Identification of Narciclasine as an in Vitro Anti-Inflammatory

Note Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

pubs.acs.org/jnp

Identification of Narciclasine as an in Vitro Anti-Inflammatory Component of Cyrtanthus contractus by Correlation-Based Metabolomics Lucie Rárová,† Bhekumthetho Ncube,‡ Johannes Van Staden,‡ Robert Fürst,§ Miroslav Strnad,⊥ and Jiri Gruz*,⊥

Downloaded via UNIV PARIS-SUD on April 1, 2019 at 19:48:57 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Š lechtitelů 27, CZ-783 71 Olomouc, Czech Republic ‡ Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa § Institute of Pharmaceutical Biology, Biocenter, Goethe University, Frankfurt/Main, Germany ⊥ Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany ASCR and Palacky University, Š lechtitelů 27, 78371 Olomouc, Czech Republic ABSTRACT: In this study, an extract from the bulbs of Cyrtanthus contractus showed strong anti-inflammatory activity in vitro. The extract was partially separated into 14 fractions and analyzed by ultrahigh performance liquid chromatography-quadrupole time-of-flight mass spectrometry metabolomics, and the correlation coefficients were calculated between biological activities and metabolite levels. As a result, the top-scoring metabolite narciclasine (1) is proposed as the active principle of C. contractus. This was confirmed by comparing the biological effect of crude extract with that of an authentic standard.

T

extracts from the bulbs of this plant collected during different months of the year varied. Extracts collected in May and September were the most active, exhibiting antimicrobial effects, antiproliferative activities in cancer cells, and inhibition of the enzymes acetylcholinesterase and cyclooxygenase. These bioactivities and the content of total alkaloids are in agreement with the use of C. contractus bulbs in traditional medicine for the treatment of relevant ailments during the above-mentioned periods.7 Narciprimine was isolated from C. contractus and has been identified in Zephyranthes, Narcissus, and Lycoris species.8 The preliminary data suggested that C. contractus may be active in anti-inflammatory bioassays. In this study, the in vitro antiinflammatory activity of the C. contractus bulb MeOH extract was evaluated, and its bioactive principle was identified using a nontargeted metabolomic approach based on partial fractionation and subsequent correlation of metabolite concentrations with expression of E-selectin. The preliminary screening of various plant extracts suggested that C. contractus may contain substances with anti-inflammatory activity. The C. contractus bulb MeOH extract applied to endothelial cells caused dose-dependent decrease in the level of

he relative dearth of currently available anti-inflammatory drugs has stimulated a search for new active substances. The plant family Amaryllidaceae serves as an important and unique source of alkaloids. The traditional use of the daffodil as a phytomedicine is known from ancient times by Hippocrates of Kos (fourth century BC) and Pedanius Dioscorides (first century AD), and they recommended narcissus oil as an antitumor treatment.1 The first identified alkaloid lycorine was isolated from the daffodil (Narcissus pseudonarcissus).2 Currently, more than 500 different alkaloids have been isolated from Amaryllidaceae.3 The Amaryllidaceae alkaloids are known for their wide spectrum of biological activities in vitro, for example, anti-invasive, antiproliferative, and apoptosis-inducing activities.4 Another interesting isocarbostyril alkaloid narciclasine (1) was discovered in the bulbs of several Narcissus species (Amaryllidaceae) in 1967.5 The anti-inflammatory effect of a 1-containing extract of Haemanthus coccineus was described in two inflammatory models in mice (kidney injury in a unilateral ureteral obstruction model and the formation of edema by arachidonic acid or croton oil).6 The present study evaluated the anti-inflammatory activity of Cyrtanthus contractus N.E. Br., which is another member of the Amaryllidaceae family. C. contractus is used widely in traditional African medicine for the treatment of various diseases, such as age-related dementia and mental illness. Ncube et al.7 reported that the bioactivities of © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 4, 2018

