SUPPLEMENTARY MATERIAL
The comparison of DPPH scavenging capacity and anti-inflammatory effects ofphenolic compounds isolated from the stems of Stewartia koreana Nakai
Manh Heun Kim, Jun Hye Jang, Myeong Hwan Oh, Jun Hyeok Heoand Min Won Lee*
College of Pharmacy,Chung-Ang University, Seoul 156-756, South Korea
The Stewartia koreana Nakai (SK) had been used in oriental traditionalmedicine as a remedy for acute gastroenteritis, liver diseases,quadriplegia, and pain. The anti-oxidantactivity guided isolation80% methyl extract from stems of SK (SKS)yielded eightphenolic compounds.We evaluated the anti-oxidative, anti-inflammatory effects of these compounds via assays of 1,1-diphenyl-2-picrylhydazyl (DPPH) radicals and inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells.The results showed that syringaresinol (6)exhibited significant DPPH radical scavenging activity as well asinhibitory effects on NO production compared with its positive controls, ascorbic acid and L-NMMA, respectively.
Keywords: Stewartia koreana;Antioxidants; Radical scavengers; Nitric oxide;
*Corresponding Author
Prof. Dr. Min Won Lee, College of Pharmacy, Chung-Ang University, Seoul 156-756, South Korea, E-mail: ; Tel: 82-2-820-5602; Fax: 82-2-822-9778
Experimental
(1)General experimental methods
The stationary phases for column chromatographic isolation were performed on Sephadex LH-20 (10-25 μM, GE Healthcare Bio-Science AB, Uppsala, Sweden), MCI-gel CHP 20P (75-150 μM, Mitsubishi Chemical, Tokyo, Japan) and ODS-B gel (40-60 μM, Daiso, Osaka, Japan). ODS-B gel also was used for the stationary phase on middle pressure liquid chromatography (MPLC), with a 110UV / VIS sample detector (Gilson, Middleton, WI) and TBP 5002 pump (Tauto Biotech, Sangai, China), for column chromatography. TLC was carried out on pre-coated silica gel 60 F254 plates (Merck, Darmstadt, Germany); spots were detected under UV radiation (254 nm) and by spraying with FeCl3 solution, 10% H2SO4and anisaldehyde-H2SO4followed by heating. The 1H- and 13C-NMR and 2D spectra were recorded on Gemini 2000 (Varian, Palo Alto, USA) and the resolution fast atom bombardment mass spectrum (LRFAB-MS) were measured with JMSAX505WA (JEOL, Tokyo, Japan).
(2) Plant material
3 kg of SKS were collected from Daea Arboretum in Jeonbuk, Korea in March 2009. A certified by professor Lee (Phamacognosy Lab, College of Pharmacy, Chung-Ang University). The voucher specimen (SKS 2009-3) was deposited at the herbarium of the college Pharmacy at Chung-Ang University.
(3) Extraction and isolation
