe 3+ complex making Fe 3+ available again.

The Stern-Volmer analysis yielded a linear representation (S3A Fig) based on this result we suggest that the metal-ligand (Fe 2+ -CT51) bond has 1:1 stoichiometry and that the quenching is 100% static. This latter assumption was verified by determining the τ/2 value in the presence and absence of the metal ion, as well as by determining the rate constant (kq) that displayed a value (in s -1 ) of 5.82 x 10 11 ± 0.4 x 10 11 kq values in this range are representative of the static quenching typical of chelators. The association constant of CT51-Fe 2+ was determined by the Benesi–Hildebrand equation, which allows for the determination of the apparent association constant (Ka) value from the slope and intercept of the plot (S3B Fig). The Ka value for Fe 2+ binding to CT51 was 6.63 x 10 𢄤 M -1 . The constant slope of the Benesi–Hildebrand plot supports the above proposal of 1:1 stoichiometry for the complex CT51-Fe +2 . The detection limit of this Fe 2+ fluorescent sensor was 1.1 x 10 𢄧 M, which allows for the detection of the labile iron pool in the cell cytoplasm that presents values in the low 10 𢄦 M range [38, 39].

Since antioxidant properties are a desired trait of neuroprotective agents, we evaluated next through chemical and biological assays the putative antioxidant capacity of CT51. To explore the antioxidant capacity of CT51, we determined the EPR spectra of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) in the absence or presence of CT51. DMPO is a spin trap widely used to study the levels of oxygen-based free radicals. Superoxide, hydroxyl and thiyl radicals form adducts with DMPO that can be detected with EPR spectroscopy. Hydroxyl radicals were generated by the Fenton reaction in the presence of DMPO, or DMPO plus Trolox or CT51 as hydroxyl radical competitors. As positive control for free radical quenching we used the antioxidant agent 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), a gold standard for oxygen radical absorbance capacity [40]. The resulting EPR spectra indicated that CT51 possesses considerable capacity for free radical quenching ( Fig 3 ). The values of the percentage reduction of the signal, calculated using the double integral of the EPR spectra, were DMPO: 0.0, Trolox: 84.8 and CT51: 99.8. In addition, voltammogram studies of CT51, determined at pH 6.8 or pH 3.0 (S4 Fig) showed that at pH 3.0, the oxidation current increased and the potential shifted from 0.71 V, indicating involvement of the phenolic proton of CT51 during the oxidation process. This proton has the capacity to neutralize ROS belonging to the free radical category [41].