A

DOI: 10.1021/acs.jnatprod.8b00973 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Note

E-selectin, which is a key player in the initiation of inflammation. Before further investigation, the toxic concentration of the C. contractus bulb MeOH extract toward human umbilical vein endothelial cells (HUVEC) as measured in a 24 h treatment was greater than 100 μg/mL (data not shown). In the subsequent assays, only non-cytotoxic concentrations were used. The C. contractus bulb MeOH extract was then tested for antiinflammatory activity using two assays in vitro in HUVECs activated by pro-inflammatory stimulus tumor necrosis factor (TNFα): (1) adhesion of THP-1 monocyte-like cell line to endothelium presented by HUVECs and (2) expression of intercellular cell adhesion molecule-1 (ICAM-1) on the cell surface of endothelial cells. The results from both assays showed that preincubation of HUVECs with the C. contractus bulb MeOH extract (3, 10, and 30 μg/mL) strongly reduced THP-1 adhesion by blocking expression of the endothelial adhesion molecule ICAM-1 in a concentration-dependent manner (Figure 1).

Figure 2. Fractions isolated from the C. contractus bulb MeOH extract reduce the relative amount of E-selectin on the cell surface of endothelial cells that were pretreated with the C. contractus bulb MeOH extract fractions for 30 min and subsequently stimulated with 10 ng/mL TNFα for 4 h. Data are means ± SDs (n = 3).

promising candidates, it was concluded that they refer to a single compound for which the elemental formula is C14H13NO7. Thus, the top-scoring metabolite in the negative mode was the same as that from the positive mode. Further analysis of its MS/ MS spectra suggested that this compound is 1 (narciclasine), for which its structure was confirmed subsequently by comparing Rt and MS/MS spectra with those of an authentic standard (Figure 3). The identification of active substances in complex mixtures is a challenging task that requires typically the isolation of an active compound. In this study, it was shown that correlation-based metabolomics is a viable alternative to compound isolation. The basic idea behind this approach is that the concentration level of a bioactive compound should be highly correlated with biological activity of the isolated fractions. A similar approach based on the analysis of different tomato accessions instead of isolated fractions was used recently to identify Nrf2-activating substances from tomato.9 Unfortunately, because of the lack of orthogonality, the number of top-scoring compounds is typically too high. In the present study, such a negative effect was reduced by combining more separation mechanisms including those based on polarity, acidity, and basicity. This resulted in a reasonably higher Pearson correlation coefficient of the topscoring 1 (r = 0.97) compared to all other compounds/features (r ≤ 0.89). Compound 1 was not only top-scoring but also a relatively abundant compound in the C. contractus bulb MeOH extract (Figure 3A). The determined levels of 1 in C. contractus (66.0 ± 4.1 μg/g dry weight) were comparable to concentrations determined previously in Narcissus species (30−200 μg/g) but notably higher than in other genera of the family Amaryllidaceae.10 By comparing the anti-inflammatory effect of C. contractus bulb MeOH extract with that of a standard solution of 1 at a corresponding concentration, it was proved that 1 is the main active component of the C. contractus bulb MeOH extract (Figure 4). The concentration of 1 (narciclasine) in C. contractus was determined by a UHPLC-MS/MS method to be 66.0 ± 4.1 μg/g dry weight, which corresponds to 7.2 nM in 30 μg/g of the C. contractus bulb MeOH extract. To prove that 1 is the active component of the C. contractus bulb MeOH extract, the in vitro anti-inflammatory activities of the extract (30 μg/mL) and pure

Figure 1. C. contractus bulb MeOH extract reduced THP-1 adhesion by blocking expression of the endothelial adhesion molecule ICAM-1. HUVECs were left untreated or were preincubated with the MeOH extract (3, 10, and 30 μg/mL) for 30 min. They were then stimulated with 10 ng/mL TNFα for 24 h. Adhesion of THP-1 cells to HUVECs or expression of ICAM-1 on the cell surface of endothelial cells by flow cytometry was determined. Data are shown as means ± standard deviations (SDs) (n = 3).