3 kg of SKS were extracted several times at room temperature with 80% aqueous CH3COCH3. The concentrated extracts were applied to preparative thin layer chromatography (TLC) using solvent systems C:M:W (Chloroform : MeOH : H2O = 80:20:2),The spots were detected under UV radiation (254 nm) and by spraying with FeCl3, 10 % H2SO4, and anisaldehyde-H2SO4, followed by heating.The aqueous layer (148g) was then applied to a Sephadex LH-20 column (25-100 μM, 2000 g, 10 × 120 cm) (Pharmacia, Uppsala, Sweden) eluted with H2O-MeOH gradient resulting in 7 sub-fractions. Repeated column chromatography of fraction 4 (3.31g) on MCI gel (50 μM, 400 g, 3 × 50 cm) with 50-100 % methanol gradient and ODS-B gel (50 μM, 150 g, 1.5 × 45 cm) with 50-100% methanol gradient yielded dibenzyltetrafurano-lignan (1, 340 mg).And fraction 3 (5.22g) on ODS-B gel (50 μM, 300 g, 3.5 × 60cm), (50 μM, 250 g, 3 × 50cm) in MPLC system (5 ml/min, 280 nm) and MCI gel (50 μM, 400 g, 3 × 50 cm), (50 μM, 150 g, 3 × 50 cm) in open column with methanol gradient (10-100 %, 20-100%, and 40-100%) followed by recrystallization yielded lariciresinol-9-O-β-D-glucopyranoside (2, 340 mg),tanegol (3, 30 mg), and syringaresinol (6, 31mg). Fraction 7 (0.39 g) on ODS-B gel (50 μM, 200g, 2.5 × 30 cm) with 50-100% methanol gradient in MPLC system (5 ml/min, 280 nm) yielded (+)-catechin (8, 10 mg) and machicendiol (5, 100 mg). Repeated column chromatography of fraction 2 (9.66g) on Sephadex-LH 20 (25-100 μM, 400 g, 3.5 × 60 cm) and ODS B gel (50 μM, 200g, 2.5 × 30 cm) with 0-100% methanol gradient in MPLC system (5 ml/min, 280 nm) and followed by recrystallization yielded syringin (7, 30 mg) and isolariciresinol 4-O-β-D-glucopyranoside (4, 30mg)
Compound 1 [dibenzyltetrafurano-lignan]
White amorphous powder
Negative FAB-MS m/z:359[M-H]-,
1H-NMR(600MHz,MeOH-d4):2.36(1H,dt,J=7.2,14.4Hz,H-8’),2.48(1H,dd,J=12.0,12.6Hz,H-7a),2.72(1H,m,H-8),2.92(1H,dd,J=4.8,13.8Hz,H-7b),3.63(1H,dd,J=6.6,10.8Hz,H-9’a),3.71(1H,dd, J=5.4,8.4Hz,H-9a),3.82(1H,dd,J=6.0,14.4Hz,H9’b),3.96(1H,dd,J=6.6, 7.8Hz,H-9b),4.74(1H,d, J=7.2,H-7’),6.63(1H,dd,J=1.2,7.8Hz,H-6),6.71(1H,d,J=8.4Hz,H-5),6.76(2H,overlapped s, H-4’,6’),6.78(1H,s,H-2),6.90(1H,s,H-2’)
13C-NMR(150MHz, MeOH-d4):32.2(C-8),42.4(C-7),52.6(C-8’),54.9(3,3’-OCH3),59.0(C-9’),72.0(C-9),82.6(C-7’),109.2(C-2’), 111.9(C-2), 114.5(C-4’), 114.7(C-5), 118.3(C-6’), 120.7(C-6),132.1(C-1),134.3(C-1’),144.3(C-4), 145.6(C5’), 147.5(C-3’,3)
Compound 2 [lariciresinol-9-O-β-D-glucopyranoside]
White amorphous powder
Negative FAB-MS m/z: 521[M-H]-
1H-NMR(300 MHz, MeOH-d4): δ2.49(1H, m, H-8’), 2.52(1H, dd, J=13.8, 6.6Hz, H-7a), 2.73(1H, m, H-8), 2.98(1H, dd, J=13.8, 4.8Hz, H-7b), 3.25(1H,m, glc-2), 3.27(1H, m, glc-5), 3.30(1H, m, glc-4), 3.36(1H, m, glc-3), 3.59(1H, dd, J=9.6, 6.0Hz, H-9’b), 3.67(1H, m, glc-6b), 3.73(1H, dd, J=8.4, 6.6Hz, H-9a), 3.87(1H, m, glc-6a), 3.96(1H, d, J=8.4, 6.6Hz, H-9b),4.20(1H, dd, J=9.6, 7.8Hz, H-9’a), 4.29(1H, d, J=7.8Hz, glc-1), 4.82(1H, d, J=7.2Hz, H-7’), 6.65(1H, d, J=9.6, 1.8Hz, H-6), 6.71(1H, d, J=8.4Hz, H-5), 6.75(1H, d, J=8.4Hz, H-5’), 6.78(1H, d, J=1.8Hz, H-2), 6.80(1H, dd, J=9.6, 1.8Hz, H-6’), 6. 92(1H, d, J=1.8Hz, H-2’)
13C-NMR(150MHz, MeOH-d4): 32.3(C-7), 42.6(C-8), 50.3(C-8’), 61.4(glc-6), 67.1(C-9’), 70.2(glc-4), 72.3(C-9), 73.7(glc-2), 76.6(glc-5), 76.8(glc-3), 82.7(C-7’), 103.2(glc-1), 109.3(C-2’), 112.1(C-2), 114.5(C-5’), 118.5(C-6’), 120.7(C-6), 132.4(C-1), 134.2(C-1’), 144.3(C-4), 145.6(C-4’), 147.5(C-3’, 3), 114.7(C-5)
Compound 3 [tanegol]
White amorphous powder
Negative FAB-MS m/z: 375[M-H]-
1H-NMR(300 MHz, MeOH-d4): δ2.26(1H, m, H-8’), 2.61(1H, m, H-8), 3.59(1H, dd, J=7.8, 9.0Hz, H-9a), 3.66-3.62(2H, dd, J=4.8, 10.8Hz, H-9a, 9b), 3.72(1H, dd, J=6.6, 9.0Hz, H-9b), 4.48(1H, d, J=9.0Hz, H-7), 4.50(1H, d, J=9.0Hz, H-7’), 6.76(1H, d, J=8.4Hz, H-5’), 6.78(1H, d, J=8.4 Hz, H-5), 6.82(1H, dd, J=1.8, 8.4Hz, H-6’), 6.83(1H, dd, J=1.8, 8.4Hz, H-6), 6.91(1H, d, J=1.8Hz, H-2’),6.99(1H, d, J=1.8Hz, H-2)
13C-NMR(150MHz, MeOH-d4): 51.4(C-8), 54.5(C-8’), 61.8(C-9’), 69.7(C-9), 76.1(C-7), 84.4(C-7’), 109.7(C-2’), 109.9(C-2), 114.5(C-5, 5’), 119.2(C-6’), 119.4(C-6), 132.5(C-1’), 134.7(C-1), 145.9(C-4), 146.0(C-4’), 147.5(C-3’), 147.6(C-3)
Compound 4 [isolariciresinol 4-O-β-D-glucopyranoside]
Pale yellow needle crystal
Negative FAB-MS m/z: 521[M-H]-
1H-NMR(300MHz, DMSO-d6+ D2O): δ1.70(1H, m, H-8’),1.85(1H, m, H-8),2.77-2.71(2H, m, H-7), 2.77(1H, m, glc-5”), 3.12(1H, m, glc-3”), 3.16(1H, m, glc-2”), 3.22(1H, m, glc-4”), 3.43-3.25(2H, m, H-9), 3.43(1H, dd, J=6.0,9.6Hz,glc-6”b),3.45-3.17(2H, m, H-9’),3.56(1H, dd, J= 4.2,10.2Hz, glc-6”a), 3.77(1H, d, J=9.8Hz, H-7’), 4.53(1H, d, J=7.2Hz, glc-1”), 6.29(1H, s, H-5), 6.49(1H, dd, J=1.5, 8,1Hz, H-6’), 6.65(1H, s, H-2), 6.66(1H, d, J=8.3Hz, H-5’), 6.67(1H, d, J=1.7Hz, H-2’)
13C-NMR(150MHz, DMSO-d6+ D2O): δ32.6 (C-7), 38.4 (C-8),45.7 (C-8’), 46.3 (C-7’), 56.1-56.0 (3’, 3-OCH3), 59.8 (C-9’), 60.4 (glc-6”), 63.9 (C-9), 68.9 (glc-4”), 73.4(glc-2”), 76.9 (glc-5”), 77.3(glc-3”), 100.5(glc-1”), 112.6(C-5’), 113.7(C-2’), 115.6(C-2), 116.9(C-5), 121.8(C-6’), 130.5(C-1), 133.0(C-6), 136.7(C-1’), 144.5(C-4’), 145.1(C-4), 147.2(C-3’), 147.6(C-3)
Compound 5 [machicendiol]
White amorphous powder
Negative FAB-MS m/z: 341 [M-H]-
1H-NMR(600MHz, DMSO-d6+ D2O): δ1.81-1.72(2H, m, H-2”), 3.52-3.42 (2H, m, H-3”), 3.94(3H, s, 7-OCH3), 4.40(1H, t, J=4.8, 5.4Hz, OH-3”), 4.71(1H, dt, J=9.0, 4.8Hz, H-1”), 5.15(1H, d, J= 4.2Hz, OH-1”), 6.07(2H, s, -O-CH2-O-),6.86(1H, d, J=1.2Hz, H-6), 7.01(1H, d, J= 8.4Hz, H-5’), 7.09(1H, d, J=0.6Hz, H-4), 7.23(1H, s, H-3), 7.38 (1H, dd, J= 7.8, 1.8Hz, H-6’), 7.41(1H, d, J=1.8Hz, H-2’)
