Suppression Effect of Purpurin Derivatives on Nitric Oxide Synthase

J. Korean Soc. Appl. Biol. Chem. 54(2), 302-307 (2011) Short Communication Suppression Effect of Purpurin Derivatives on Nitric Oxide Synthase Hoi-Seon Lee* Department of Bioenvironmental Chemistry and Institute of Agricultural Science & Technology, College of Agriculture & Life Science, Chonbuk National University, Jeonju 561-756, Republic of Korea Received September 1, 2010; Accepted September 9, 2010 The inhibitory effects of Rubia tinctorum root-derived materials against nitric oxide (NO) production were assessed by evaluation of NO production and inducible NO synthase (iNOS) expression, and compared to those of trihydroxyanthraquinone derivatives. The inhibitory effect of 1,2,4-trihydroxyanthraquinone (purpurin) from R. tinctorum roots against NO production was 60.7 and 38.5% at 2.5 and 1.25 μg/mL, respectively. Inhibitory effect of 1,2,7-trihydroxyanthraquinone was 52.5% at 2.5 μg/mL. Suppression effects of 1,2,4-trihydroxyanthraquinone on iNOS expression were confirmed by western blot analysis. Key words: functional foods, inducible nitric oxide synthase, NO production, Rubia tinctorum, 1,2,4-trihydroxyanthraquinone Medicinal plants may prove to be an alternative to the murine peritoneal macrophages following stimulation inducible nitric oxide synthase (iNOS) inhibitors presently with LPS. in use, as the plants constitute a rich source of active Acrylamide, ε-amino-n-caproic acid, bovine serum chemicals [Lee et al., 2000; 2002; Lee, 2011]. Because albumin, brilliant blue G-250, 1,3,8-trihydroxyanthraquinone, these plant-derived compounds are largely free from 6-methyl-1,3,8-trihydroxyanthraquinone, ethylene diamine adverse effects, studies may facilitate the development of acid, leupeptin, lipopolysaccharide, N,N'-methylene-bis- safer iNOS inhibitors. Therefore, a great deal of effort has acrylamide, sodium dodecyl sulfate, sodium nitrite, been focused on the use of medicinal plants as lead sulfanilamide, and trypsin inhibitor type II (soybean) compounds. The present authors reported that 30 medicinal were purchased from Sigma Chemical (St. Louis, MO). plant species exhibited an inhibitory response to nitric 1,2,7-Trihydroxyanthraquinone, 1,4,5-trihydroxyanthraquinone, oxide (NO) production against iNOS [Lee et al., 2000; and 2,7,8-trihydroxyanthraquinone were purchased from 2002]. The roots of Rubia tinctorum L. used in these Aldrich (Milwaukee, WI), Chemos (Regenstauf, Germany), studies are utilized in dyeing of textiles and treatment of and Fluka (Buchs, Switzerland), respectively. Fetal bovine kidney and bladder stones. Furthermore, R. tinctorum is serum, penicillin, RPMI 1640, skim milk dehydrate, and not only important as a source of antifungal activity, but is streptomycin were supplied by Gibco (Gaithersburg, also considered in conjunction with possible medicinal NM). Goat anti-rabbit IgG (H+L)-AP conjugate was properties of foods, such as anticancer, antimalarial, obtained from Bio-Rad Laboratories (Hercules, CA) and antimicrobial, and antioxidant activities [Yang et al., rabbit anti-mouse macNOS from Transduction Laboratories 2003; Kim et al., 2004; Manojlovic et al., 2005; Faith et (Rockville, MD). All other chemicals were of reagent al., 2006; Galindo et al., 2008]. However, thus far grade. relatively little work has been conducted on the inhibitory The R. tinctorum roots (5 kg) were ground in a blender, responses of R. tinctorum root-derived materials extracted twice with methanol (MeOH) (15 L) for 2 days against NO and iNOS induced either by bacterial at 25-27oC, and filtered. The combined filtrate was then lipopolysaccharide (LPS) or a variety of cytokines. The concentrated in vacuo at 45oC to yield 7.3% (365 g). The present study was conducted to evaluate the inhibitory combined filtrate (20 g) was partitioned into hexane (1.9 effects of R. tinctorum root-derived materials on iNOS in g), chloroform (4.1 g), ethyl acetate (2.4 g), butanol (2.5 g), and water-soluble fractions (9.1 g). This step was *Corresponding author repeated 18 times with the organic solvent. The organic Phone: +82-63-270-2544, 2546; Fax: +82-63-270-2550 E-mail: [email protected] solvent fractions were concentrated to dryness via rotary evaporation at 45oC, and the water fraction was freeze- doi:10.3839/jksabc.2011.048 dried. The yield of each fraction was; hexane (34.2 g), Nitric oxide synthase inhibitor and purpurin 303 chloroform (73.8 g), ethyl acetate (43.2 g), butanol were washed twice and resuspended in RPMI 1640 (45 g), and water-soluble (163.8 g). Owing to its excellent containing 10% heat-inactivated FBS, 2 mM glutamine, inhibitory activity against NO production, the chloroform penicillin (200 IU/mL), and streptomycin (200 IU/mL). (8 g) fraction was chromatographed on a 90×5.5 cm (ID) Peritoneal exudate cells were seeded at densities of 1×105 silica gel column (Merck 70-230 mesh, 760 g, Nahway, cells/well in 96-well tissue