Because of the promising in vitro anti-inflammatory activity of the C. contractus bulb MeOH extract, a search for its active component was started by separating the crude extract into 14 fractions. Eight fractions were prepared using reversed-phase solid phase extraction (SPE) (C0, C1, C2, C3, C5, C6, C7, and C9), three fractions using cation-exchange SPE (K0, K1, and K2), and three using anion-exchange SPE (A0, A1, and A2). All isolated fractions were tested for their ability to decrease the expression of E-selectin on the cell surface of endothelial cells following their induction by TNFα (Figure 2). These data showed that biological activity was concentrated into fractions C2 and K1. In parallel, the chemical composition of each fraction was analyzed by nontargeted ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS), which resulted in a list of 31 323 features (14 447 in the negative mode and 16 876 in the positive mode). All features were correlated further with antiinflammatory activity in vitro (expression of E-selectin) across all fractions, to obtain a list of candidates for an active component of the C. contractus bulb MeOH extract. Manually identified molecular ions derived from candidate features are summarized in Table 1, where the features represent identified molecular ions, that is, [M + H]+ and [M − H]−. In this case, the Pearson correlation coefficients of the best ranked two molecular ions (r = 0.97) were much higher than those of the rest (r ≤ 0.89). From a preliminary examination of the two most B



Note

Table 1. Top 10 Candidate Features out of 4397 According to Pearson Correlation Coefficient (r) rank

measureda m/z

theoretical m/z

Δppm

mode

Rt (min)

rb

molecular formula

1 2 3 4 5 6 7 8 9 10

306.0615 308.0775 304.1185 246.0982 207.1390 151.0399 521.1880 505.1556 308.0407 403.1240

306.0614 308.0770 304.1185 246.0978 207.1385 151.0395 521.1870 505.1557 308.0406 403.1240

0.3 1.6 0.0 1.6 2.4 2.6 1.9 −0.2 0.3 0.0

negative positive positive negative positive negative positive negative negative negative

7.52 7.50 5.58 7.02 6.79 7.87 6.16 7.62 7.72 7.68

0.974 0.966 0.891 0.890 0.888 0.883 0.878 0.876 0.875 0.867

C14H13NO7 (1)c C14H13NO7 (1)c C16H17NO5 C10H17NO6 C13H18O2 C8H8O3 (2-OH-phenylacetic acid)d C22H32O14 C21H30O14 C13H11NO8 C17H24O11

The given ions are [M − H]− and [M + H]+ for the negative and positive modes, respectively. bPearson correlation coefficient between peak area and anti-inflammatory activity in vitro. cIdentification based on authentic standard. dPutative identification based on MS/MS spectra published in the Metlin database (https://metlin.scripps.edu/). a

Figure 3. UHPLC-electrospray ionization-MS chromatograms of a crude extract from C. contractus recorded in the negative-ion mode. Chromatogram A shows a base peak chromatogram (m/z 70−1500) while chromatogram B shows m/z 306 (±0.5 Da) with a dominant peak of 1 (7.35 min). (inset) A fragmentation spectrum of 1 at a collision energy of 20 eV (negative mode).

1 (7.2 nM) were compared. This comparison clearly demonstrated that both samples reduce the expression of Eselectin to a similar extent (Figure 4). It also suggested that the effect of 1 is not dramatically affected by other compounds present in the C. contractus bulb MeOH extract and that 1 itself is the main bioactive principle for the observed in vitro antiinflammatory activity of the extract. To understand the mechanism of the C. contractus bulb MeOH extract activity, different steps in NF-κB signaling pathway were analyzed, including expression of endothelial leukocyte adhesion molecule-1 (ELAM/E-selectin), intercellular cell adhesion molecule-1 (ICAM-1), and NF-κB translocation. The signaling pathway in endothelial cells was induced by TNFα, which triggers the translocation of NF-κB from the cytoplasm into the nucleus, where transcription of target genes is