13C-NMR(150MHz, DMSO-d6+D2O): δ47.9(C-2”), 60.9(7-OCH3 ), 63.2(C-3”), 75.1(C-1”), 106.4(C-3), 106.6(-O-CH2-O-),110.1(C-2’), 110.1(C-6), 114.0(C-5’), 114.9(C-4), 123.9(C-6’), 129.2(C-1’), 135.2(C-9), 147.2(C-8), 147.9(C-5), 149.4(C-7), 152.9(C-4’), 153.1(C-3’), 160.4(C-2)
Compound 6 [syringaresinol]
Pale yellow amorphous powder
Negative FAB-MS m/z: 417 [M-H]-
1H-NMR(300MHz, DMSO-d6 + D2O): δ3.02(2H, m, H-1,5), 3.72 (12H, 3’,5’-OMe,s), 3.76(2H, dd, J=6.0, 6.6Hz, H-4,8ax), 4.14(2H, dd, J=2.1, 6.6Hz, H-4,8eq), 4.59(2H, d, J= 3.6Hz, H-2,6), 6.57(4H, s, H-2’,6’)
13C-NMR(150MHz, DMSO-d6+D2O): δ54.0(C-1,5), 56.4(q,3’,5’-OCH3), 71.4(C-4,8), 85.7(d,C-2, 6), 103.9(C-2’,6’), 131.9(C-1’), 135.0(C-4’), 148.2(C-3’,5’)
Compound 7 [syringin]
White amorphous powder
Negative FAB-MS m/z: 371[M-H]-
1NMR(600MHz, MeOH-d4): 3.21(1H,m, glc-5”), 3.43-3.41(2H, m, glc-3”,4”), 3.49-3.47(1H, dd, J=2.4,7.8Hz, glc-2”), 3.67(1H,dd,J=5.4,12.0Hz, glc-6”b), 3.78(1H,dd,J=2.4, 12.0Hz, glc-6”a), 4.22(2H, dd,J=1.2,5.4Hz, H-3’), 4.87(1H, d, J=7.8Hz, glc-1”), 6.35-6.30(1H,dt,J=5.4,11.4,15.6Hz,H-2’), 6.56-6.53(1H,d,J=15.6Hz,H-1’), 6.75(2H,s,H-2,6)
13C-NMR(150MHz, MeOH-d4): 55.6-55.5(3, 5-OCH3), 62.1(C-3’, glc-6”), 69.8(glc-4”),74.2(glc-2”),76.3(glc-3”),76.8(glc-5”), 104.0(C-3,9, glc-1”),133.8(C-1’,2’),134.3(C-1,4),152.8(C-2,6)
Compound 8 [(+)-catechin]
Brown amorphous powder
EI-MS m/z:290.0631[M]+
1H-NMR(300MHz, MeOH-d4): δ2.50(1H,dd,J=5.4, 16.2Hz,H-4ax), 2.85(1H,dd,J= 5.7, 16.5Hz,H4eq),3.97(1H,ddd,J=5.7, 7.5, 8.4Hz,H-3),4.56(1H,d,J=7.5Hz,H-2),5.85(1H,d,J=2.1Hz,H-6),5.92(1H,d,J=2.4Hz,H-8),6.72(1H,dd,J=1.8,8.1Hz,H-6’),6.76(1H,d,J=8.1Hz,H-5’),6.83(1H,d,J=1.8Hz,H-2’)
13C-NMR(75MHz, MeOH-d4): δ28.5(C-4), 68.8(C-3), 82.9(C-2), 95.5(C-8), 96.3 (C-6), 100.8(C-10), 115.3(C-2’), 116.1(C-5’), 120.1(C-6’), 132.2(C-1’), 146.3(C-3’,4’), 157.6(C-9), 157.8(C-5), 157.9(C-7)
(4) Measurement of DPPH radical scavenging activity
The anti-oxidant activity was determined on the basis of stable DPPH free radical scavanging (Sigma, St. Louis, USA) (Hatano et al., 1989). Twenty μl of each sample in absolute ethanol was added to 180 μl of 0.2 mM DPPH solution. After the reaction was mixed gently and allowed to stand for 30 min at room temperature, the optical density was measured at 518 nm using a microplate reader (TECAN, Salzburg, Austria). The free radical scavenging activity was calculated as the inhibition rate (%) = [1-(sample O.D. / control O.D.)] × 100 and IC50 values were determined. L-ascorbic acid was used as a positive control.
