TY - JOUR
AU1 - Hamed, Ahmed R
AU2 - El-Hawary, Seham S
AU3 - Ibrahim, Rana M
AU4 - Abdelmohsen, Usama Ramadan
AU5 - El-Halawany, Ali M
AB - Abstract Six halophytes, namely, Aptenia cordifolia var. variegata, Glottiphyllum linguiforme, Carpobrotus edulis, Ferocactus glaucescens, F. pottsii and F. herrerae were investigated for chemopreventive effect. Prioritization of most promising plant for further investigation was carried out through an integrated liquid chromatography–high resolution electrospray ionization mass spectrometry profiling—bioassay guided approach. NAD(P)H: quinone oxidoreductase-1 (NQO-1) induction in cultured murine hepatoma cells (Hepa-1c1c7) and inhibition of nitric oxide (NO) production in lipopolysaccharide-activated macrophages (RAW 264.7) were carried out to investigate chemopreventive effect. Bioassay data revealed that F. herrerae, A. cordifolia, C. edulis and F. glaucescens were the most active with 2-, 1.7-, 1.6- and 1.5-folds induction of NQO-1 activity. Only F. glaucescens exhibited >50% inhibition of NO release. LCMS profiling of the F. glaucescens revealed its high content of flavonoids, a known micheal acceptor with possible NQO-1 induction, as proved by quantitative high-performance liquid chromatography analysis. Thus, the extract of F. glaucescens was subjected to chromatographic fractionation leading to the isolation of four compounds including (i) 2S-naringenin, (ii) trans-dihydrokaempferol (aromadendrin), (iii) 2S-naringenin-7-O-β-d-glucopyranoside and (iv) kaempferol-7-O-β-d-glucopyranoside (populnin). The current study through an LCMS dereplication along with bio guided approach reported the activity of populnin as NO inhibitor and NQO-1 inducer with promising chemopreventive potential. Introduction Chemoprevention is a strategy to slow or prevent the process of cancer development by intervening in the process of carcinogenesis (1). Due to the adverse effects produced by chemotherapy and radiation therapy, plant derived phytochemicals with chemoprotective effects have received considerable attention in recent years (2). Plant phytochemicals are in demand as they are safer for long-term use in cancer patients and reduce the side effects of conventional cancer therapy (3). The reactive oxygen species (ROS) are physiological metabolites generated as part of the body’s normal metabolic process (4). A low physiological level of ROS can be scavenged efficiently by the cellular antioxidant mechanisms (5). However, the overproduction of ROS converts into a state of oxidative stress (6). Oxidative stress contributes to carcinogenesis by directly attacking DNA causing a modification of gene expression or by modulating signal transduction pathways (7). The induction of phase II detoxification enzymes, such as NAD(P)H: quinone reductase (NQO1) is associated with the metabolic detoxification of carcinogens, alleviating oxidative stress and decreasing the incidence or progression of cancer (8). Excessive production of nitric oxide (NO) during chronic inflammation is considered a causative factor of cellular injury and cancer (9). Sustained exposure to NO can mediate DNA damage, cause tumor suppressor gene mutations and affect tumor biology, including angiogenesis and metastasis (10). Furthermore, increased expression of inducible NO synthase (iNOS) has been observed during tumorigenesis (11). Consequently, inhibition of iNOS and NO-releasing agents could be an important strategy in cancer chemoprevention beside preventing inflammatory diseases (12). Halophytes are salt-resistant plants, mainly grown in deserts and adapted to harsh environmental conditions (water deficiency, salinity, high temperature and hard soil) (13). Such conditions may trigger oxidative stress in plants by generating excessive amounts of ROS (14). Thus, halophytes have developed highly effective defense systems, to withstand and quench these toxic ROS (15). Several desert plants were reported to exhibit anticancer effects indicating that the same defensive secondary metabolites protecting them against the harsh environment can be used as anticancer or cancer chemopreventive agents (16). Interestingly, phenolic antioxidant compounds contained in halophytes exhibit strong biological activity sometimes exceeding synthetic antioxidants (17). Therefore, it is rational to investigate the underutilized halophytes for their chemical composition, health benefits and biological importance (18). Aptenia cordifolia (L.f.) Schwantes and Carpobrotus edulis (L.) N.E. Br. (Aizoaceae) are widely used as ground-covers and ornamentals as well as for their biological activities (19). C. edulis was used in the African traditional medicine for the treatment of many diseases e.g., diarrhea, sinusitis, infantile eczema and tuberculosis (20). Several in vitro biological activities were reported for this species including antioxidant (19), antimicrobial (21), immune modulating (22), anticholinesterase, anticancer (23) and anti-neuroinflammatory activities (24). The phenolic content of the shoots was evaluated using the colorimetric method and high-performance liquid chromatography (HPLC) analysis (25). The procyanidins and propelargonidins content of C. edulis were characterized by LC/ESI-MS/MS (26). Nevertheless, few studies have examined the phenolic profile of this plant from Egypt. A. cordifolia was used by traditional healers in the treatment of nervous and sexual complaints, as well as pleurisy and dropsy (27). Leaves and stems were reported to relief the inflammation of swollen joints, sore throats and tonsillitis (28). Various classes of compounds have been separated from A. cordifolia like sterols, flavonoids (29), oxyneolignans, phenolic acids, lignans, amides (30, 31) and alkaloids (28). However, no phytochemical or biological studies were found about the variegated variety of A. cordifolia. Also, no reports were traced concerning the phytochemical composition or the biological effects of Glottiphyllum linguiforme (L.) N.E. Br. (Aizoaceae), Ferocactus glaucescens (DC.) Britton & Rose, F. pottsii (Salm-Dyck) Backeb and F. herrerae (J.G. Ortega) (Cactaceae) species. In the present study, the six halophytes, abundantly cultivated as ornamental plants in Egypt were selected for metabolic profiling using liquid chromatography–high resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS) followed by quantification of their main components using high-performance liquid chromatography with ultraviolet (HPLC-UV). In addition to investigating the chemopreventive potential of the ethanolic extracts of these halophytes using cellular bioassays. Experimental Instrumentation LC-HR-MS analyses Analysis was performed using Thermo Scientific Accela HPLC (Bremen, Germany) coupled with Accela UV–vis and Thermo Fisher Scientific Exactive (Orbitrap) mass spectrometer (Bremen, Germany). The analysis was achieved on HiChrom, ACE (Theale, Berkshire, UK) C18column (particle size 5 μm, 75 × 3.0 mm Ø). HPLC determination of flavonoids. Analysis was performed on Agilent 1100 series HPLC system (Agilent Technologies, Palo Alto, CA) equipped with a quaternary pump G1311A, degasser G1322A, UV detector