initiated. The vascular endothelium activated by pro-inflammatory mediators (tumor necrosis factor, TNFα) strongly increases the expression of cell adhesion molecules (ICAM-1, VCAM-1, E-selectin) as a crucial step for the extravasation of leukocytes into inflamed tissue.11 In this study, the treatment with the C. contractus bulb MeOH extract led to a reduction in Eselectin and ICAM-1 expressions, although the translocation of NF-κB into the nucleus and expression of COX-2 was not affected (Figure 5). This suggests that the C. contractus bulb MeOH extract may interfere with mRNA production, shuttling, or stability12 but not with accumulation of NF-κB in the nucleus. For instance, flavopiridol can alter transcription elongation in the nucleus by inhibition of CDK9.12 A similar effect toward the NF-κB pathway was observed when pure 1, an antiinflammatory substance identified in Haemanthus coccineus, C



Note

death receptor pathway in cancer cells by 1 has been reported. Activation of this receptor regulates endothelial cell apoptosis, differentiation, proliferation, and inflammation.13 Natural compounds isolated from various plants serve as an important pool for potential new anticancer and antiinflammatory drugs.14 Compound 1 also showed potent biological activities, such as in cytotoxicity, anti-inflammatory, antiviral, and Alzheimer’s disease models.15 New anti-inflammatory agents with fewer undesirable properties are being actively sought, due to the number of side-effects of current antiinflammatory drugs.16 In this study, it was found that the C. contractus bulb MeOH extract is non-cytotoxic toward noncancer cells (HUVECs; data not shown). This finding is also supported by previous studies showing that 1, the main active component of the C. contractus bulb MeOH extract, is noncytotoxic toward both normal human fibroblasts and endothelial cells despite its potent cytotoxic effects against a panel of cancer cell lines.13,17 Thus, not only pure 1 (narciclasine) but also C. contractus extract can potentially be used as a remedy for inflammation if no safety issues are discovered.

