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29

F.SIMARD ET AL

Exlruction and isolation. The air-dried (.1 wcek) pow-dered wood froni P. resinosa (9.2 kg) was sucecssivcty exirac(cd with hcxane, dichloromeihane, mclhanol and water using a Soxhlcl npparatus (5 L cach for 24 h). Evaporation under rcduced pressure al a temperaturo not higher than 45 °C yielded a mclhanol extract (101 g) and a water cxtracl (38 g).

Part of ihc mclhanol cxtracl front /’. resinosa (71 g) was parlilionod bciwcen cthyl acetale and water. The ElOAc-solublo CKtract (20 g) gave 15 fraclions by opca column chromatography on silica gcl with a CHCly-McOH gradient (100:0 -» 0:100). Fraction 3 (285.9 mg) was subjeeted to rcvcrsed-phase column chromalogra-phy using MeOH-H_:0 7:3 as oluent to give purc 1 (114.6 mg). Fuithcr silica gcl column chromatography of fraction 8 (6S9 mg) cluled with a hcxa»e-EtOAc gradient (90:10 -» 40:60) affotdcd compound 4 (31 mg) and impure 5. This compound was purified on rcversed-phase column chromatography (MeOH-HjO, 6:4) to give purc 5 (100 mg). Fraction 10 was scparaied by open column chromatography on silica gcl with a hcxanc-ElOAc gradient (20:80 —> 70:30) to givc purc 2 (5253 mg).

To isolatc known compound 3, the hexnnc cxtrnct (310 g) was defatted as dcscribcd by Barrero et al. (1991). Pan of the defatted hcxanc cxtract (100 g) was fractionatcd by open column chromatography on silica gcl with a CHOr-McOH gradient (1<X):0 -» 0:100) to givc scvcn fractions. Compound 3 (13 ntg) was obtaincd from opon column chromatography of fraction 2 (31.8 g) using a hexane-£tOAc gradient (98:2 -+ (>0:40).

Pinosvlvin monomclhvl cllter (1): Ycllowish powder, nip 118-121 °C: R₍ - 0.65 (Si()₂. CHCI,-McOH 80:l); HR-ESI-MS (positive) m/z = 226.0994 (C„H_U0₂, (Mj‘, rct|uircs 226.0994). IR. ’H and ^UC NMR data werc simi-lar to published values (iNgo et ul., 1998).

Pinosylvin (2): Ycllowish powder, mp 153-154 °C; R, = 0.27 (Si0₂, CMOrMcOM 40:1); HR-ES1-MS (positivc) m/z = 212.0832 (C_mH_i;0₂, [M]*, rcquires 212.0837). IR. ’H and ^lłC NMR data werc similar to published values (Ngo and Brown. 1998).

Pinosylvin dimcthyl ether (3): Amorphous solid. R₍ = 0.36 (SiO>. Hex-ElÓAc 97:3); HR-ESI-MS (positivc) m/z = 240*1152 (C^Hm.Oj, [M]\ requires 240.1150). IR. H and ^l-'C NMR data werc similar to published valucs (Ngo and Brown. 1998).

Pinobanksin (4); Ycllow solid, mp 164-166 ^C‘Q. (aJo²* = +10.1 (c =0.5, acetonu); R_t = 0.29 (SiO-. CHClj-McOH 80;l); HR-ESl-MS (posilivc) m/z = 272.0681 (C\<H_pO,. [MJ*, rcquircs 272.0685). IR, 'H and ''C NMR data werc similar to published valucs (Kuroyanagi et al.. 1982).

(-)-Nortrachelogenin (5): Amorphous solid. [r/J₀^:5 = -38.3 (c- 0.5. acctone); R<= 0.27 (SiO„ CI łCK-MeOM 80:1): HR-ESI-MS (posilivc) m/z = 374.1365 (Ć*,! U₂0,. (MJ*. rcquircd 374.1366). IR. *H and ^UC NMR dala werc similar to published valucs (Sefkow. 2001).