and Agilent Chem Stations software. NMR analysis The isolated compounds were analyzed using Bruker Ascend™ 400/R Nuclear magnetic resonance (NMR) spectrometer (1H-NMR, 400 MHz and 13C-NMR, 100 MHz). Materials and Reagent Plant materials Three Ferocactus plants from family Cactaceae (F. glaucescens (DC.) Britton & Rose, F. pottsii (Salm-Dyck) Backeb and F. herrerae J.G. Ortega) and three Aizoaceae plants (A. cordifolia var. variegata (L.f.) Schwantes, G. linguiforme (L.) N.E.Br and C. edulis (L.) N.E. Br), were obtained from Helal Cactus Farm, Shibin El-Qanater, Qalyubia Governorate. They were collected in the flowering stage in May 2016 (1 kg for each plant). Their Identities were verified morphologically by Botany specialist, Dr. Mohamed El-Gibali, former researcher of Botany, Department of Botany, National Research Centre (NRC) and by Mrs. Tereez Labib, Consultant of Plant Taxonomy at Ministry of Agriculture and former director of Orman botanical garden, Giza, Egypt. Voucher specimens of the plants were kept at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University. Dried powdered plants were repeatedly extracted by cold percolation with 70% ethanol till exhaustion and the combined extracts, in each case, were evaporated in vacuo to obtain a residue of 100 g of each plant used for biological screening, liquid chromatography–mass spectrometry (LC/MS) analysis and phytochemical study. LC-HR-ESI-MS analysis of the plants The dried ethanolic extract of each plant was prepared at a concentration of 1 mg/mL in methanol. Mobile phase consisting of purified water (A) and acetonitrile (B) with 0.1% formic acid in each solvent with a flow rate of 300 μL/min was used. The gradient elution was started with 10% B then linearly increased to 100% B within 30 min and remained isocratic for 5 min then linearly decreased to 10% B for 1 min. The mobile phase was equilibrated for 9 min prior to the next injection. The injection volume was 10 μL. The tray temperature was maintained at 4°C whereas the column oven was controlled at 20°C. HR-MS was carried out with a spray voltage at 4.5 kV and capillary temperature at 320°C in both positive and negative ionization modes. The mass range was set from m/z 100–2000 using in-source collision-induced dissociation mechanism. LC/MS spectra were viewed using ThermoXcalibur 2.1 (Thermo Scientific, Germany). The RAW data files were converted by ProteoWizard into mzML format then imported to the MZmine 2.8 (data mining software). Mass ion peaks were isolated with a centroid detector threshold with MS level of 1 and noise level set to 1.0 × 102. Chromatogram builder was used with a minimum time span set to 0.1 min, minimum height to 5 × 103 and m/z tolerance to 0.001 m/z or 5.0 ppm. Chromatogram deconvolution was performed using baseline cut-off algorithm with minimum peak height: 5 × 103, peak duration range (0–0.4 min) and baseline level: 5 × 102. The separated peaks were then deisotoped using isotopic peaks grouper (m/z tolerance: 0.001 m/z or 5.0 ppm, retention time tolerance: 0.2 absolute (min), maximum charge: 2 and representative isotope: most intense). The parameters for data filtering, gap-filling and the RT normalizer were set to m/z tolerance: 0.001 m/z or 5.0 ppm, retention time tolerance: 0.2 absolute (min) and minimum standard intensity: 5 × 103. The peak lists were then aligned together using the join aligner with m/z tolerance: 0.001 m/z or 5.0 ppm, weight for m/z: 20, RT tolerance: 0.2 absolute (min), weight for RT: 20. Adduct search was performed for Na+, K+, NH+4, formate and ACN+ (retention time tolerance: 0.2 absolute (min), m/z tolerance: 0.001 m/z or 5.0 ppm, max relative adduct peak height: 50%). The processed data were then subjected to formula prediction by selecting atoms C, H, N, O and S, adjusting the three suboptions of heuristics element count and the parameters of isotope pattern filter to work with all isotope peaks. Finally, peak identification to search for unidentified peaks using LipidMaps online database. The data were then converted into a CSV file, consequentially providing ID number, m/z, retention time and peak area for each peak in all samples. Quantitative determination of main constituents using HPLC-DAD The separation was carried out on a LiChrospher® RP-18 HPLC column 250 × 4.6 mm, 5 μm; Merck, Germany). The mobile phase and HPLC conditions were the same as those for liquid chromatography coupled to high-resolution mass spectrometry (LC-HR-MS) analysis. The analysis was carried out in triplicates at room temperature, and the detection wavelength was set at 280 nm. The concentration of each ethanolic extract was 10 mg/mL in methanol. Each standard compound (p-coumaric acid, syringic acid, naringenin, rutin and naringenin 7-glucoside) was dissolved in methanol and used to establish calibration curves at concentration range of 500–31.25 μg/mL (Figures S4 and S5). Cell culture and treatment Murine macrophage RAW264.7 cells (ATCC®) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin sulphate (100 μg/mL) and l-glutamine (4 mM) in a humidified 5% CO2 atmosphere. For subculture and treatments, cells were scrapped off the flasks using sterile scrappers (Greiner Bio-one, Frickenhausen, Germany). Murine hepatoma Hepa-1c1c7 cells (ATCC®, USA) were grown as a monolayer culture in α-modified minimum essential medium Eagle supplemented with 10% (v/v) heat-and charcoal–inactivated FBS, 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin sulphate and routinely maintained in humidified incubator (Sartorius CMAT, Germany, 37°C, 5% CO2/95% air). At ~80% confluence, cells were routinely subcultured with Trypsin EDTA solution. Inhibition of LPS-induced NO release RAW264.7 Cells (5 × 105 cells/mL) were seeded onto 96-well microplates overnight. Cells were treated with either 0.1% v/v DMSO (negative control lipopolysaccharide [LPS−]), 100 ng/mL LPS+ (Sigma-Aldrich, from Escherichia coli serotype O111: B4) or LPS in the presence of 30 μg/mL of the extracts or 7.5, 15 or 30 μM of isolated compounds. Griess assay (32) was employed to determine NO in quadrates of culture supernatants following 24 h exposure time in all groups. Briefly 100 μL of culture supernatant from each well were mixed with equal volume of Griess reagent, mixed at room temperature and absorbance was measured at 540 nm on a Tristarlb 942® microplate reader (Berthold, Germany). Inhibition (%) was calculated relative to the LPS only group (LPS+), normalized to viable cell number as determined with MTT reduction assay. NO assay Nitrite level was used as an indicator of NO production in the cell culture medium by the Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylene diamine dihydrochloride and 2.5% phosphoric acid) (32, 33). From each well, culture supernatant was mixed with equal volume of Griess reagent for 15 min, and optical density was measured at 540 nm. % NO production was determined relative to the positive control (LPS only treated group). iNOS western blot analysis Overnight culture of RAW264.7 (6-well plates, initially seeded as 1.5 × 106 cells/well) were treated with compound 1 and 2 (7.5, 15 and 30 μM) or indomethacin (positive control, 250 μM). Following 24 h exposure, cells were washed using ice-cold PBS and scrapped in RIPA lysis buffer. After incubation for 20 min on ice, cell lysates were centrifuged at 15 000×g for 10 min at 4 °C and protein concentration was determined on a Thermo® nano-drop spectrophotometer. Proteins in cell lysates were resolved on 10% PAGE gel (Bio-Rad Tetra Cell®) and transferred onto nitrocellulose membrane using a Trans-blot mini module (Bio-rad). The membrane was blocked using 5% skim milk for 1 h at room temperature, followed by an overnight incubation at 4 °C with 1:1000 dilution of iNOS primary antibody (Merck, MA, USA). Following four washes, the membranes were incubated with 1:10 000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Membrane proteins were detected using the ECL western blotting detection substrate. Assessment of the induction of NQO1 in Hepa-1c1c7 cells The induction of NQO1 in Hepa-1c1c7 cells was assessed. Briefly, cells (3 × 105 cells/mL) were seeded onto 6-well plates and left overnight to adhere and form semiconfluent monolayers. Monolayers were treated with either vehicle (final concentration 0.1% v/v DMSO), plant ethanolic extracts (100 μg/mL) or isolated compounds (100 μM) for additional 24 h. In parallel, sulforaphane was used as a positive control for NQO1 induction. After aspiration of treatment media, monolayers were washed with ice-cold Dulbecco’s PBS (2 mL/well). Cells were then scrapped in ice-cold lysis buffer (25 mM Tris–HCl, pH 7.4, 250 mM sucrose and 5 μM FAD) and transferred to labeled microcentrifuge tubes. Cell suspensions were then sonicated on ice for 5 s (20% amplitude). Sonicates were then centrifuged (15 000×g for 10 min) and the supernatants (cytosolic fractions) were aliquoted and stored at −80 °C freezer until assayed for enzyme activity. Kinetic DCPIP reduction assay for NQO1 The dicoumarol-sensitive NQO1 activity was measured in cell lysate according to previously reported methods (34, 35) as optimized in our laboratory for measurement using 96-well microplates. Briefly, the reaction mixture contained the following reagents in a final volume of 200 μL: 25 mM Tris buffer (pH 7.4), 0.7 mg/mL bovine serum albumin, 0.2 mM β-NADH, 20 μM of 2, 6-dichlorophenolindophenol (DCPIP) in the absence (total reductase activity) or the presence of 20 mM dicoumarol (inhibited non-NQO1 activity). The kinetic determination of enzyme activity was carried out using a Tristar2 lb 942 multimode reader (Berthold, Germany), monitoring the enzymatic reduction of DCPIP at 600 nm within 1 min. Using an extinction coefficient of 21 000 for DCPIP, reaction rates were normalized to the total protein contents in each sample determined at a micro-scale using a Thermo® spectrophotometer. The enzyme activity was expressed as folds of specific NQO1 activity in tested samples over vehicle-treated control cell sonicates based on triplicate assays. Isolation of main constituents from F. glaucescens The ethanolic extract (100 g) was subjected to liquid–liquid fractionation with n-hexane (55 × 200 mL), methylene chloride (5 × 200 mL), ethyl acetate (6 × 200 mL) to obtain their corresponding fractions. The methylene chloride fraction (25 g) was chromatographed on a silica gel H vacuum liquid chromatography column (VLC) (12.5 × 10 cm), eluting with CH2Cl2 and followed by a gradient of CH2Cl2-EtOAc up to 100% EtOAc and EtOAc-MeOH up to 100% MeOH to afford nine fractions (I–IX). Fraction VII was subjected to a VLC silica gel RP-18 column (3 × 20 cm), eluting with 0–50% of MeOH in H2O to afford three subfractions (VII-1, VII-2 and VII-3). Subfraction VII-2 was purified on Sephadex LH-20 column (2 × 20 cm) eluting with 50% aqueous MeOH to obtain compound 1 (30 mg). Subfraction VII-3 was purified on a silica gel 60 A column (25 × 25 cm) using n-hexane: EtOAc (80:20 v/v) to afford compound 2 (25 mg). The ethyl acetate fraction (20 g) was chromatographed on a polyamide column (250 g, 4 × 35 cm) eluting with 0–100% MeOH in water to afford 9 fractions (1–9). Fraction 4 was loaded onto a sephadex LH-20 column (2 × 15 cm) using 50% aqueous methanol followed by a silica gel RP-18 column (2 × 15 cm) eluting with methanol: water (30:70 v/v) to give compound 3 (12 mg). Fraction 5 was purified on a silica gel RP-18 column (2 × 15 cm) eluting with methanol: water (30:70 v/v) to give compound 4 (20 mg). Results LC-HR-ESI-MS metabolite profiling The chemical constituents of the 70% ethanol extracts were analyzed via LC-HR-ESI-MS in both positive and negative ionization modes to better interpret the diversity of available phytochemicals within the six plants. All compounds characterized in A. cordifolia var. variegata, C. edulis, G. linguiforme, F. glaucescens, F. herrerae and F. pottsii ethanolic extracts, including metabolite class, retention times, experimental m/z, molecular formulas, fragment ions, as well as putative compounds are summarized in Tables S1–S7 (supplementary materials). The detection of the metabolites by LC/MS was accomplished by acquisition of full-scan HR-MS and product ion spectral datasets for all metabolites in the extracts followed by data mining, determination of empirical formulas using MZmine 2.8 software. A total of 268 compounds were identified based on their accurate MS, fragment ions and by searching online databases; lipidMaps, dictionary of natural products (DNP, 2015) and the reported literature. The representative LC/MS base peak chromatograms of the six extracts are shown in (Figure S1A–L, supplementary material). Identified metabolites belonged to 6 classes including 31 phenolic acids and their conjugates, 98 flavonoids, 28 anthocyanins/procyanidins, 56 fatty acids, 14 alkaloids and 27 sterols/triterpenes, along with 14 miscellaneous compounds. Identification of phenolic acids and their conjugates Simple phenolic acids e.g., hydroxycinnamic acids as well as their conjugates with quinic acid, malic acid, glucose and benzoic acid were detected in this study. In the MS spectra, the predominant fragments of 191, 163 and 179 amu for quinic acid, coumaric acid and caffeic acid, respectively, are diagnostic for hydroxycinnamic acid derivatives (Table S1) (36). Phenolic acid amides e.g., N-Trans-feruloyl tyramine (peak 164) and N-Trans-feruloyl octopamine (peak 240) were identified in the ethanolic extract of A. cordifolia var. variegata as reported in previous paper (30). Identification of flavonoids The identified flavonoids generally occur as sugar conjugates, principally as O-glycosides (Table S2). In MS analyses, the nature of the sugars could be detected from the loss of the sugar residue, that is, hexose (−162 amu), rhamnose (146 amu) and pentose (−132 amu). All glycosides identified based on their mass spectra were flavones e.g., kaempferol hexoside (peak 245), apigenin O-hexoside (peak 143), Myricetin pentoside (peak 132) or flavanone derivatives e.g., naringin (peak 158) and aromadendrin (peak 129). Two prenylated flavonoids, prenyl naringenin (peak 242) and prenyl apigenin (peak 244) were also identified by comparing their data with previous reports (37, 38). Figure 1 Open in new tabDownload slide Histogram