Figure 4. Expression of E-selectin on the cell surface of endothelial cells pretreated with the C. contractus bulb MeOH extract, 1, and curcumin (10 μM) for 30 min and then stimulated with 10 ng/mL TNFα for another 4 h. The standard solution of 1 was prepared according to its concentration determined by UHPLC-UV in the C. contractus bulb MeOH extract. Thus, the concentration of 7.2 nM of 1 was applied in both the extract and 1 treatments.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Metabolomic analyses were performed using UHPLC (Acquity Ultra Performance LC) coupled to QTOF-MS (Synapt G2-Si). Western blots were analyzed using a charged coupled device (CCD) camera (Fujifilm). Absorbance and fluorescence were recorded using a microplate reader (Infinite 200, TECAN). Flow cytometric analysis was performed using a cytometer (FACS Verse, Becton Dickinson). Plant Material. Bulbs from a naturally growing population of Cyrtanthus contractus in a grassland area adjacent to the School of Life Sciences at the University of KwaZulu-Natal (UKZN), Pietermaritzburg, South Africa, were randomly collected in May 2012. A voucher specimen (NCUBE 05 NU), identified by Dr. C. Potgieter, was deposited at the Bews Herbarium (NU) at UKZN, Pietermaritzburg. Extraction and Isolation. Aliquots (30 mg) of lyophilized samples were homogenized in 1 mL of 80% methanol using an oscillation ball mill (MM 301, Retsch). Microcentrifuge tubes containing finely ground plant material were sonicated for 15 min, vortexed for 5 min, and then subjected to centrifugation for 10 min at 20 000g and 7 °C (Avanti 30, Beckman). The supernatant was evaporated under nitrogen, dissolved in 0.5 mL water, and used as a C. contractus extract for biological testing and LC-MS analysis. This crude extract was further fractionated into 14 fractions by using three types of SPE cartridges. Fractions C0, C1, C2, C3, C5, C6, C7, and C9 were obtained by elution of the crude extract from an SPE cartridge (Spe-ed C18, 0.5 g/6 mL, Applied Separations) with 8 × 2 mL of aqueous methanol of gradually increasing concentrations (0, 10, 20, 30, 50, 60, 70, and 90% methanol). Fractions K0, K1, and K2 were separated on mixed-mode cation-exchange SPE (Oasis MCX, 0.15 g/6 mL, Waters) by subsequent elutions with 2% HCOOH (K0), 100% methanol (K1), and 5% NH4OH in methanol (K2). Fractions A0, A1, and A2 were separated on mixed-mode anionexchange SPE (Oasis MAX, 0.15 g/6 mL, Waters) by subsequent elutions with 5% NH4OH (A0), 100% methanol (A1), and 2% HCOOH in methanol (A2). The eluent was always evaporated under nitrogen and redissolved in 20% MeOH prior to UHPLC-MS analysis. UHPLC-QTOF-MS. All samples were filtered through a 0.2 μm cellulose membrane microfilter (Grace) before analysis by UHPLC. Samples were injected onto a reversed-phase column (BEH C18, 1.7 μm, 2.1 × 150 mm, Waters) maintained at 30 °C and eluted by a binary gradient consisting of acetonitrile (A) and 5 mM HCOOH (B). The gradient was as follows: 0 min 5% A, 1 min 10% A, 12 min 35% A, 17 min 70% A, 17.5 min 100% A. The separated compounds were ionized in an electrospray ion source (capillary 2.5 kV, cone 25 V) and analyzed by QTOF-MS, recording in the range of m/z 70−1500. Resulting chromatograms were processed by several in-house Matlab algorithms to obtain a list of features. The Pearson correlation coefficients between

Figure 5. Endothelial cells treated for 30 min with 30 μg/mL of the C. contractus bulb MeOH extract or 7.2 nM 1 were stimulated subsequently with 10 ng/mL TNFα for 30 min. Cells incubated with only TNFα or without TNFα were used as a control. (A) Translocation of NF-κB into the nucleus of endothelial cells after exposure to TNFα. Fixed cells were stained using primary and fluorescence-labeled secondary antibodies. The experiment was repeated three times. (B) Quantification of the level of IκBα and COX-2 in HUVECs treated with 1 and the C. contractus bulb MeOH extract. Signal intensities of bands were quantified by MultiGauge Software (FujiFilm) and normalized to loading (β-actin).

was studied in vitro.6 An extract of this plant decreased NF-κBdependent gene transcription in the endothelium, although it did not interact with the NF-κB activation cascade, including p65 nuclear translocation, IκBα degradation, and NF-κB DNAbinding activity. Besides the NF-κB pathway, 1 can also influence the cell at a molecular level by other mechanisms of action. Induction of apoptosis by inducing the initial caspases of D



Note

washed with phosphate-buffered saline (PBS), fixed on the slides with 10% formaldehyde (v/v) for 10 min, labeled with primary antibody (Cell Signaling Technology) and fluorescently conjugated secondary antibody (Thermo Fisher Scientific), and mounted using medium FluorSave (Calbiochem, Merck Millipore). The cells were then visualized using a fluorescence microscope (IX51, Olympus).