(5) Cell culture
RAW 264.7 macrophagecell lines were purchased from the Korean Cell Line Bank. The RAW 264.7 macrophage cells were grown at 37oC in a humidified atmosphere (5% CO2) in DMEM (Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum, 10 IU/ml penicillin G and 100 μg/ml streptomycin (Gibco BRL, Grand Island, NY, USA).
(6) Measurement of cell viability (MTT)
After culturing of RAW 264.7 macrophage(3 × 104 cells/ 200 μl medium) in 96-well plates and incubating for 2 h, in 96-well plates and incubating for 24 h, the cells were treated with the test samples. The cells were incubated for an additional 24 h, and the medium was replaced with fresh medium containing 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) (Sigma, St. Louis, MO, USA). Incubation continued for 4 h at 37 oC. The medium was then removed and the MTT-formazan produced was dissolved in dimethyl sulfoxide (DMSO). The extent of the reduction of MTT to dark purple crystals within the cells was quantified by measuring the absorbance at 540 nm using the microplate reader(TECAN, Salzburg, Austria) (Mosmann, 1983). Cytotoxicity was calculated as cell viability (%) = sample O.D. / blank O.D. × 100.
(7) Measurement of inhibition of NO production
RAW 264.7 macrophage cells (3 × 104 / 200 μl medium) were seeded onto 96-well plates and incubated for 2 h at 37oC in a humidified atmosphere (5% CO2). The cells were then incubated in medium containing 0.1 μg/ml lipopolysaccharide (LPS) (Sigma, St. Louis, MO, USA) and test samples. After incubating for an additional 18 h, Griess reagent (0.1% naphthylethylene-diamine and 1% sulfanilamide in 5% H3PO4 solution) (Sigma, St. Louis, MO, USA) was added to each supernatant from the cells treated with the samples. The absorbance of the samples was then read at 540 nm using a microplate reader (TECAN, Salzburg, Austria) and the amount of nitrite in the samples was calculated from a sodium nitrite standard curve(Green et al., 1982). Inhibitory of NO production was calculated as inhibition rate (%) = [1-(sample O.D.- blank O.D.) / (control O.D. - blank O.D.)] × 100, and IC50 values were determined. L-NMMA was used as the positive control.
(8) Statistical analysis
All data are expressed as mean ± S.D. Values were performed by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls (S-N-K) test using the SPSS software package; values were considered significantly different when the p value was less than 0.05. Values bearing different superscripts in the same column are significantly different.
References
Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R. (1982). Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids.Analytical biochemistry,126, 131–138.
Hatano, T., Edamatsu, R., Hiramatsu, M., Mori, A., Fujita, Y., Yasuhara, T., (1989). Effects of the interaction of tannins with co-exist substances. IV. Effects of tannins and ralated polyphenols on superoxide anion radical, and on 1,1-diphenyl-2-picrylhydrazyl radical. Chemical & Pharmaceutical Bulletin, 37, 2016-2021.
Mosmann, T. (1983). Rapid colorimetric assay for the cellular growth and survival. Journal of Immunological Methods, 65, 55–63.