Chemical Constituents of Abies koreana Leaves with Inhibitory ...

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cm −1 ; 1 H-NMR (600 MHz, CD 3 OD) : δ 8.1 (2H, d,1510= 9.0 Hz, H-2' and H-6'), 6.9 (2H, d, J = 9.0 Hz, H-3'JH-5'), 6.4 (1H, d, J = 1.8 Hz, H-8), 6.2 (1H, d, J = 1.8andH-6), 5.3 (1H, d, J = 7.8 Hz, H-1''), 3.2~3.9 (5H, m,Hz,158.6 (C-2), 137.3 (C-3), 132.4 (C-2'), 132.4 (C-(C-9),122.9 (C-1'), 116.2 (C-3'), 116.2 (C-5'), 104.3 (C-10),6'),(C-1''), 94.9 (C-8), 78.6 (C-3''), 78.2 (C-5''), 75.9100.071.5 (C-4''), 62.8 (C-6'').(C-2''),(7) − Brownish oil; FAB MS m/z : 361Isolariciresinol +; [α] 20 D : 68.0 (c 1.0, Me 2 CO); UV λ max (MeOH)[M+H]: 220, 285; IR ν max (KBr): 3350, 1600, 1500 cm −1 ; 1 H-nm(600 MHz, CD 3 OD) : δ 6.7 (1H, d, J = 8.0 Hz, H-NMR6.7 (1H, d, J = 1.4 Hz, H-2), 6.6 (1H, s, H-2'), 6.6 (1H,5),J = 1.5, 8.0 Hz, H-6), 6.2 (1H, s, H-5'), 3.8 (1H, d,dd,= 10.7 Hz, H-7), 3.8 (3H, s, 3'-OCH 3 ), 3.8 (3H, s, 3-J3 ), 3.7 (1H, m, H-9'), 3.7 (3H, dd, J = 3.8, 10.8 Hz,OCHb -9), 3.4 (1H, dd, J = 4.0, 11.2 Hz, H a -9), 2.8 (2H, d,H= 7.7 Hz, H-7'), 2.0 (1H, m, H-8'), 1.8 (1H, m, H-8);JC-NMR (150 MHz, CD13OD) : δ 149.1 (C-3), 147.3 (C-3146.0 (C-4), 145.4 (C-4'), 138.7 (C-6'), 134.3 (C-1),3'),(C-1'), 123.3 (C-6), 117.5 (C-5'), 116.1 (C-2), 113.9129.1112.5 (C-2'), 66.1 (C-9'), 62.4 (C-9), 56.5 (OCH 3 ),(C-5),(OCH 3 ), 48.2 (C-8), 48.13 (C-7), 40.14 (C-8'), 33.756.5(C-7').(8) − Brownish oil; FAB MS m/z :Secoisolariciresinol[M + H] + ; [α 20 D : -16.0 (c 0.10, MeOH); UV λ max363nm : 226, 281; IR ν max (KBr): 3425, 1516 cm −1 ;(MeOH)1(400 MHz, CD 3 OD) : δ 6.7 (2H, d, J = 7.9 Hz,H-NMRand H-5'), 6.6 (2H, d, J = 1.9 Hz, H-2 and H-2'), 6.5H-5dd, J = 7.9, 1.9 Hz, H-6 and H-6'), 3.7 (3H, s,(2H,3 ), 3.6 (4H, m, H-9 and H-9'), 2.6 (2H, dd, J = 13.6,OCHHz, H b -7 and H-7'), 2.5 (2H, dd, J = 13.6, 7.7 Hz, H a -7.0and H-7'), 1.9 (2H, m, H-2 and H-3); 13 C-NMR7MHz, CD 3 OD) : δ 149.5 (C-3), 149.5 (C-3'), 146.1(100146.1 (C-4'), 134.6 (C-1), 134.6 (C-1'), 123.4 (C-(C-4),123.4 (C-6'), 116.6 (C-5), 116.6 (C-5'), 113.9 (C-2),6),(C-2'), 62.8 (C-9), 62.8 (C-9'), 57.0 (OCH 3 ), 57.0113.93 ), 44.8 (C-8), 44.8 (C-8'), 36.7 (C-7), 36.7 (C-7').(OCH(9) − Whitish powder; EIMS m/z : 166Rhododendrol +; UV λ max (MeOH) nm : 214.5, 276; IR ν max (KBr):[M]H-7 and H-9), 3.8 (1H, m, H-2), 2.6 (1H, t, J = 7.0Hz,H-4), 1.7 (1H, m, H-3), 1.2 (3H, d, J = 6.3 Hz, CH 3 );Hz,C-NMR (75 MHz, CD13OD) : δ 157.1 (C-8), 135.2 (C-3131.0 (C-6), 131.0 (C-10), 116.8 (C-7), 116.8 (C-9),5),(C-2), 43.2 (C-4), 33.0 (C-3), 24.3 (CH 3 ).68.7acid (10) − Yellowish powder; EI MS m/z :FerulicMHz, CD 3 OD) : δ 7.6 (1H, d, J = 15.9 Hz, H-7), 7.1(300d, J = 2.0 Hz, H-2), 7.0 (1H, dd, J = 8.1, 1.8 Hz, H-(1H,6.9 (1H, d, J = 8.1 Hz, H-5), 6.3 (1H, d, J = 15.9 Hz,6),2.9 (3H, s, OCH 3 ); 13 C-NMR (75 MHz, CD 3 OD) : δH-8),(C-9), 153.9 (C-3), 150.5 (C-7), 149.1 (C-4), 131.5173.3125.1 (C-6), 119.1 (C-5), 117.3 (C-8), 115.0 (C-2),(C-1),(OCH 3 ).58.8(11) − Brownish oil;4-(4-hydroxyphenyl)butan-2-oneMS m/z : 164 [M] + ; UV λ max (MeOH) nm : 224, 