feature area and expression of E-selectin were calculated by using Excel (Microsoft). Highly correlated features were manually processed by using Masslynx 4.1 (Waters) to obtain a list of candidate molecular ions. Elemental composition and accurate mass were determined by using built-in functionality of Masslynx 4.1 (Waters). Compound 1 was quantified in the negative-ion mode using its authentic standard. Internal deuterated standard of [2,3,5,6-2H4]-4-OH-benzoic acid was added at a final concentration of 100 μM to compensate for matrix effects. Cell Culture. Test materials were dissolved in dimethyl sulfoxide (DMSO) to the concentration of 200 mg/mL or 10 mM. Endothelial Cell Proliferation Medium (ECPM, Provitro) supplemented with 10% heat-inactivated fetal bovine serum (HyClone, GE Healthcare) was used for the cultivation of HUVECs. The HUVECs were a kind gift of Prof. J. Ulrichová (Faculty of Medicine and Dentistry, Palacky University, Olomouc). THP-1 (a monocyte-like cell line, derived from the peripheral blood of a childhood case of acute monocytic leukemia cells) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with 2 mM L-glutamine, 1% penicillin-streptomycin, and 10% fetal bovine serum (Sigma-Aldrich) under standard cell culture conditions in a humid environment at 5% CO2 and 37 °C. The standard trypsinization procedure twice or three times a week was used for cultivation of cells. THP-1 cells were kindly provided by the Leibniz Institute for German Collection of Microorganisms and Cell Cultures (DSMZ). Cell-Surface ELISA CD62E (E-Selectin, ELAM). Enzyme-linked activity assay (ELISA) was used to detect the levels of cell adhesion molecule ELAM on HUVECs after 30 min of incubation with tested extracts, fractions, or compounds and 4 h of stimulation with TNFα as described earlier.18 Experiments were repeated three times in triplicate. Cytotoxicity Testing. Calcein AM (Molecular Probes, Invitrogen) cytotoxicity assays after 4 or 24 h of treatment in the HUVECs were used to measure the cytotoxicity of extracts, fractions, or compounds for ELAM expression assay as described previously.18 Triplicates of at least three independent experiments were used. THP-1 Adhesion Assay. Adhesion of THP-1 cells on TNFαstimulated HUVECs after the pretreatment for 30 min with test extracts, fractions, or pure compounds and stimulation with TNFα for 24 h, and 1 h of adherence was determined as described earlier.6 Four different biological replicates were performed in triplicate. Flow Cytometric Analysis of Cell Adhesion Molecule ICAM-1. The level of ICAM-1 on the cell surface of HUVECs after the pretreatment for 30 min with extract, fractions, or compounds and stimulation with TNFα for 24 h was measured using flow cytometry as described previously.18 At least three different sets of experiments were performed in triplicates. SDS−Poly(acrylamide) Gel Electrophoresis and Immunoblotting. Cells were pretreated for 30 min with the test extract, fractions, or compounds and stimulated with TNFα for another 30 min. Treated cells were scratched and lysed in RIPA protein extraction buffer (20 mM Tris-HCl, pH 7.4, 5 mM ethylenediaminetetraacetic acid (EDTA), 2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′N′tetraacetic acid (EGTA), 100 mM NaCl, 2 mM NaF, 0.2% Nonidet P40, 30 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 10 mg/mL of aprotinin and leupeptin). Proteins were quantified using the Bradford assay (Bio-Rad Laboratories) according to the manufacturer’s protocol, separated on 10% or 12% SDS-PAGE gels, transferred to nitrocellulose membranes (Bio-Rad Laboratories), blocked with 5% (w/v) nonfat dry milk and 0.1% Tween-20 in TBS for 1 h, and probed with specific primary and horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology; Santa Cruz Biotechnology). Chemiluminiscence was visualized with West Pico Supersignal chemiluminescent detection reagent (Thermo Fisher Scientific), and the signal was detected by CCD camera. Immunodetection of anti-β-actin monoclonal antibody (Santa Cruz Biotechnology) was performed to confirm equal protein loading. Experiments were repeated three times, and protein expression in treated cells was compared to untreated controls. Immunofluorescence Labeling Methods. Cells were treated as described above. Then, the cells in eight-well ibidi μ-slides (ibidi) were

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +420 585632173. E-mail: [email protected]. ORCID

Johannes Van Staden: 0000-0003-0515-1281 Jiri Gruz: 0000-0002-8546-9697 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The excellent technical assistances from O. Hustáková and L. Slobodianová are gratefully acknowledged. We thank Prof. Ulrichová for the kind gift of HUVEC cells. This work was supported by the National Research Foundation, Pretoria (No. 88455), and ERDF project “Development of preapplied research in nanotechnogy and biotechnology” (No. CZ.02.1.01/0.0/0.0/17_048/0007323).