representing the effect of the six ethanolic extracts on the activity of NQO1 enzyme in cultured murine hepatoma cells (Hepa-1c1c7) compared to sulforaphane as a positive control. Figure 1 Open in new tabDownload slide Histogram representing the effect of the six ethanolic extracts on the activity of NQO1 enzyme in cultured murine hepatoma cells (Hepa-1c1c7) compared to sulforaphane as a positive control. Identification of anthocyanins and procyanidins The ethanolic extracts of the six halophytes were found to contain anthocyanins especially C. edulis as reported previously (26). The identified anthocyanins (Table S3) showed similar mass spectral patterns in terms of having an anthocyanidin (cyanidin, m/z 287; delphinidin, m/z 303, pelargonidin, m/z 271, petunidin, m/z 317 and malvidin, m/z 331) conjugated to one or more sugar moieties. Some anthocyanins peaks showed mass data due to loss of acetoyl, coumaroyl and malonyl groups beside the sugar unit e.g., delphinidin malonyl hexoside (peak 48), malvidin acetyl hexoside (peak 51) and petunidin coumaroyl hexoside (peak 73). Proanthocyanidins were identified as free flavan-3-ols e.g., catechin (peak 23), epicatechin (peak 38), (epi) afzelechin (peak 135) or as galloylated derivatives e.g., gallocatechin (peak 13), epigallocatechin (peak 18), epigallocatechin gallate (peak 33) and the glycosylated derivatives e.g., catechin rhamnosyl hexoside (peak 25), catechin hexoside (peak 77). Procyanidin dimer (peak 32) and trimer (peak 41) were also identified by comparing their MS data with published reports (39). Identification of sterols and triterpenes Mass spectra of triterpenes showed characteristic fragment ions resulting from loss of one and/or two water molecules (−18 amu) (Table S4) (40). Triterpenes belonging to different classes were identified in the ethanolic extracts of the selected halophytes mainly, uvaol (peak 193), betulin (peak 180) and amyrin (peak 212). Some MS signals were also assigned to triterpenic acids e.g., oleanolic (peak 214) and betulinic acid (peak 236) with major fragment ions due to loss of CO2 group (−44 amu) (41). Several sterols, e.g., campesterol (peak 175), sitosterol (peak 251) and stigmasterol (peak 262) were readily identified by their fragment ions due to loss of water molecule, steroidal glycosides e.g., campesteryl hexoside (peak 118) and stigmasteryl hexoside (peak 191) gave ions due to loss of the sugar moiety (−162 amu) (42). Identification of fatty acids The normalized abundances for the C6H6 (−78 amu), C5H6O2 (−98 amu) and C8H8O2 (−136 amu) fragment ions from the molecular ion peaks are characteristic in the MS spectra of fatty acids (43). Besides, the fragment ions formed by the neutral loss of carbon dioxide (−44 amu) and fragment ion corresponding to the loss of water molecule (−18 amu) (44) (Table S5). Several unsaturated fatty acids, e.g., octadecenoic acid (peak 151) and hexadecatrienoic acid (peak 264) were readily identified. Some MS signals were assigned to saturated fatty acids, e.g., arachidic acid (peak 172) and hydroxylated fatty acids e.g., hydroxy linolenic acid (peak 157). Identification of alkaloids Mesembrine-type alkaloids were reported in Aptenia species (45). Alkaloids are better ionized in positive mode compared to flavonoids that exhibit better ionization in the negative mode (46). Thus, 14 known alkaloids were exclusively identified in the MS spectra obtained by the positive mode from the ethanolic extract of A. cordifolia var. variegata for the first time (Figure S1B, Table S6). Four alkaloids were of mesembrine-type, namely, mesembrine (peak 37), mesembranol (peak 102), epi-mesembranol (peak 80) and 4′-O-desmethyl mesembranol (peak 82). Five peaks were identified as ∆4-mesembrine-type, namely, ∆4-mesembrenone (peak 72), O-acetyl mesembrenol (peak 196), 4′-O-demethyl mesembrenol (peak 39), sceletenone (peak 140) and 4′-O-methylsceletenone (peak 113). One compound was of ∆7-mesembrine-type and identified as ∆7-mesembrenone (peak 71). Three alkaloids were of joubertiamine-type, namely, dihydrojoubertiamine (peak 52), O-methyl-dehydrojoubertiamine (peak 101) and 3′-Methoxy-4′-O-methyljoubertiamine (peak 176). Besides, the non-mesembrine alkaloid, hordenine (peak 16). All identified alkaloids exhibited the same fragmentation patterns as reported in previous literature (47–49). The structures of identified alkaloids are depicted in (Figure S2). The fragmentation pattern of mesembrine-type alkaloids is demonstrated in (Figure S3A) with mesembrine as representative compound for this class. Mesembrine (peak 37, Table S6) showed molecular ion peak (M+H)+ at m/z 290 and main fragment ion due to the formation of the aryl conjugated pyrrolidinium ion at m/z 217. Besides, the other fragment ions formed by the radical loss of methyl group, loss of hydrogen, cyclopropanone group and by the elimination of N-methyl aziridine to give several less abundant fragment ions as seen in (Figure S3A). In similar way mesembranol (peak 37), epi-mesembranol and desmethyl mesembranol (peak 82) were identified (Table S6). The introduction of a double bond in the 4, 5-position of the mesembrine-type alkaloids (Figure S2A and B) causes a drastic change in the appearance of the mass spectrum of the ∆4-mesembrine-type comparing to the mesembrine alkaloid as shown in the fragmentation scheme (Figure S3B). ∆4-Mesembrenone (peak 72) exhibited a molecular ion peak at m/z 288, accompanied by fragment ions at m/z 274 and m/z 262 correponding to [(M+H) − CH3] and [(M+H) − CO], respectively. Other ions formed from loss of N-methyl iminium ion and N-methyl aziridinium ion at m/z 248 and m/z 230, respectively. The acetyl derivative, O-acetyl mesembrenol (peak 196), 4′-O-desmethyl mesembrenol (peak 39) and sceletenone (peak 140) a mono-oxyaryl derivative were also identified. ∆7-Mesembrenone (peak 71, Figure S2C, Table S6) exhibited the same molecular ion peak as ∆4-mesembrenone with m/z of 288 but with a different fragmentation pattern. The mass spectrum of ∆7-mesembrenone showed fragment ion at m/z 274 due to loss of methyl radical and another ion at m/z 258 due to loss of ethylene group followed by loss of methyl radical to give fragment ion at m/z 244 as depicted in Figure S3C. The seco-mesembrine (joubertiamine-type, Figure S2D) alkaloids showed mass fragmentation mainly due to the cleavage of N, N-dimethyl amino ethyl side chain to give fragment ions due to loss of N, N-dimethyl iminium ion (−58 amu) and N, N-dimethyl aziridinium ion (−72 amu) as shown in (Figure S3D). Besides, these main fragments other ions may be formed from the loss of hydrogen, ethylene radical and/or methyl group. Dihydrojoubertiamine (peak 52), showed molecular ion peak at m/z 262 and major fragment ions due to loss of N, N-dimethyl iminium ion (−58 amu) to give an ion at m/z 204 and N, N-dimethyl aziridinium ion (−72 amu) to give an ion at m/z 177. O-Methyl-dehydrojoubertiamine (peak 101), and 3′-Methoxy-4′-O-methyl joubertiamine (peak 176) were also identified. Finally, the non mesembrine alkaloid hordenine (phenyl ethylamine-type, peak 16, Figure S2E, Table S6), was identified by its accurate mass 166.1240 (mass difference equals to 0.0007 m/z). Other chemical compounds identified in the ethanolic extracts Finally, lignans e.g., secoisolariciresinol (peak 96), lariciresinol (peak 142) and magnolol (peak 151), coumarins e.g., umbelliferone (peak 9) and esculin (201), dihydrochalcones e.g., phloretin (peak 31) and its glycoside phloridzin (peak 59) along with many other metabolites were identified in the ethanolic extracts of the six halophytes (Table S7). HPLC quantitation of major compounds According to the qualitative results, a quantitative analysis method for the ethanolic extracts of the six plants was established by high-performance liquid chromatography with a diode-array detector (HPLC-DAD). Five representative compounds (p-coumaric acid, syringic acid, naringenin, rutin and naringenin 7-glucoside) unequivocally identified were chosen as marker components. Peaks in the chromatograms were identified by comparing the retention times and UV spectra with those of the standards. The HPLC-UV detection profiles are illustrated in supplementary data (Figure S6). p-Coumaric acid was the main component with concentration varied from 3.55 ± 0.02 to 4.74 ± 0.01 followed by syringic acid with content ranged from 0.62 ± 0.01 to 0.73 ± 0.03 mg/g of the dried ethanolic extract. Rutin was detected in three extracts at concentrations equaled to 1.01 ± 0.03, 0.99 ± 0.07 and 2.32 ± 0.91 in F. glaucescens, F. pottsii and C. edulis, respectively. Naringenin was detected in F. glaucescens and G. linguiforme at concentration levels of 2.76 ± 0.03 and 2.44 ± 0.01, respectively. Finally, naringenin-7-glucoside was marker component in F. glaucescens and F. pottsii and its amounts was 2.54 ± 0.01 and 2.82 ± 0.01, respectively. NQO1 induction and NO inhibition bioassays Among the six halophytic plants, F. herrerae, A. cordifolia var. variegata, C. edulis and F. glaucescens were the most active with 2-, 1.7-, 1.6- and 1.5-folds induction of NQO1 enzyme activity over the control, respectively. Although, G. linguiforme exhibited less effect with 1.2-fold increase in NQO1 activity and F. pottsii did not exert any remarkable activity, compared to sulforaphane as a positive control with 2.08-fold induction (Figure 1). On the other hand, only F. glaucescens extract exhibited 50% inhibition of NO release in LPS-induced RAW 264.7 macrophage cells, compared to indomethacin as positive control (74% inhibition). Isolation of main flavonoids from F. glaucescens The LC/MS analyses showed that F. glaucescens was the richest among other plants in flavonoids (30 identified flavonoids, Table S2). Literature data reported that flavonoids exhibited cancer chemopreventive activity through various pathways including the induction of phase II carcinogen metabolizing enzymes e.g., (NQO1) (50). Flavonoids were also reported as modulators of pro-inflammatory gene expression e.g., COX-2 and iNOS expressions (51). F. glaucescens exhibited moderate activity with 50% inhibition of NO release at concentration of 100 μg/mL. The extract also increased NQO1 activity by 1.5-fold over the control at the same concentration (Table I). Combining both bioassay and LC/MS results encouraged the investigation and isolation of major compounds from F. glaucescens. Table I Amounts of Five Markers Determined in the Ethanolic Extracts of the Six Plants Using HPLC-UV Analysis Plant . Concentration (mg/g, mean ± SD, n = 3) . Syringic acid . p-Coumaric acid . Rutin . Naringenin . Naringenin-7-glucoside . FG 0.66 ± 0.0058 — 1.01 ± 0.036 2.76 ± 0.032 2.54 ± 0.015 FH 0.64 ± 0.0057 4.7 ± 0.021 — — — FP 0.62 ± 0.01 4.6 ± 0.046 0.99 ± 0.076 — 2.82 ± 0.016 CA — 3.55 ± 0.025 2.32 ± 0.91 — — AP 0.73 ± 0.037 4.74 ± 0.017 — — — GL 0.67 ± 0.006 4.56 ± 0.011 — 2.44 ± 0.01 — Plant . Concentration (mg/g, mean ± SD, n = 3) . Syringic acid . p-Coumaric acid . Rutin . Naringenin . Naringenin-7-glucoside . FG 0.66 ± 0.0058 — 1.01 ± 0.036 2.76 ± 0.032 2.54 ± 0.015 FH 0.64 ± 0.0057 4.7 ± 0.021 — — — FP 0.62 ± 0.01 4.6 ± 0.046 0.99 ± 0.076 — 2.82 ± 0.016 CA — 3.55 ± 0.025 2.32 ± 0.91 — — AP 0.73 ± 0.037 4.74 ± 0.017 — — — GL 0.67 ± 0.006 4.56 ± 0.011 — 2.44 ± 0.01 — Notes: —: Not detected; FG, F. glaucescens; FH, F. herrerae; FP: F. pottsii; CA: C. edulis; AP: A. cordifolia var. variegata; GL: G. linguiforme. Open in new tab Table I Amounts of Five Markers Determined in the Ethanolic Extracts of the Six Plants Using HPLC-UV Analysis Plant . Concentration (mg/g, mean ± SD, n = 3) . Syringic acid . p-Coumaric acid . Rutin . Naringenin . Naringenin-7-glucoside . FG 0.66 ± 0.0058 — 1.01 ± 0.036 2.76 ± 0.032 2.54 ± 0.015 FH 0.64 ± 0.0057 4.7 ± 0.021 — — — FP 0.62 ± 0.01 4.6 ± 0.046 0.99 ± 0.076 — 2.82 ± 0.016 CA — 3.55 ± 0.025 2.32 ± 0.91 — — AP 0.73 ± 0.037 4.74 ± 0.017 — — — GL 0.67 ± 0.006 4.56 ± 0.011 — 2.44 ± 0.01 — Plant . Concentration (mg/g, mean ± SD, n = 3) . Syringic acid . p-Coumaric acid . Rutin . Naringenin . Naringenin-7-glucoside . FG 0.66 ± 0.0058 — 1.01 ± 0.036 2.76 ± 0.032 2.54 ± 0.015 FH 0.64 ± 0.0057 4.7 ± 0.021 — — — FP 0.62 ± 0.01 4.6 ± 0.046 0.99 ± 0.076 — 2.82 ± 0.016 CA — 3.55 ± 0.025 2.32 ± 0.91 — — AP 0.73 ± 0.037 4.74 ± 0.017 — — — GL 0.67 ± 0.006 4.56 ± 0.011 — 2.44 ± 0.01 — Notes: —: Not detected; FG, F. glaucescens; FH, F. herrerae; FP: F. pottsii; CA: C. edulis; AP: A. cordifolia var. variegata; GL: G. linguiforme. Open in new tab The CH2Cl2 and EtOAc-soluble fractions of F. glaucescens were the most potent in inducing NQO1 (Table I). Also, both fractions showed higher anti-inflammatory activity with 50.8% and 53.7% inhibition of NO release, respectively. Thus, the methylene chloride and ethyl acetate fractions were subjected to further purification leading to the isolation of four compounds, 1–4 (Figure 2). Their identities were established as; 2S-naringenin (1), trans-dihydrokaempferol (2), 2S-naringenin-7-O-β-d-glucopyranoside (3) and kaempferol-7-O-β-d-glucopyranoside (populnin)(4) (52–58) (Supplementary data). Figure 2 Open in new tabDownload slide Isolated compounds from F. glaucescens DC. (1), 2S-naringenin; (2), trans-dihydrokaempferol; (3), 2S-naringenin-7-O-β-d-glucopyranoside; (4), kaempferol-7-O-β-d-glucopyranoside (populnin). Figure 2 Open in new tabDownload slide Isolated compounds from F. glaucescens DC. (1), 2S-naringenin; (2), trans-dihydrokaempferol; (3), 2S-naringenin-7-O-β-d-glucopyranoside; (4), kaempferol-7-O-β-d-glucopyranoside (populnin). Isolated compounds (30 μM) were tested for NO inhibition, naringenin (1) and populnin (4) recorded 61.8 and 58.6% inhibition of NO release, respectively (data not shown). Therefore, these compounds were assessed for their ability to inhibit the protein expression of iNOS as shown in Figures 3 and 4. Co-treatment of RAW 264.7 cells with 100 ng/mL LPS and serial dilutions of 2S-naringenin (1) or populnin (4) at (0, 7.5, 15 or 30 μM) resulted in a concentration dependent inhibition of iNOS protein expression as revealed with western blotting analysis. Figure 3 Open in new tabDownload slide The protein expression of iNOS and β-actin was detected by western blotting. RAW 264.7 cells were treated with 100 ng/mL of LPS only or with 7.5–30 μM of naringenin (compound 1) or