Celi ciillure. The following human celi lines werc uscd for this study: A549 (lung carcinomn), DLD-1 (coloreclal adenocareinoma) and WSI (normal skin fibroblast). AU ccii lines were obtaincd from the American Typo Cullure Collcclion (ATCC. Manassas. USA). The A549, DLD-1 and WSI ccii lines werc grown in minimum csscntial medium with Earle‘s salts. Medium was supplemented with 10% fetal calf serum, L-glulamine (IX). solution of yilamins (IX). sodium

pyruvatc (IX), non-esscntial amino acids (IX). ponicil-lin (1(X)11J) and streptomycin (100pg/tnL). The cclls werc culiurcd in a humidined almosphcrc at 37 °C in 5% C0₂.

C\tot«xicily assay. Exponentially growing cclls were platcd at a den sity of 5 x 10' cclls per wcII in 96-wcll microplatcs in lOOuL of culturc medium and werc allowed to ndhere for 16 h beforc ircalmcnl. Thon, 100 pL of inereasing conccnlralions of extract or purc compounds dissoWcd in Ihc appropriatc solvcnt werc added. The finał conccntration of $oIvcnt in the culturc medium was maintained at 0.5% (v/v) to avoid solvcnl toxicity. The cclls were ineubated for 48 h in the abscncc or in the prescncc of cxtraci. Gytotoxicity was assessed using the resnzurin rcduction test as dcscribcd by 0'Biicn (0’Bricn et ul.. 2(XX)). Fluorcsccncc was mensured al an excitation wavelcngtl) of 530 nm and an cmission \vavelength of 590 nm. Cytotoxicily was expressed as the conccntration of estract or compound inhibiting celi growth by 50% (IG*,).

RESULTS AND DlSCUSSlON

Lipophilic compounds (5.2%) were rcmoved from plant materiał using hcxanc followcd by DCM cxlractions. Tire extraction yield obtaincd with DCM was Iow suggesting iha! lipophilic compounds were officicnlly oxtraeted by hexanc. Thcn, the biomass was cxtraeled again with MeOH and water with a yield of about 1.1% and 0.4%. rcspcctivcly. The cvtotoxicity of the McOH and the aqucous extracts w'crc tested against human lung carcinomn (A549), human colorcctal adcnocarci-noma (DLD-1) and normal libroblasls (WSI). The rc-sulls prcscnlcd in Tablc 1 show thnt the MeOH cxtracl is modcralcly cytotoxic against A549 (1C_H, 41 ± 6pg/ mL) bul significanlly scleciive toward cancer cclls in contparison with healthy cclls. WSI (1Gb, 130 ± 11 pg/ mL). Similar cylotoxicity or the cihanol exiract from knotwood of Pintts resinosa was previously reported against murinc hepaticccll lino. Hcpa-1 (Viilimaa etui.. 2007). The water extract did noi show cytotoxicily against A549. DLD-1 and WSI at lOOpg/mL. As illustraicd in Fig. 1. Ihc purc cytotoxic compounds

Pmus resirosa MeOtl cstnc, (activcl* |S<4wni ptrjiMtł

r , , i

ElOAc (łłtUYc)’ ;    H;0 (inacuvc)*

| Opm trtunwi

Ftaciion J <activę)' | | Fraciion 8 (acthef		| Ftaciion 10 taaMf
Open coiumc chrcfTtilrfrtpby	coi-irwi Opał toiuit* chromtlocjTf^y cbf«nalcęr*pby
1 (Ktivcf 4 (inaclivc)^k	5 (2 r« bvcf

* Estrad and fraction werc considcrcd inxiivc when IC»> 50 |ig/ml. ^kA purc compound was considcrcd inactivc when IC« > 20 pg/ml.

Figura 1. Bioassay-guided isolation of compounds 1. 2, 4 and 5.

Copyright O 2008 Julu* Wiley A Sons. Lut.    Phrtoihcr. Ha. (2008)

DOI: 10.1lKl2/p«r