278;EIν max (KBr): 3360, 3020, 2920, 2870, 1685, 1620,IR1510, 1440, 1365, 1320, 1290, 1225, 1170, 1105,1600,960, 875, 830, 765, 730 cm −1 ; 1 H-NMR (500 MHz,1040,3 OD) : δ 7.0 (2H, d, J = 8.3, H-6 and H-10), 6.8 (2H,CDJ = 8.3, H-7 and H-9), 2.8-2.7 (4H, m, H-3 and 4), 2.1d,s, CH 3 ); 13 C-NMR (125 MHz, CD 3 OD) : δ 210.0 (C-(3H,154.5 (C-8), 132.6 (C-5), 129.5 (C-6, 10), 115.6 (C-7,2),45.6 (C-3), 30.3 (CH 3 ), 29.1 (C-4).9),of BV2 microglial cells − BV2 microglialCulturewere provided by Prof. Sun-yeou Kim, at KyungcellsUniversity (Suwon, Korea). The cell line wasHeein DMEM containing 10% FBS withmaintained(100 IU/mL) and streptomycin (10 mg/mL) atpenicillinoC in a humidified atmosphere of 95% air-5% CO 2 .37for inhibition of nitric oxide production − ToAssayany trace of phenol red, the cell cultures wereremoveand the medium was replaced with Griesswashedand further incubated with test samples and LPS.medium24 hrs incubation, 100 ul aliquots of sample wereAfterwith 100 µl of Griess reagent (1% sulfanilamidemixed0.1% naphtylethylenediamine dihydrochloride, 2%andacid) in a 96 well plate and incubated at roomphosphoricfor 15 min. The absorbance at 550 nm wastemperatureon a microplate reader. The concentration wasmeasuredusing a nitrite standard curve (Dawson et al.,determined1994)viability measurements using the MTT assay −Cell100 µl aliquots of sample were collected for GriessAfterMTT (0.2 mg/mL) was directly added to cultures,assay,by incubation at 37 o C for 3 hrs. The supernatantfollowedthen aspirated and 100 µl of DMSO was added towasthe formazan. After insoluble crystals weredissolvedissolved, absorbance (abs) at 540 nm wascompletelyusing a microplate reader. Data were expressedmeasuredpercent cell viability relative to control cultures.as178 Natural Product Sciences[M] + ; UV λ max (MeOH) nm : 242, 293, 323; IR ν max1943427, 2921, 1620, 1516, 1432 cm −1 ; 1 H-NMR(KBr):3'', 4'', 5", 6''); 13 C-NMR (150 MHz, CD 3 OD) : δH-2'',(C-4), 166.1 (C-7), 163.2 (C-5), 161.7 (C-4'), 159.2179.71595, 1500 cm −1 ; 1 H-NMR (500 MHz, CD 3 OD) : δ3330,(2H, d, J = 8.4 Hz, H-6 and H-10), 6.7 (2H, d, J = 8.47.0Cell viabiltity (%) = 100 ×(Abs of LPS-treated or LPS + saple-treated)Abs of control
2. Effect of each fraction from A. koreana leaves on LPS-induced NO production in BV-2 microgliaFig.microglia were washed with DMEM and incubated with test fractions for 1 hr. The cultures were then stimulated by 100 ng/mL ofBV2for 24 hrs. After incubation, NO production was measured by the Griess reaction and sodium nitrite was used as a standard. NOLPS(NP) of control and LPS-treated cultures were 1.76 ± 1.6 and 22.48 ± 1.2 µM, respectively. Relative production (%) wasproductionas (NP of sample treated - NP of control)/(NP of LPS-treated - NP of control) × 100. Mean value is significantly differentcalculated< 0.05, **p < 0.01, ***p < 0.001) from the value of the LPS-treated.