{Anti-inflammatory activity of synthetic and natural glucoraphanin} J. Serb. Chem. Soc. 84 (5) 445–453 (2019) UDC 635.4:547.458.34:616.006.03/04:615.276 JSCS–5197 Original scientific paper 445 Anti-inflammatory activity of synthetic and natural glucoraphanin QUAN V. VO1,2*, PHAM C. NAM3**, THUC N. DINH4, ADAM MECHLER5 and THI T. V. TRAN6 1Department for Management of Science and Technology Development, Ton Duc Thang University, Ho Chi Minh City, Vietnam, 2Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, Vietnam, 3Department of Chemical Engineering, University of Da Nang – University of Science and Technology, Vietnam, 4Faculty of Natural Sciences, Hong Duc University, Thanh Hoa, Vietnam, 5La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia and 6Department of Chemistry, Hue University of Science, 77 Nguyen Hue, Hue, Vietnam (Received 18 May, revised 13 November, accepted 3 December 2018) Abstract: Glucoraphanin is one of the best known glucosinolates because of its health benefits. The compound is known to eliminate carcinogens in tissue and hence is frequently studied for its cancer preventative properties. In this work, the total synthesis of α- and β-glucoraphanin epimers was attempted. β-Glu- coraphanin potassium salt was successfully synthesized in high overall yield, whereas the α-epimer was found to be unstable as it decomposed in the final step of the total synthesis. The anti-inflammatory activity of the synthetic glu- coraphanin was determined by inhibition of the release of tumor necrosis factor alpha (TNF-α) secretion in lipopolysaccharide-stimulated THP-1 cells. It was shown that in the presence of either the synthetic or natural glucoraphanin, TNF-α secretion was significantly reduced (≈52 % inhibition) at a concen- tration of 15 μM. Keywords: broccoli; TNF-α; sulforaphane; glucosinolates; synthesis; THP-1. INTRODUCTION Glucoraphanin (4-methylsulfinylbutyl glucosinolate, GRP) is the most abun- dant glucosinolate in common culinary brassica species, such as broccoli (Bras- sica oleracea)1 or hoary cress (Cardaria draba).2 When ingested, it is hydro- lyzed by the enzyme myrosinase to sulforaphane that imparts numerous health benefits: it was shown to eliminate carcinogens in living tissue3,4 and it is an inducer of phase 2 enzymes (glutathione S-transferase and quinone reductase)5 that are linked to cancer protection.6,7 Consistently it is possible, and desirable, *,** Corresponding authors. E-mail: (*)vovanquan@tdtu.edu.vn; (**)pcnam@dut.udn.vn https://doi.org/10.2298/JSC180518108V ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. 446 VO et al. to create a GRP-based nutraceutical that retains the health advantages of sulfo- raphane and natural GRP. The evaluation of the biosafety and anti-tumor activity of purified and semi- -purified GRP was reported previously.8,9 The studies were performed on male F344 rats by testing the effect of GRP on ethoxyresorufin O-deethylase and/or NQO1 activity. The response to GRP was similar for the purified and semi-puri- fied GRP preparations, suggesting that semi-purified preparations could be used in supplements and to fortify foods. However, there are no reports of control stu- dies of GRP bioactivity using synthetic GRP that is a better defined source with much reduced and easily identifiable impurities. Furthermore, there are indicat- ions that the activity of GRP is more diverse and/or it exerts a protective function via an anti-inflammatory effect, similar to the pathway reported for aromatic and indole glucosinolates (GLs).10,11 There is a link between chronic inflammation and carcinogenesis,12–14 and thus a study of anti-inflammatory activity could be a simple and inexpensive step to initially evaluate the anticancer activity of glu- coraphanin. Therefore, in this study, a comprehensive study of the total synthesis of α- and β-GRP potassium salts and the measurements of their anti-inflammatory activity in comparison with other typical glucosinolates are reported. EXPERIMENTAL General methods Melting points (m.p.) were recorded on a hot stage apparatus and are uncorrected. Opti- cal rotations were measured at the stated temperatures in the stated solvent on a polarimeter at the sodium d-line (589 nm); [α]D values are given in 10-1° cm2 g-1. Infrared spectra (νmax) were recorded on a FT-IR spectrometer. The samples were analyzed as KBr discs (for solids) or as thin films on NaCl plates (for liquids/oils). Unless otherwise specified, the proton (1H) and carbon (13C) NMR spectra were recorded on a 300 MHz spectrometer operating at 300 MHz for protons and 75 MHz for carbon nuclei. Chemical shifts were recorded as δ values in ppm. The spectra were acquired in deuterated chloroform (CDCl3), methanol-d4 (CD3OD) or deuterium oxide (D2O) at 300 K unless otherwise stated. For the 1H-NMR spectra recorded in CDCl3, CD3OD and D2O, the peaks due to residual CHCl3, CD3OD and D2O (δH 7.24, 3.28 and 4.65 ppm, respectively) were used as the internal reference, while the central peaks (δC 77.0 and 49.0 ppm) of CDCl3 and CD3OD were used as the reference for the proton- -decoupled 13C-NMR spectra. Low-resolution mass spectra were measured on a mass spectro- meter at 300 °C at a scan rate of 5500 m/z per s using water/methanol/acetic acid in a volume ratio of either 0/99/1 or 50/50/1 as the mobile phase. Accurate mass measurement was by mass spectrometry with a heated electrospray ionization (HESI) source. The mass spectro- meter was operated with full scan (50–1000 amu) in the positive or negative FT mode (at a resolution of 100,000). The analyte was dissolved in water/methanol/acetic acid in a volume ratio of 0/99/1 or 50/50/1 and infused via syringe pump at a rate of 5 µL/min. The heated cap- illary was maintained at 320 °C with a source heater temperature of 350 °C and the sheath, auxiliary and sweep gases were at 40, 15 and 8 units, respectively. The source voltage was set to 4.2 kV. The solvents were dried over standard drying agents and freshly distilled before use. Ethyl acetate and hexane used for chromatography were distilled prior to use. All solvents ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. ANTI-INFLAMMATORY ACTIVITY OF GLUCORAPHANIN 447 were purified by distillation. Reactions were monitored by thin layer chromatography (TLC) on silica gel 60 F254 plates with detection by ultra-violet (UV) fluorescence or charring with a basic potassium permanganate stain. Flash column chromatography was performed on silica gel 60 particle size 0.040–0.063 mm (230–400 mesh). General procedures for the synthetic compounds 1-Thio-α- or β-D-glucopyranoze 2,3,4,5-tetraacetate 1-[(1Z)-N-hydroxy-5-(methylsul- finyl)pentanimidate] (2a or 2b). To a suspension of 1 (150 mg, 0.9 mmol) in DCM (10 mL) was added pyridine (0.09 mL, 0.95 mmol) and then N-chlorosuccinimide (120 mg, 0.63 mmol). The mixture was stirred for 2.5 h at r.t. under a nitrogen atmosphere, then 1-thio-α-D- -glucopyranose 2,3,4,6-tetraacetate or 1-thio-β-D-glucopyranose 2,3,4,6-tetraacetate (0.33 g, 0.9 mmol) in DCM (5 mL) was added. The resulting mixture was treated with triethylamine (0.75 mL, 5.4 mmol). The reaction mixture was stirred for 2 h at r.t. under a nitrogen atmo- sphere then acidified with aqueous 1 M H2SO4 (7 mL/mmol of sugar). The mixture was left to stand for about 10 min and then separated. The aqueous phase was extracted with DCM (3×30 mL). The combined organic layers were dried over MgSO4, filtered and the filtrate was con- centrated under reduced pressure. Compound 2a (189 mg, 40 %) was obtained as a foam by flash column chromatography on silica gel eluting with 10 % MeOH/DCM. The characteriz- ation data for 2a are given in the Supplementary material to this paper. Compound 2b (150 mg, 47 %) was obtained as a foam by flash column chromatography eluting with 90 % DCM/MeOH. The characterization data for compound 2b were identical to literature values.10 Potassium salt of 1-thio-α- or β-D-glucopyranoze 2,3,4,5-tetraacetate 1-[(1Z)-5-(methyl- sulfinyl)-N-(sulfooxy)pentanimidate] (3a or 3b). To a stirred solution of the thiohydroximate (2a or 2b, 120 mg, 0.2 mmol) in dry pyridine (5 mL) was added pyridine–sulfur trioxide complex (95.0 mg, 0.6 mmol). After stirring at r.t. under N2 for 24 h, an additional portion of the pyridine–sulfur trioxide complex (19.0 mg, 0.1 mmol) was added and stirring was con- tinued for 2 h. Subsequently, a solution of KHCO3 (850 mg, 8.4 mmol) in water (10 mL) was added, the mixture stirred for 30 min and then concentrated under reduced pressure. The resi- due was dissolved in water and extracted with chloroform (3×40 mL) and then 20 % MeOH/ /CHCl3 (2×30 mL). The organic layers were dried (MgSO4), filtered and concentrated under reduced pressure. To remove excess pyridine, the mixture was co-distilled several times with toluene. Compound 3a was obtained as a white solid (74 mg, 50 %). Compound 3b was obtained by flash chromatography eluting with 80 % DCM/MeOH as a colorless solid (103 mg, 73 %). The characterization data for compound 3b were identical to literature values.10 β-Glucoraphanin potassium salt (4b). The β-GRP potassium salt 4b was prepared from 3b following literature methods and the data were identical to literature values.10 Spectral and analytical data of the compounds are given in Supplementary material to this paper. Anti-inflammatory assays The anti-inflammatory assays were conducted following the literature.11,15 Human monocytic leukaemia THP-1 cells were obtained from the American Type Culture Collection. The cells were grown in 10 % heat-deactivated fetal bovine serum and Invitrogen RPMI-1640 containing 2 mM L-glutamine. The cytokine (TNF-α) ELISA kit including the reagents was obtained from BD Bioscience (R&D systems). All compounds were dissolved in sterile distilled water then further diluted in Invitrogen DMEM (Dulbecco’s modified eagle medium). The cells were grown in a 75 mL flask and ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. 448 VO et al. maintained at 37 °C in a humidified 5 % CO2 atmosphere. The experiments were performed once the cells had reached 105 cells mL-1. Phorbol 12-myristate 13-acetate (PMA) was dis- solved in DMSO to a concentration of 1 mg mL-1 and then further diluted before use. The cells were plated out to a cell density of 10×104 cell mL-1, at 100 µl well-1 in a 96-well plate then treated with PMA to a final concentration of 50 nM for 24 h at 37° under a humidified 5 % CO2 atmosphere. Lipopolysaccharides (LPS) were dissolved in sterile water to a concentration of 5 mg mL-1 and then further diluted to the working stock of 10 µg mL-1. The THP-1 cells were challenged with various compounds at a concentration ranging from 0.1–15 µM. They were stimulated with LPS at a final concentration of 50 ng mL-1. The supernatants were collected after 4 h incubation and stored at –20 °C until enzyme-linked immunosorbent assay (ELISA) analysis. A sandwich ELISA was used to screen the supernatants for the release of cytokine TNF-α. The ELISA plates were coated with a capture antibody (1:250) which was diluted in coating buffer and left at 4 °C overnight. The ELISA plates were aspirated and washed 3 times with 1×PBST (phosphate buffered saline with Tween-20 (0.05 % Tween-20, pH 7.4) before adding 200 μL well-1 assay diluent and incubated at room temperature for 1 h. Standards were pre- pared by 2-fold serial dilutions to the range from 500–7.8 pg mL-1 in assay buffer diluent. Standards and sample were added in quadruplicate into appropriate wells and incubated at room temperature for 2 h. After the 2 h incubation, the plates were aspirated and washed for a total of 5 washes. The detection antibody and HRP reagent were added (100 μL well-1) and incubated at room temperature for 1 h. The plates were aspirated and washed again, this time for a total of 7 washes and were soaked for 30 s between each wash. The substrate solutions were added (100 μL well-1) and incubated at room temperature for 30 min in the dark. The reaction was stopped by adding 50 μL well-1 of kit stop solution then read at 450 nm with a plate reader within 30 min with a λ correction at 570 nm. Isolation and purification of GRP from broccoli seeds Natural GRP was isolated from broccoli seeds and purified following the literature.16 To approximately 9 g of broccoli seeds 90 mL of boiling water was added, and the mixture boiled for 5 min. The bulk of the water was decanted and the seeds transferred to a mortar with 15 mL of water. The seeds were ground to a paste. The resultant slurry was transferred to a 200 mL volumetric flask with deionized water, made to the mark and sonicated for 5 min. The ext- ract was filtered under vacuum through Whatman No. 4 filter paper. Mega Bond Elut C18 car- tridges (3 g) were activated with methanol and washed with water. Mega Bond Elut NH2 cartridges (3 g) were activated with methanol and equilibrated with 1 % acetic acid in water. The C18 and NH3+ cartridges were connected in series and 30 mL of the extract loaded onto the C18 cartridge. The cartridges were washed with 18 mL of deionized water, the C18 car- tridge discarded and the NH3+ cartridge washed with 18 mL of methanol. The glucosinolate fraction was removed from the NH3+ cartridge with 30 mL of freshly prepared 2 % solution of concentrated NH4OH solution in methanol. The solution was evaporated to dryness under a stream of nitrogen at room temperature. GRP was obtained from the crude residue by HPLC (mobile phase 1 vol. % CH3CN, 99 % aqueous 0.1 % formic acid)16 as a colorless liquid (35.7 mg). The NMR and MS data of the isolated GRP matched with the literature values.10,17 The isolated GRP was then used for anti-inflammatory assays. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. ANTI-INFLAMMATORY ACTIVITY OF GLUCORAPHANIN 449 RESULTS AND DISCUSSION Synthesis of α- or β-glucoraphanin potassium salts It was shown that the oxime 1 could be synthesized from the 5-chloropen- tanol (Scheme 1).10 Based on previous work, the thiohydroxymates 2a or 2b were formed by the coupling of oxime 1 and 1-thio-α-D-glucopyranose 2,3,4,6- -tetraacetate18 (or 1-thio-β-D-glucopyranose 2,3,4,6-tetraacetate11) following the Vo method.10 Thus, oxime 1 was treated with N-chlorosuccinimide (NCS) in the presence of pyridine in dichloromethane (DCM) to form the hydroximoyl chlo- ride, which was directly coupled with the α-thiol (or β-thiol) in triethylamine to yield the thiohydroxymates 2a (or 2b) in 47 % (or 40 %) yield in a one-pot reaction (Scheme 1). Scheme 1. Synthesis of α- or β-GRP potassium salts. Sulfation of 2a (or 2b) was accomplished with pyridine-sulfur trioxide com- plex in pyridine (Pyr) (Scheme 1).19 The resulting potassium salts 3a or 3b (73 % and 40 % yields) were isolated by flash column chromatography on silica gel. De-O-acetylations were performed by dissolving 3a (or 3b) in MeOH in the pre- sence of MeOK as catalyst. The final β-GRP potassium salt 4b (17 % overall yield over seven steps) was successfully purified by flash column chromato- graphy on silica gel.10 Unfortunately, the final α-epimer could not be obtained. The mass spec- trogram of the reaction solution showed no peak for the [M–K]– of the α-GRP potassium salt, which was usually observed as the base peak ion in the spectra of α-GLs.18 Attempts to achieve the α-isomer by using different de-O-acetylation conditions20 were unsuccessful. Thus, only the potassium salt of 1-Thio-α- or β-D-glucopyranoze 2,3,4,5-tetraacetate 1-[(1Z)-5-(methylsulfinyl)-N-(sulfooxy)- pentanimidate] 3a was obtained for the first time in 11.4 % yield over 6 steps. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. 450 VO et al. Anti-inflammatory activity of the synthetic β-GRP To compare the anti-inflammatory activity of the synthetic β-GRP (4b) with natural GRP and typical indole and aromatic glucosinolates,11,21 the anti-inflam- matory properties of the synthetic β-GRP were tested via an in vitro assay deve- loped based on the THP-1 cell line following the literature.11,15,21 The results are summarized in Fig. 1 and Table I. Fig. 1. Comparison of TNF-α released by LPS alone and in addition to GLs at different concentrations. LPS stimulates the release of TNF-α. The % inhibition is calculated from the difference between TNF-α released with LPS alone and in combination with the GLs (from the average of the three replicates). Moderate activity was observed for the majority of the GLs at low micromolar levels,17 a11, b21. It was shown that in the presence of synthetic β-GRP, TNF-α secretion was significantly inhibited (> 50 % inhibition) at a concentration of 15 μM, while synthetic β-GRP exhibited higher inhibition than neoglucobrassicin at all testing concentrations, reaching nearly the same point as glucobrassicin but higher than 4-methoxyglucobrassicin at a concentration of 15 μM (> 50 %, Fig. 1).11 Com- parison with the positive control (catechin) showed that at concentrations lower than 10 μM, most of the synthetic GLs showed lower inhibition than catechin, but at a concentration of 15 μM, β-GRP exhibited higher activity than catechin (52 % inhibition for β-GRP compared with 48 % inhibition for catechin). The trend was almost opposite in comparison with potassium 1-thio-β-D-glucopyra- nose 1-[[C(Z)]-3,4-dimethoxy-N-(sulfooxy)benzenecarboximidate] (the glucosin- ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. ANTI-INFLAMMATORY ACTIVITY OF GLUCORAPHANIN 451 olate with the highest anti-inflammatory activity in the aromatic glucosinolate family18). The results showed that synthetic β-GRP reached nearly the same inhi- bition of natural GRP at a concentration of 15 μM (52 % inhibition by β-GRP, 54 % inhibition by natural GRP); the trends were similar at higher investigated concen- trations. Thus, it was clearly demonstrated that the synthetic β-GRP has signific- ant anti-inflammatory activity at low concentrations (≈50 % inhibition at a con- centration of 15 μM). The results of the biological activity of synthetic GRP, con- sistent with previous studies,8,9 provide ample evidence for the potential of syn- hetic GRP for medicinal uses. TABLE I. Effects of synthetic GLs on TNF-α secretion in LPS-stimulated THP-1 cells Treatment Content of TNF-α secretion (SD)a, pg mg-1 LPS (50 μg L-1) 488.68 (15.81)b LPS + 15.00 μM catechin 255.94 (33.77) LPS + 10.00 μM catechin 273.02 (21.53) LPS + 1.00 μM catechin 301.41 (20.14) LPS + 0.10 μM catechin 373.30 (25.14) LPS (50 μg L-1) 236.16 (65.15)b LPS + 15.00 μM β-GRP 113.07 (39.42) LPS + 10.00 μM β-GRP 137.63 (52.90) LPS + 1.00 μM β-GRP 178.98 (47.48) LPS + 0.10 μM β-GRP 204.98 (52.34) LPS (50 μg L-1) 253.15 (45.53)b LPS + 15.00 μM GRP 115.38 (14.45) LPS + 10.00 μM GRP 137.69 (32.82) LPS + 1.00 μM GRP 170.26 (41.76) LPS + 0.10 μM GRP 199.33 (29.51) aThe results are for 3 different experiments run in duplicate; bp ≤ 0.07, compared with control CONCLUSIONS The total synthesis of α- or β-GRP (potassium salts) was attempted. While the β-epimer was successfully synthesized in high overall yield (17 % over seven steps), α-GRP was found to be unstable as it decomposed in the final step. By observing the inhibition of the release of TNF-α in LPS-stimulated THP-1 cells, it was shown that the synthetic β-GRP and natural GRP have similar, significant anti-inflammatory activity at low concentrations. The obtained results raise the possibility of developing a GRP-based nutraceutical for therapeutic and/or pre- ventive medicine purposes. SUPPLEMENTARY MATERIAL Analytical and spectral data of the compounds are available electronically at the pages of journal website: http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgement. This research is funded by The Vietnam National Foundation for Sci- ence and Technology Development (NAFOSTED) under Grant No. 104.06–2016.03. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. 452 VO et al. И З В О Д АНТИ-ИНФЛАМАТОРНА АКТИВНОСТ СИНТЕТИЧКОГ И ПРИРОДНОГ ГЛУКОРАФАНИНА QUAN V. VO1,2, PHAM C. NAM3, THUC N. DINH4, ADAM MECHLER5 и THI T. V. TRAN6 1 Department for Management of Science and Technology Development, Ton Duc Thang University, Ho Chi Minh City, Vietnam, 2 Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, Vietnam, 3 Department of Chemical Engineering, University of Da Nang – University of Science and Technology, Vietnam, 4 Faculty of Natural Sciences, Hong Duc University, Thanh Hoa, Vietnam, 5 La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia и 6 Department of Chemistry, Hue University of Science, 77 Nguyen Hue, Hue, Vietnam Глукорафанин је због својих корисних лековитих карактеристика један од најпоз- натијих глукозинолата. Познато је да деловањем једињења долази до уклањања канце- рогена у ткивима, и услед тога су често изучаване превентивна антиканцерска својства једињења. Успешно је синтетисана калијумова со β-глукорафанина, у високом укупном приносу, док је утврђено да је α-епимер нестабилан и да се разграђује у финалном ко- раку синтезе. Антиинфламаторна активност глукорафанина је испитана одређивањем инхибиције ослобађања фактора некрозе тумора-алфа (TNF-α) из THP-1 ћелија стиму- лисаних липополисахаридима. Показано је да је у присуству синтетичког или природног глукорафанина, при концентрацијама 15 μM значајно инхибирана (≈ 52 % инхибиције) секреција TNF-α. (Примљено 18. маја, ревидирано 13. новембра, прихваћено 3. децембра 2018) REFERENCES 1. L. G. West, K. A. Meyer, B. A. Balch, F. J. Rossi, M. R. Schultz, G. W. Haas, J. Agric. Food Chem. 52 (2004) 916 (https://doi.org/10.1021/jf0307189) 2. E. E. Powell, G. A. Hill, B. H. Juurlink, D. J. Carrier, J. Chem. Technol. Biotechnol. 80 (2005) 985 (https://doi.org/10.1002/jctb.1273) 3. J. W. Fahey, A. T. Zalcmann, P. Talalay, Phytochemistry 56 (2001) 5 (https://doi.org/10.1016/S0031-9422(00)00316-2) 4. Y. Zhang, P. Talalay, C. G. Cho, G. H. Posner, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 2399 (https://doi.org/10.1073/pnas.89.6.2399) 5. R. Iori, R. Bernardi, D. Gueyrard, P. Rollin, S. Palmieri, Bioorg. Med. Chem. Lett. 9 (1999) 1047 (https://doi.org/10.1016/S0960-894X(99)00136-5) 6. G. Kiddle, R. N. Bennett, N. P. Botting, N. E. Davidson, A. A. B. Robertson, R. M. Wallsgrove, Phytochem. Anal. 12 (2001) 226 (https://doi.org/10.1002/pca.589) 7. F. M. V. Pereira, E. Rosa, J. W. Fahey, K. K. Stephenson, R. Carvalho, A. Aires, J. Agric. Food Chem. 50 (2002) 6239 (https://pubs.acs.org/doi/abs/10.1021/jf020309x) 8. R. H. Lai, A. S. Keck, M. Wallig, L. West, E. Jeffery, Food Chem. Toxicol. 46 (2008) 195 (https://doi.org/10.1016/j.fct.2007.07.015) 9. N. Zhu, M. Soendergaard, E. H. Jeffery, R. H. Lai, J. Agric. Food Chem. 58 (2010) 1558 (https://doi.org/10.1021/jf9034817) 10. Q. V. Vo, C. Trenerry, S. Rochfort, A. B. Hughes, Tetrahedron 69 (2013) 8731 (https://doi.org/10.1016/j.tet.2013.07.097) 11. Q. V. Vo, C. Trenerry, S. Rochfort, J. Wadeson, C. Leyton, A. B. Hughes, Bioorg. Med. Chem. 22 (2014) 856 (https://doi.org/10.1016/j.bmc.2013.12.003) 12. N. Juge, R. F. Mithen, M. Traka, Cell. Mol. Life Sci. 64 (2007) 1105 (https://doi.org/10.1007/s00018-007-6484-5) ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. ANTI-INFLAMMATORY ACTIVITY OF GLUCORAPHANIN 453 13. H. Ohshima, H. Bartsch, Mutat. Res., Fundam. Mol. Mech. Mutagen 305 (1994) 253 (https://doi.org/10.1016/0027-5107(94)90245-3) 14. V. Dobričić, B. M. Francuski, V. Jaćević, M. V. Rodić, S. Vladimirov, O. Čudina, D. Francuski, J. Serb. Chem. Soc. 80 (2015) 1481 (http://www.doiserbia.nb.rs/img/doi/0352- 5139/2015/0352-51391500067D.pdf) 15. U. Singh, J. Tabibian, S. K. Venugopal, S. Devaraj, I. Jialal, Clin. Chem. 51 (2005) 2252 (https://doi.org/10.1373/clinchem.2005.056093) 16. Q. V. Vo, S. Rochfort, P. C. Nam, T. L. Nguyen, T. T. Nguyen, A. Mechler, Carbohydr. Res. 455 (2018) 45 (https://doi.org/10.1016/j.carres.2017.11.004) 17. M. H. Benn, Can. J. Chem. 41 (1963) 2836 (https://doi.org/10.1139/v63-415) 18. P. Rollin, A. Tatibouët, C. R. Chim. 14 (2011) 194 (https://doi.org/10.1016/j.crci.2010.05.002) 19. Q. V. Vo, C. Trenerry, S. Rochfort, J. Wadeson, C. Leyton, A. B. Hughes, Bioorg. Med. Chem. 21 (2013) 5945 (https://doi.org/10.1016/j.bmc.2013.07.049) 20. S. Rochfort, D. Caridi, M. Stinton, V. C. Trenerry, R. Jones, J. Chromatogr. A 1120 (2006) 205 (https://doi.org/10.1016/j.chroma.2006.01.046) 21. S. J. Rochfort, V. C. Trenerry, M. Imsic, J. Panozzo, R. Jones, Phytochemistry 69 (2008) 1671 (https://doi.org/10.1016/j.phytochem.2008.02.010). ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. 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