culture plates or 1×107 cells/ NJ) and eluted successively with a stepwise gradient of dish on 6-cm tissue culture dishes, and the macrophages petroleum ether/chloroform (10, 20, 30, 40, and 50%, were allowed to adhere for 2 h at 5% humidified v/v). The active 20% fraction (4.2 g) evidenced inhibitory atmosphere. The nonadherent cells were removed by activity against NO production. This fraction was pouring off the medium and rinsing the wells twice with chromatographed further on a silica gel column and pre-warmed medium. The adherent cells were incubated eluted with petroleum ether/chloroform (7:1, v/v). Column under the conditions deemed appropriate for individual fractions were analyzed via thin-layer chromatography experiments. (TLC; silica gel 60 F254, petroleum ether/chloroform, Peritoneal macrophages were incubated in 96-well 10:1, v/v), and fractions with similar TLC patterns were tissue culture plates (1×106 cells/mL) or on 6-cm tissue combined. The active fraction (3.1 g) evidenced inhibitory culture dishes (1×107 cells/dish) with LPS (1 μg/mL) for o activity against NO production. The bioactive fraction 24 h at 37 C in 5% CO2-air incubator [Lee et al., 2002]. was then chromatographed over a Sephadex LH-20 The supernatant was harvested and assayed for nitrite column (Pharmacia, 800× 49 mm, Uppsala, Sweden) production. using chloroform/acetone/methanol (20:2:1, v/v/v). This NO production was measured by estimating the stable procedure was repeated three times. The active fraction NO metabolite, nitrite, in a conditioned medium via (1.8 g), which evidenced inhibitory activity against NO Griess reaction [Billiar et al., 1989; Lee et al., 2000; production, was chromatographed over a Polyclar AT 2002]. The cell-free supernatant (100 μL) was mixed column (Touzart and Matignon, 100 g, Vitry sur Seine, with 100 μL of Griess reagent [Lee et al., 2000; 2002] France), packed with chloroform/acetone (20:2, v/v), and and incubated for 15 min at room temperature. then eluted with an increasing ratio of chloroform/acetone Subsequently, the absorbance of the wells was (20:2, 20:3, 20:4, 20:5, 20:8, v/v). The active fraction determined with a microplate reader equipped with a 540- (879 mg) exhibiting inhibitory activity against NO nm filter. Nitrite concentrations were determined from a production was finally purified on a Sephadex LH-20 linear regression analysis of standards (sodium nitrite) column (Pharmacia, 800×49 mm) eluted with petroleum generated for each plate. The cytosolic protein contents ether/chloroform (6:4, v/v) and cellulose (Merck, Darmstadt, were measured by the Bradford method [Bradford, 1976], Germany) eluted with petroleum ether/chloroform (6:4, using bovine serum albumin as a standard. v/v). Finally, the active component (379.3 mg) was RAW 264.7 cells were stimulated with LPS (1 μg/mL) isolated. The active isolate was structurally determined and IFN-γ (10 units/mL) with or without samples via spectral analysis. The 1H- and 13C-NMR spectra were [Krizsán et al., 1996; Lee et al., 2002]. The supernatants recorded in deuteriochloroform with a JNM-LA 400F7 were then harvested and assayed for nitrite production. spectrometer (JNM-ECA600, JEOL Ltd, Tokyo, Japan), The cells were incubated on 6-cm culture dishes, at 600 and 150 MHz (TMS as an internal standard), scrapped, and collected. The cells were then resuspended respectively, and chemical shifts are expressed in δ (parts with 500 μL of sonication buffer. The cells were disrupted per million). Unambiguous 1H- and 13C-NMR chemical via sonication (10 s), and the sonicate was centrifuged for shifts were obtained using DEPT, 1H-1H COSY, and 1H- 10 min at 12,000 rpm at 4oC. The supernatant was 13C COSY spectra. UV spectra were obtained in chloroform employed as the cytosol fraction in the Western blot with a Jasco V-550 spectrometer (Jasco Ltd, Tokyo, analysis. Japan), IR spectra on a Bio Rad FT-80 spectrophotometer, The sonicated cells were subjected to electrophoresis and mass spectra on a JEOL JMS-DX 30 spectrometer. on 1.5-mm thick 15% polyacrylamide gels. The separated Macrophages were purified from peritoneal exudate proteins were transferred to PVDF membranes using cells in accordance with the previously established Trans-Blot [Lee et al., 2000; 2002]. The membranes were protocols [Kirikae et al., 1996; Lee et al., 2002]. blocked for 30 min at room temperature with 5% skim Thioglycollate-elicited peritoneal exudate cells were milk in 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, and obtained from 8- to 10-week-old ICR male mice via 0.05% Tween 20 and was incubated with anti-iNOS intraperitoneal injection of 1 mL Brewer Thioglycollate antibody (1:2000 dilution) in blot buffer (50 mM Tris- broth (4.05%, w/v) and lavage of the peritoneal cavity HCl, pH 7.5, 200 mM NaCl, 5% skim milk and 0.05% with RPMI 1640 medium (5 mL) 3 days later. The cells Tween 20) overnight at 4oC.

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