(*psearch of inhibitory activity against NO productionInnatural sources, 80% MeOH extract of Abiesfromdisplayed moderate inhibitory activity againstkoreanainduced NO production in BV-2 microglia inLPSdependent manner (Fig. 2). The 80%concentrationextract of A. koreana was partitioned with n-hexane,MeOHacetate, n-buthanol and water. The ethyl acetate solubleethylshowed potent inhibitory activity against NOfractionat concentrations of 50 and 100 µg/mL. Theproductionacetate soluble fraction of the 80% MeOH extractethylA. koreana was chromatographed by repetitivefromchromatography using diverse solid absorbentscolumnsilica gel, HP-20, and RP-C 18 resin to yieldincludingcompounds (1 - 11). Isolates were identified byeleven1 H-, 13 C-NMR and MS spectroscopic datacomparingliterature values as 3-hydroxy-2-methyl-4-pyrone (1,withet al., 2001), maltol-3-O-β-D-glucoside (2, Li et al.,Kim(−)-epicatechin (3, Khallouki et al., 2007), naringenin2004),(4, Choi et al., 1990), naringenin-7-O-β-D-glucopyranoside(5, Akiyama et al., 2000), kaempferol7-O-rhamnoglucoside(6, Kovganko et al., 2002), (+)-3-O-β-D-glucopyranoside(7, Baderschneider et al., 2001), secoisola-isolariciresinol(8, Fonseca et al., 1978), rhododendrol (9, Dasriciresinolal., 1993), ferulic acid (10, Anselmi et al., 2006) and 4-etbutan-2-one (rheosmin) (11, Smith et(4-hydroxyphenyl)1996). Among eleven isolates, compounds 3, 5, andal.,cells are known as the phagocytic immuneMicroglialof the brain, and excessive activation of these cellscellsassociated with neurodegenerative disorders includingaredisease, Parkinson’s disease and ischemia.Alzheimer’striggered by various stimuli, microglial cells produceWhencytokines (interleukin-1β, interleukin-6,proinflammatorytumor necrosis factor-α (TNF-α), reactive oxygenand(ROS), and nitric oxide (NO), leading tospeciesand neurodegeneration [Eguchi et al.,neuroinflammationTherefore, it is suggested that suppression of2008].microglial cells may mitigate the progressionoveractivatedneuroinflammatory and neurodegenerative diseases.of1 - 11 were tested in vitro for theirCompoundseffects against LPS induced nitric oxideinhibitoryof microglia BV2 cells. Among the isolates,production1, 7 and 11 showed inhibitory activitiescompoundsLPS induced NO production in BV-2 microglia inagainstconcentration dependent manner.aet al. (2006) demonstrated that compound 1Kangfrom four year old Panax ginseng inhibited NOisolatedfrom nitroprusside as well as simple phenolicsproductionas p-coumaric and vanillic acid. In addition, maltolsuchwere markedly increased by heat processing oflevelsginseng. Even though there was a difference inPanaxconcentration, the present study is consistent withmaltolreported data. (+)-Isolariciresinol (7) waspreviouslyto exert a potent in vitro inhibitory effect onreportedof a pro-inflammatory cytokine, TNF-a (ChoproductionVol. 17, No. 3, 2011 179Results and discussion7-11 were isolated for the first time from Abies koreana.et al., 2001; Esra et al., 2003). It was reported that
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Page 3: Table 1. The effect of compounds is

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