■

REFERENCES

(1) Riddle, J. M. Isis 1985, 76, 319−330. (2) Gerrard, A. W. Pharm. J. 1877, 8, 214−215. (3) Christenhusz, M. J. M.; Byng, J. W. Phytotaxa 2016, 261, 201− 217. (4) Evidente, A.; Kireev, A. S.; Jenkins, A. R.; Romero, A. E.; Steelant, W. F.; et al. Planta Med. 2009, 75, 501−507. (5) Ceriotti, G. Nature 1967, 213, 595−596. (6) Fuchs, S.; Hsieh, L. T.; Saarberg, W.; Erdelmeier, C. A.; Wichelhaus, T. A.; Schaefer, L.; Koch, E.; Fürst, R. J. Cell Mol. Med. 2015, 19, 1021−1032. (7) Ncube, B.; Nair, J. J.; Rarova, L.; Strnad, M.; Finnie, J. F.; van Staden, J. S. Afr. J. Bot. 2015, 97, 69−76. (8) Nair, J. J.; Aremu, A. O.; van Staden, J. J. Ethnopharmacol. 2011, 137, 1102−1106. (9) van Eekelen, H. D. L. M.; Gijsbers, L.; Maliepaard, C. A.; Vreeburg, R. A. M.; Finkers, R.; Tikunov, Y. M.; Gomez Roldan, V. M.; de Haan, L. H. J.; de Vos, R. C. H.; Aarts, J. M. M. J. G.; Rietjens, I. M. C. M.; Bovy, A. G. Metabolomics 2015, 11, 302−311. (10) Piozzi, F.; Marino, M. L.; Fuganti, C.; Di Martino, A. Phytochemistry 1969, 8, 1745−1748. (11) Trepels, T.; Zeiher, A. M.; Fichtlscherer, S. Endothelium 2006, 13, 423−429. (12) Schmerwitz, U. K.; Sass, G.; Khandoga, A. G.; Joore, J.; Mayer, B. A.; Berberich, N.; Totzke, F.; Krombach, F.; Tiegs, G.; Zahler, S.; Vollmar, A. M.; Fürst, R. Arterioscler., Thromb., Vasc. Biol. 2011, 31, 280−288. (13) Dumont, P.; Ingrassia, L.; Rouzeau, S.; Ribaucour, F.; Thomas, S.; Roland, I.; Darro, F.; Lefranc, F.; Kiss, R. Neoplasia 2007, 9, 766− 776. (14) Orlikova, B.; Legrand, N.; Panning, J.; Dicato, M.; Diederich, M. Cancer Treat. Res. 2014, 159, 123−143. (15) Fürst, R. Planta Med. 2016, 82, 1389−1394. (16) Tabas, I.; Glass, C. K. Science 2013, 339, 166−172. (17) Ingrassia, L.; Lefranc, F.; Dewelle, J.; Pottier, L.; Mathieu, V.; Spiegl-Kreinecker, S.; Sauvage, S.; El Yazidi, M.; Dehoux, M.; Berger, W.; Van Quaquebeke, E.; Kiss, R. J. Med. Chem. 2009, 52, 1100−1114. (18) Morrogh-Bernard, H. C.; Foitová, I.; Yeen, Z.; Wilkin, P.; de Martin, R.; Rárová, L.; Doležal, K.; Nurcahyo, W.; Olšanský, M. Sci. Rep. 2017, 7, 16653.

E


Identification of Narciclasine as an in Vitro Anti-Inflammatory

Recommend Documents