indomethacin (250 μM) for 24 h. Figure 3 Open in new tabDownload slide The protein expression of iNOS and β-actin was detected by western blotting. RAW 264.7 cells were treated with 100 ng/mL of LPS only or with 7.5–30 μM of naringenin (compound 1) or indomethacin (250 μM) for 24 h. Figure 4 Open in new tabDownload slide The protein expression of iNOS as detected by western blot. The cells were treated with100 ng/mL of LPS only or with 7.5–30 μM of Kaempferol-7-glucoside (Compound 4) or indomethacin (250 μM) for 24 h. Figure 4 Open in new tabDownload slide The protein expression of iNOS as detected by western blot. The cells were treated with100 ng/mL of LPS only or with 7.5–30 μM of Kaempferol-7-glucoside (Compound 4) or indomethacin (250 μM) for 24 h. 2S-Naringenin (1), aromadendrin (2) and populnin (4) were found to induce NQO1 activity with 1.57-, 1.52- and 1.55-folds, respectively, at concentration of 100 μM. Whereas, 2S-naringenin-7-O-β-d-glucopyranoside showed less activity. These results are in agreement with previous reports of naringenin effects against oxidative stress through induction of NQO1 enzyme activity (59). Discussion Naringenin is a flavanone with reported antioxidant, anti-inflammatory, anticancer and chemopreventive effects (60). The functional properties of naringenin e.g., antioxidant activity are attributed to the presence of three hydroxyl groups substituted on its aromatic rings. Although, glycosides of naringenin e.g., naringenin-7-glucoside is more hydrophilic and may require transporter proteins for its cellular uptake and thus exerted less biological effects (61, 62). Even though, aromadendrin contain no Michael reaction center, yet it can exert marked NQO1 induction and inhibit LPS-stimulated iNOS activity that suggested a redox mechanism of action instead of the Keap1/Nrf2/ARE mechanism of classic phase II inducers (63). On the other hand, populnin has beside the hydroxyl groups in its aromatic rings, an α, β-unsaturated carbonyl structure (Michael addition/acceptor site) and extensively conjugated double bond system (63). Herein, the glycosylation site on the 7-position retained the 3-OH free that is required for activity (64). Inhibition of LPS-stimulated iNOS production is less clearly correlated with the structure, despite the fact that different chemical classes of phase II enzyme inducers have showed remarkable anti-inflammatory potencies, suggesting common molecular targets (65). Interestingly, it is the first biological reports for populnin as anti-inflammatory agent and phase II enzyme inducer. Conclusion This study contributed to the identification and quality control of the six halophytes through the assessment of their marker components using HPLC-UV. In addition, it presents first chemical investigation of the genus Ferocactus and first reports for the identification of populnin and aromadendrin as chemopreventive agents. Conflict of interest The authors declare no conflict of interest. References 1. Lee , K.W. , Lee , H.J.; The roles of polyphenols in cancer chemoprevention ; BioFactors , ( 2006 ); 26 ( 2 ): 105 – 121 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Singh , R.P. , Dhanalakshmi , S., Agarwal , R.; Phytochemicals as cell cycle modulators a less toxic approach in halting human cancers ; Cell Cycle , ( 2002 ); 1 ( 3 ): 155 – 160 . Google Scholar Crossref Search ADS WorldCat 3. Hosseini , A. , Ghorbani , A.; Cancer therapy with phytochemicals: Evidence from clinical studies ; Avicenna Journal of Phytomedicine , ( 2015 ); 5 ( 2 ): 84 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 4. Cairns , R.A. , Harris , I.S., Mak , T.W.; Regulation of cancer cell metabolism ; Nature Reviews Cancer , ( 2011 ); 11 ( 2 ): 85 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Circu , M.L. , Aw , T.Y.; Reactive oxygen species, cellular redox systems, and apoptosis ; Free Radical Biology & Medicine , ( 2010 ); 48 ( 6 ): 749 – 762 . Google Scholar Crossref Search ADS PubMed WorldCat 6. Boulaaba , M. , Tsolmon , S., Ksouri , R., Han , J., Kawada , K., Smaoui , A. et al. ; Anticancer effect of Tamarix gallica extracts on human colon cancer cells involves Erk1/2 and p38 action on G 2/M cell cycle arrest ; Cytotechnology , ( 2013 ); 65 ( 6 ): 927 – 936 . Google Scholar Crossref Search ADS PubMed WorldCat 7. Dong , Z. , Surh , Y.J.; Dietary modulation of cell signaling pathways . CRC Press , United States , ( 2008 ). Google Scholar Crossref Search ADS Google Preview WorldCat COPAC 8. Surh , Y.-J. , Kundu , J.K., Na , H.-K.; Nrf2 as a master redox switch in turning on the cellular signaling involved in the induction of cytoprotective genes by some chemopreventive phytochemicals ; Planta Medica , ( 2008 ); 74 ( 13 ): 1526 – 1539 . Google Scholar Crossref Search ADS PubMed WorldCat 9. De Flora , S. , Ferguson , L.R.; Overview of mechanisms of cancer chemopreventive agents ; Mutation Research, Fundamental and Molecular Mechanisms of Mutagenesis , ( 2005 ); 591 ( 1–2 ): 8 – 15 . Google Scholar Crossref Search ADS WorldCat 10. Rao , C.V. ; Nitric oxide signaling in colon cancer chemoprevention ; Mutation Research, Fundamental and Molecular Mechanisms of Mutagenesis , ( 2004 ); 555 ( 1–2 ): 107 – 119 . Google Scholar Crossref Search ADS WorldCat 11. Ahn , B. , Ohshima , H.; Suppression of intestinal polyposis in ApcMin/+ mice by inhibiting nitric oxide production ; Cancer Research , ( 2001 ); 61 ( 23 ): 8357 – 8360 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 12. Lee , S. , Lee , J., Song , D., Pan , C., Nho , C., Kim , M., et al. ; Cancer chemopreventive effects of Korean seaweed extracts ; Food Science and Biotechnology , ( 2008 ); 17 ( 3 ): 613 – 622 . Google Scholar OpenURL Placeholder Text WorldCat 13. Khan , M.A. , Ungar , I.A., Showalter , A.M.; The effect of salinity on the growth, water status, and ion content of a leaf succulent perennial halophyte, Suaeda fruticosa (L.) Forssk ; Journal of Arid Environments , ( 2000 ); 45 ( 1 ): 73 – 84 . Google Scholar Crossref Search ADS WorldCat 14. Ksouri , R. , Megdiche , W., Falleh , H., Trabelsi , N., Boulaaba , M., Smaoui , A. et al. ; Influence of biological, environmental and technical factors on phenolic content and antioxidant activities of Tunisian halophytes ; Comptes Rendus Biologies , ( 2008 ); 331 ( 11 ): 865 – 873 . Google Scholar Crossref Search ADS PubMed WorldCat 15. Oueslati , S. et al. ; Phenolic content, antioxidant, anti-inflammatory and anticancer activities of the edible halophyte Suaeda fruticosa Forssk ; Food Chemistry , ( 2012 ); 132 ( 2 ): 943 – 947 . Google Scholar Crossref Search ADS WorldCat 16. Harlev , E. , Nevo , E., Solowey , E., Lee , S.B.; Cancer preventive and curative attributes of plants of the Cactaceae family: A review ; Planta Medica , ( 2013 ); 79 ( 09 ): 713 – 722 . Google Scholar Crossref Search ADS PubMed WorldCat 17. Ksouri , W.M. , Medini , F., Mkadmini , K., Legault , J., Magne , C., Abdelly , C. et al. ; LC–ESI-TOF–MS identification of bioactive secondary metabolites involved in the antioxidant, anti-inflammatory and anticancer activities of the edible halophyte Zygophyllum album Desf ; Food Chemistry , ( 2013 ); 139 ( 1–4 ): 1073 – 1080 . Google Scholar Crossref Search ADS PubMed WorldCat 18. Joseph , D. , Chakraborty , K., Subin , C., Vijayan , K.K.; Halophytes of chenopodiaceae and aizoaceae from south-east coast of India as potential sources of essential nutrients and antioxidants ; Journal of Food and Nutrition Research , ( 2013 ); 1 ( 5 ): 97 – 107 . Google Scholar OpenURL Placeholder Text WorldCat 19. Meddeb , E. , Charni , M., Ghazouani , T., Cozzolino , A., Fratianni , F., Raboudi , F. et al. ; Biochemical and molecular study of Carpobrotus edulis bioactive properties and their effects on Dugesia sicula (Turbellaria, Tricladida) regeneration ; Applied Biochemistry and Biotechnology , ( 2017 ); 182 ( 3 ): 1131 – 1143 . Google Scholar Crossref Search ADS PubMed WorldCat 20. Martins , M. , Ordway , D., Kristiansen , M., Viveiros , M., Leandro , C., Molnar , J. et al. ; Inhibition of the Carpobrotus edulis methanol extract on the growth of phagocytosed multidrug-resistant mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus ; Fitoterapia , ( 2005 ); 76 ( 1 ): 96 – 99 . Google Scholar Crossref Search ADS PubMed WorldCat 21. Falleh , H. , Oueslati , S., Guyot , S., Dali , A.B., Magne , C., Abdelly , C. et al. ; Polyphenol content and biological activities of Mesembryanthemum edule organs after fractionation ; Industrial Crops and Products , ( 2013 ); 42 : 145 – 152 . Google Scholar Crossref Search ADS WorldCat 22. Ordway , D. , Hohmann , J., Viveiros , M., Viveiros , A., Molnar , J., Leandro , C. et al. ; Carpobrotus edulis methanol extract inhibits the MDR efflux pumps, enhances killing of phagocytosed S. aureus and promotes immune modulation ; Phytotherapy Research , ( 2003 ); 17 ( 5 ): 512 – 519 . Google Scholar Crossref Search ADS PubMed WorldCat 23. Martins , A. , Vasas , A., Schelz , Z.S., Viveiros , M., Molnar , J., Hohmann , J. et al. ; Constituents of Carpobrotus edulis inhibit P-glycoprotein of MDR1-transfected mouse lymphoma cells ; Anticancer Research , ( 2010 ); 30 ( 3 ): 829 – 835 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 24. Rocha , M. , Rodrigues , M.J., Pereira , C., Pereira , H., da Silva , S.B., da Rosa Neng , N. et al. ; Biochemical profile and in vitro neuroprotective properties of Carpobrotus edulis L., a medicinal and edible halophyte native to the coast of South Africa ; South African Journal of Botany , ( 2017 ); 111 : 222 – 231 . Google Scholar Crossref Search ADS WorldCat 25. Falleh , H. , Ksouri , R., Boulaaba , M., Guyot , S., Abdelly , C., Magne , C.; Phenolic nature, occurrence and polymerization degree as marker of environmental adaptation in the edible halophyte Mesembryanthemum edule ; South African Journal of Botany , ( 2012 ); 79 : 117 – 124 . Google Scholar Crossref Search ADS WorldCat 26. Falleh , H. , Ouestali , S., Guyot , S., Dali , A.I., Magne , C., Abdelly , C. et al. ; LC/ESI-MS/MS characterisation of procyanidins and propelargonidins responsible for the strong antioxidant activity of the edible halophyte Mesembryanthemum edule L ; Food Chemistry , ( 2011 ); 127 ( 4 ): 1732 – 1738 . Google Scholar Crossref Search ADS WorldCat 27. Crouch , N. , Smith , G., Smith , M.T.; Aptenia cordifolia (L. f.) Schwantes (Mesembryanthemaceae) in Zulu traditional medicine - an overview ; Haseltonia , ( 2000 ); 7 : 30 – 36 . Google Scholar OpenURL Placeholder Text WorldCat 28. Gaffney , C.D. ; A study of Mesembryanthemaceae alkaloids , in Chemistry . University of Johannesburg : Nexus Current and Completed Projects , ( 2006 ), p. 134 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 29. Elgindi , D. , Mabry , T.; Flavonoids of Aptenia cordifolia ; Asian Journal of Chemistry , ( 1999 ); 11 ( 4 ): 1525 . Google Scholar OpenURL Placeholder Text WorldCat 30. DellaGreca , M. , Previtera , L., Purcaro , R., Zarrelli , A.; Cinnamic acid amides and lignanamides from Aptenia cordifolia ; Tetrahedron , ( 2006 ); 62 ( 12 ): 2877 – 2882 . Google Scholar Crossref Search ADS WorldCat 31. DellaGreca , M. , Di Marino , C., Previtera , L., Purcaro , R., Zarelli , A.; Apteniols A–F, oxyneolignans from the leaves of Aptenia cordifolia ; Tetrahedron , ( 2005 ); 61 ( 50 ): 11924 – 11929 . Google Scholar Crossref Search ADS WorldCat 32. Stone , W.L. , Yang , H., Qui , M.; Assays for nitric oxide expression, in Mast cells . Springer , New York , ( 2006 ), pp. 245 – 256 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 33. Sun , J. , Zhang , X., Broderick , M., Fein , H.; Measurement of nitric oxide production in biological systems by using Griess reaction assay ; Sensors , ( 2003 ); 3 ( 8 ): 276 – 284 . Google Scholar Crossref Search ADS WorldCat 34. Benson , A.M. , Hunkeler , M.J., Talalay , P.; Increase of NAD (P) H: Quinone reductase by dietary antioxidants: Possible role in protection against carcinogenesis and toxicity ; Proceedings of the National Academy of Sciences , ( 1980 ); 77 ( 9 ): 5216 – 5220 . Google Scholar Crossref Search ADS WorldCat 35. Siegel , D. , Kepa , J.K., Ross , D.; Biochemical and genetic analysis of NAD (P) H: quinone oxidoreductase 1 (NQO1) ; Current Protocols in Toxicology , ( 2007 ); 32 ( 1 ): 4.22. 1 – 4.22. 21 . Google Scholar Crossref Search ADS WorldCat 36. Parveen , I. , Threadgill , M., Hauck , B., Donnison , I., Winters , A.; Isolation, identification and quantitation of hydroxycinnamic acid conjugates, potential platform chemicals, in the leaves and stems of Miscanthus× giganteus using LC–ESI-MSn ; Phytochemistry , ( 2011 ); 72 ( 18 ): 2376 – 2384 . Google Scholar Crossref Search ADS PubMed WorldCat 37. Yuan , Y. , Qui , X., Nikolic , D., Dahl , J.H., van Breemen , R.B. et al. ; Method development and validation for ultra-high-pressure LC/MS/MS determination of hop prenylflavonoids in human serum ; Journal of AOAC International , ( 2012 ); 95 ( 6 ): 1744 – 1749 . Google Scholar Crossref Search ADS PubMed WorldCat 38. Qiao , X. , Wang , Q., Song , S., Guo , D., Ye , M. et al. ; HPLC-DAD-MS n analysis and HPLC quantitation of chemical constituents in the traditional Chinese medicine formula Ya-tong-yi-li-wan ; Analytical Methods , ( 2013 ); 5 ( 19 ): 5241 – 5247 . Google Scholar Crossref Search ADS WorldCat 39. Gavrilova , V. , Kajdzanoska , M., Gjamovski , V., Stefova , M. et al. ; Separation, characterization and quantification of phenolic compounds in blueberries and red and black currants by HPLC−DAD−ESI-MS n ; Journal of Agricultural and Food Chemistry , ( 2011 ); 59 ( 8 ): 4009 – 4018 . Google Scholar Crossref Search ADS PubMed WorldCat 40. Rhourri-Frih , B. , Chaimbault , P., Claude , B., Lamy , C., Andre , P., Lafosse , M. et al. ; Analysis of pentacyclic triterpenes by LC–MS. a comparative study between APCI and APPI ; Journal of Mass Spectrometry , ( 2009 ); 44 ( 1 ): 71 – 80 . Google Scholar Crossref Search ADS PubMed WorldCat 41. Guo , S. , Duan , J., Tang , Y., Yang , N., Qian , D., Su , S. et al. ; Characterization of triterpenic acids in fruits of Ziziphus species by HPLC-ELSD-MS ; Journal of Agricultural and Food Chemistry , ( 2010 ); 58 ( 10 ): 6285 – 6289 . Google Scholar Crossref Search ADS PubMed WorldCat 42. Rozenberg , R. , Ruibal-Mendieta , N.L., Petitjean , G., Cani , P., Delacroix , D.L., Delzenne , N. et al. ; Phytosterol analysis and characterization in spelt (Triticum aestivum ssp. spelta L.) and wheat (T. aestivum L.) lipids by LC/APCI-MS ; Journal of Cereal Science , ( 2003 ); 38 ( 2 ): 189 – 197 . Google Scholar Crossref Search ADS WorldCat 43. Thomas , M.C. , Dunn , S.R., Altvater , J., Dove , S.G., Nette , G.W.; Rapid identification of long-chain polyunsaturated fatty acids in a marine extract by HPLC-MS using data-dependent acquisition ; Analytical Chemistry , ( 2012 ); 84 ( 14 ): 5976 – 5983 . Google Scholar Crossref Search ADS PubMed WorldCat 44. Yang , K. , Zhao , Z., Gross , R., Han , X.; Identification and quantitation of unsaturated fatty acid isomers by electrospray ionization tandem mass spectrometry: A shotgun lipidomics approach ; Analytical Chemistry , ( 2011 ); 83 ( 11 ): 4243 – 4250 . Google Scholar Crossref Search ADS PubMed WorldCat 45. Gaffney , C.D. ; A study of Mesembryanthemaceae alkaloids . University of Johannesburg , South Africa ( 2008 ). Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 46. Hill , C.B. , Taylor , J.D., Edwards , J., Mather , D., Langridge , P., Bacic , A. et al. ; Detection of QTL for metabolic and agronomic traits in wheat with adjustments for variation at genetic loci that affect plant phenology ; Plant Science , ( 2015 ); 233 : 143 – 154 . Google Scholar Crossref Search ADS PubMed WorldCat 47. Meyer , G.M. , Wink , C.S., Zapp , J., Maurer , H.H.; GC-MS, LC-MS n, LC-high resolution-MS n, and NMR studies on the metabolism and toxicological detection of mesembrine and mesembrenone, the main alkaloids of the legal high “Kanna” isolated from Sceletium tortuosum ; Analytical and Bioanalytical Chemistry , ( 2015 ); 407 ( 3 ): 761 – 778 . Google Scholar Crossref Search ADS PubMed WorldCat 48. Patnala , S. , Kanfer , I.; Investigations of the phytochemical content of Sceletium tortuosum following the preparation of “Kougoed” by fermentation of plant material ; Journal of Ethnopharmacology , ( 2009 ); 121 ( 1 ): 86 – 91 . Google Scholar Crossref Search ADS PubMed WorldCat 49. Patnala , S. , Kanfer , I.; Medicinal use of Sceletium: Characterization of phytochemical components of Sceletium plant species using HPLC with UV and electrospray ionization–tandem mass spectroscopy ; Journal of Pharmacy & Pharmaceutical Sciences , ( 2015 ); 18 ( 4 ): 414 – 423 . Google Scholar Crossref Search ADS WorldCat 50. Galati , G. , O'brien , P.J.; Potential toxicity of flavonoids and other dietary phenolics: Significance for their chemopreventive and anticancer properties ; Free Radical Biology and Medicine , ( 2004 ); 37 ( 3 ): 287 – 303 . Google Scholar Crossref Search ADS PubMed WorldCat 51. García-Lafuente , A. , Guillamon , E., Villares , A., Rostagno , M.A., Martinez , J.A.; Flavonoids as anti-inflammatory agents: Implications in cancer and cardiovascular disease ; Inflammation Research , ( 2009 ); 58 ( 9 ): 537 – 552 . Google Scholar Crossref Search ADS PubMed WorldCat 52. Xu , S. , Shang , M.Y., Liu , G.X., Xu , F., Wang , X., Shou , C.C. et al. ; Chemical constituents from the rhizomes of Smilax glabra and their antimicrobial activity ; Molecules , ( 2013 ); 18 ( 5 ): 5265 – 5287 . Google Scholar Crossref Search ADS PubMed WorldCat 53. Liao , C.R. , Kuo , Y.H., Ho , Y.L., Wang , C.Y., Yang , C.S., Lin , C.W. et al. ; Studies on cytotoxic constituents from the leaves of Elaeagnus oldhamii maxim. In non-small cell lung cancer A549 cells ; Molecules , ( 2014 ); 19 ( 7 ): 9515 – 9534 . Google Scholar Crossref Search ADS PubMed WorldCat 54. Mabry , T.J. , Markham , K.R., Thomas , M.B.; The systematic identification of flavonoids . Springer-Verlag , New York , ( 1970 ). Google Scholar Crossref Search ADS Google Preview WorldCat COPAC 55. Harborne , J.B. , Mabry , T.J.; The flavonoids: Advances in research . Springer US , ( 2013 ). Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 56. Agrawal , P.K. ; Carbon-13 NMR of flavonoids . Elsevier Science , Amsterdam , ( 2013 ). Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 57. Zhang , H. , Li , X., Wu , K., Wang , M., Liu , P., Wang , X. et al. ; Antioxidant activities and chemical constituents of flavonoids from the flower of Paeonia ostii ; Molecules , ( 2016 ); 22 ( 1 ): 5 . Google Scholar Crossref Search ADS WorldCat 58. Markham , K.R. , Chari , V.M.; Carbon-13 NMR spectroscopy of flavonoids. In Harborne , J.B., Mabry , T.J. (eds). The flavonoids: Advances in research . Springer US , Boston, MA , ( 1982 ), pp. 19 – 134 . Google Scholar Crossref Search ADS Google Preview WorldCat COPAC 59. Ramprasath , T. , Senthamizharasi , M., Vasudevan , V., Sasikumar , S., Yuvaraj , S., Selvam , G.S.; Naringenin confers protection against oxidative stress through upregulation of Nrf2 target genes in cardiomyoblast cells ; Journal of Physiology and Biochemistry , ( 2014 ); 70 ( 2 ): 407 – 415 . Google Scholar Crossref Search ADS PubMed WorldCat 60. Bodduluru , L.N. , Kasala , E.R., Madhana , R.M., Barua , C.C., Hussain , M., Haloi , P. et al. ; Naringenin ameliorates inflammation and cell proliferation in benzo (a) pyrene induced pulmonary carcinogenesis by modulating CYP1A1, NFκB and PCNA expression ; International Immunopharmacology , ( 2016 ); 30 : 102 – 110 . Google Scholar Crossref Search ADS PubMed WorldCat 61. Manach , C. , Scalbert , A., Morand , C., Remesy , C., Jimenez , L.; Polyphenols: Food sources and bioavailability ; The American Journal of Clinical Nutrition , ( 2004 ); 79 ( 5 ): 727 – 747 . Google Scholar Crossref Search ADS PubMed WorldCat 62. Day , A.J. , Dupont , M.S., Ridley , S., Rhodes , M., Rhodes , M.J., Morgan , M.R.A. et al. ; Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver β-glucosidase activity ; FEBS Letters , ( 1998 ); 436 ( 1 ): 71 – 75 . Google Scholar Crossref Search ADS PubMed WorldCat 63. Tsuji , P.A. , Stephenson , K.K., Wade , K.L., Liu , H., Fahey , J.W.; Structure-activity analysis of flavonoids: Direct and indirect antioxidant, and antiinflammatory potencies and toxicities ; Nutrition and Cancer , ( 2013 ); 65 ( 7 ): 1014 – 1025 . Google Scholar Crossref Search ADS PubMed WorldCat 64. Uda , Y. , Prince , K.R., Williamson , G., Rhodes , M.J.; Induction of the anticarcinogenic marker enzyme, quinone reductase, in murine hepatoma cells in vitro by flavonoids ; Cancer Letters , ( 1997 ); 120 ( 2 ): 213 – 216 . Google Scholar Crossref Search ADS PubMed WorldCat 65. Liu , H. , Dinkova-Kostova , A.T., Talalay , P.; Coordinate regulation of enzyme markers for inflammation and for protection against oxidants and electrophiles ; Proceedings of the National Academy of Sciences , ( 2008 ); 105 ( 41 ): 15926 – 15931 . Google Scholar Crossref Search ADS WorldCat © The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Identification of Chemopreventive Components from Halophytes Belonging to Aizoaceae and Cactaceae Through LC/MS—Bioassay Guided Approach
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmaa112
DA - 2020-12-23
UR - https://www.deepdyve.com/lp/oxford-university-press/identification-of-chemopreventive-components-from-halophytes-belonging-TeO7PS7WbY
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -