Copyright © 2007 John Wiley & Sons, Ltd. Phytother. Res. 21, 406–409 (2007)DOI: 10.1002/ptr

406 H-K. LEE ET AL.

Copyright © 2007 John Wiley & Sons, Ltd.

PHYTOTHERAPY RESEARCHPhytother. Res. 21, 406–409 (2007)Published online 18 January 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ptr.2082

Received 19 July 2006Revised 9 November 2006

Accepted 9 November 2006

* Correspondence to: Byung-Sun Min, College of Pharmacy, CatholicUniversity of Daegu, Gyeongbuk 712-702, Korea.E-mail: bsmin@cu.ac.krContract/grant sponsor: Plant Diversity Research Center of 21st FrontierResearch Program funded by the Ministry of Science and Technology ofthe Korean Government; contract/grant number: PF0300401-00.

INTRODUCTION

Angelica species (Umbelliferae) are endemic toNortheast Asia and are important medicinal plantsthat have attracted considerable attention on accountof their biological and chemical diversity. Studies ofthe chemical components of Angelica have led tothe identification of many compounds including cou-marins, chromones, phenylpropanoids, sesquiterpenesand polyacetylenes (An et al., 2005). Some of themhave been shown to have 5α-reductase type I activity(Seo et al., 2002), antiproliferative activity (Fujiokaet al., 1999), anti-HIV-1 activity (Zhou et al., 2000),antiacetylcholinesterase activity (Kim et al., 2002) andactivity in inhibiting prostaglandin E2 (Ban et al., 2003)and TNF-α production (Cho et al., 1998). Angelicapurpuraefolia Chung is one substitute source of theChinese herbal drug ‘Gangwhal’ (Lee, 1996). The rootsof this plant have been used to treat the common cold,headache, neuralgia and arthralgia (Woo et al., 1982).As part of an ongoing study into the antitumor activityof compounds from natural sources, the compounds ofA. purpuraefolia were examined. This paper presentsthe structure elucidation of hydroxylomatin (1) and theabsolute configuration at C-1 of compound 2, as well asthe antitumor activity of khellactone (3).

MATERIAL AND METHODS

General instrumental equipment. Optical rotationswere measured with a JASCO DIP-370 automaticdigital polarimeter in CHCl3. The NMR spectra wererecorded on a Varian NMR System 400 MHz, withchemical shifts being represented in ppm andtetramethylsilane used as an internal standard. TheFAB-MS was measured on a JMS-HX 110/110A massspectrometer (JEOL). ESI-MA was measured on aHP5989 DIP mass spectrometer. Medium pressureliquid chromatography (MPLC) separations wereperformed over LiChroprep RP C-18 (Merck, size B).The spots were detected under UV radiation and byspraying with 10% H2SO4, followed by heating.

Plant material. The roots of A. purpuraefolia werecollected during September 2003 at Chirisan, Korea,and dried at room temperature. A voucher specimen(PBS-2358.1) was deposited at Plant Extract Bank,Korea Research Institute of Bioscience and Biotech-nology, Daejeon, Korea.

Extraction and isolation. The dried and powdered rootsof A. purpuraefolia (10 kg) were extracted with MeOH(3 × 10 L) for 7 days at 25 °C. The combined extractswere concentrated under reduced pressure. The residue(652 g) was diluted with water (1 L), and then parti-tioned successively with CHCl3 (3 × 1 L) and EtOAc (3× 1 L) to afford the CHCl3-(340 g) and EtOAc-solublefractions (10 g), respectively. The CHCl3-soluble fractionwas chromatographed on a silica gel column. The columneluted using a stepwise gradient of hexane and EtOAc

Isolation of Coumarins and Ferulate from theRoots of Angelica purpuraefolia and theAntitumor Activity of Khellactone

Hyeong-Kyu Lee1, Sei-Ryang Oh1, Ok-Kyoung Kwon1, Kyoung-Seop Ahn1, Joongku Lee1,Jin-Cheol Kim2, Byung-Sun Min3* and Hyouk Joung1

1Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea2Korea Research Institute of Chemical Technology, Daejeon 305-600, Korea3College of Pharmacy, Catholic University of Daegu, Gyeongbuk 712-702, Korea

A new coumarin, hydroxylomatin (1), was isolated from the CHCl3-soluble fraction of the roots ofAngelica purpuraefolia, along with one ferulate (2) and three other known coumarins (3–5) including khellac-tone (3). The structure of hydroxylomatin (1) was determined to be 3′βββββ ,5′-dihydroxy-3′,4′-dihydroseselin(1) by spectroscopic means including 2D-NMR. The modified Mosher’s method was used to determinethe chiral center at C-1 of compound 2. Khellactone (3) is a major compound of the roots of A.purpuraefolia. This study also examined the antitumor activity of khellactone (3) using a LLC mouselung carcinoma in the BDF-1 mice and a NCI-H460 human lung carcinoma in a human tumor xenograftmodel in nude mice. This compound (3) inhibited LLC tumor growth with a T/C (mean value of treated group/mean value of control group) value of 12.9% at a dose of 5 mg/kg and 33.2% at a dose of 10 mg/kg, respec-tively, in a dose-dependent manner. In addition, it suppressed the growth of NCI-H460 tumor cells, accountingfor 81.4% at a dose of 10 mg/kg in nude mice. Copyright © 2007 John Wiley & Sons, Ltd.

Keywords: Angelica purpuraefolia; Umbelliferae; khellactone; antitumor activity; hydroxylomatin.

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Copyright © 2007 John Wiley & Sons, Ltd. Phytother. Res. 21, 406–409 (2007)DOI: 10.1002/ptr

to give nine fractions (Fr. 1–9; 4.6 g, 19.3 g, 6.2 g, 93.2 g,137.1 g, 11.3 g, 11.0 g, 5.1 g, 9.0 g). Of nine fractions,fractions 7 and 8 exhibited antitumor activity againstLLC tumor cells in BDF-1 mice with a T/C (mean valueof treated group/mean value of control group) value of25.1% and 25.2% at a dose of 125 mg/kg, respectively.Fraction 7 was loaded on to a silica gel column, whichwas eluted with hexane:EtOAc (50:1 → 1:1, v/v) to givesix sub-fractions (sub-fr. 1–6; 0.044 g, 0.218 g, 4.73 g,1.87 g, 1.06 g, 0.22 g). Repeat column chromatographyof sub-fraction 3 on silica gel (hexane:EtOAc) andMPLC (RP C-18, 45% acetonitrile) afforded 3 (294 mg)and 4 (15 mg). Fraction 8 was chromatographed on asilica gel column with hexane:EtOAc (3:1 → 1:1) andCHCl3:MeOH (9:1) to yield five sub-fractions (sub-fr.1–5; 0.159 g, 6.564 g, 2.051 g, 2.225 g, 3.713 g). Columnchromatography of sub-fraction 2 over silica gel(hexane:EtOAc, 3:2), RP C-18 MPLC (40% acetonitrile)and HPLC (ODS, 20% → 44% acetonitrile) resulted in1 (14 mg), 2 (10.4 mg) and 5 (8.0 mg).

Hydroxylomatin (1). White amorphous powder; mp135–136 °C; [α]D: −30.3° (c = 0.2, CHCl3); ESI-MS m/z:263.1 [M+H]+; HREIMS m/z: 262.0842 ([M]+, Calcdfor C14H14O5: 262.0841); 1H-NMR (400 MHz, CDCl3):δ 7.83 (1H, d, J = 9.6 Hz, H-4), 7.38 (1H, d, J = 8.4 Hz,H-5), 6.77 (1H, d, J = 8.4 Hz), 6.16 (1H, d, J = 9.6 Hz,H-3), 5.02 (1H, t, J = 9.4 Hz, H-3′), 3.72 (1H, d, J =11.0 Hz, H-5′), 3.53 (1H, d, J = 11.0 Hz, H-5′), 3.34 (2H,m, H-4′); 13C-NMR (100 MHz, CDCl3): δ 165.7 (C-7),163.2 (C-2), 152.5 (C-9), 146.3 (C-4), 130.3 (C-5), 115.1(C-8), 114.3 (C-10), 112.2 (C-3), 108.0 (C-6), 89.2(C-3′), 74.7 (C-2′), 67.7 (C-5′), 27.5 (C-4′), 20.4 (C-6′).

(1S)-2-O-E-Feruloyl-1-(4-hydroxyphenyl)ethane-1,2-diol(2). White amorphous powder; mp 70–71 °C; ESI-MSm/z: 331.1 [M+H]+; HREIMS m/z: 330.1106 ([M]+, Calcdfor C18H18O6: 330.1103); 1H-NMR (400 MHz, MeOH-d4): δ 7.63 (1H, d, J = 16.4 Hz, H-7′), 7.25 (2H, d,J = 8.6 Hz, H-4,8), 7.18 (1H, d, J = 1.6 Hz, H-3′), 7.07(1H, dd, J = 8.4, 1.6 Hz, H-6′), 6.81 (1H, d, J = 8.4 Hz,H-5′), 6.79 (2H, d, J = 8.4 Hz, H-5,7), 6.37 (1H, d,J = 16.4 Hz, H-8′), 4.72 (1H, m, H-1), 4.22 (2H, m,H-2), 3.89 (3H, s, OCH3). 13C-NMR (100 MHz, MeOH-d4): δ 169.1 (C-9′), 158.3 (C-6), 150.7 (C-4′), 149.4(C-3′), 147.1 (7′), 133.1 (C-3), 128.7 (C-4,8), 127.7(C-1′), 124.2 (C-6′), 116.5 (C-5′), 116.2 (C-5,7), 115.3(C-8′), 111.7 (C-2′), 72.7 (C-1), 70.1 (C-2), 56.4(C-OCH3).

(R)-MTPA ester of 2 (2a). (+)-MTPA chloride (1.5 mg)and DMAP (10 mg) in pyridine (0.2 mL) were addedto a solution of 2 (2.0 mg) in CCl4 (0.2 mL). Afterstirring at room temperature for 12 h, the mixture waspoured into water (10 mL) and extracted with EtOAc(10 mL × 2). The EtOAc extract was concentratedin vacuo and purified by preparative thin layer chro-matography (hexane:EtOAc, 2:1) to give the (R)-MTPAester (2a) as an oil; 1H-NMR (CDCl3): δ 7.58 (1H, d,J = 16.2 Hz, H-8′), 7.43 (2H, d, J = 8.6 Hz, H-4,8), 7.17(2H, d, J = 8.6 Hz, H-5,7), 6.27 (1H, d, J = 16.2 Hz,H-7′), 4.40 (2H, m, H-2).

(S)-MTPA ester of 2 (2b). (−)-MTPA chloride (1.5 mg)and DMAP (10 mg) in pyridine (0.2 mL) were addedto a solution of 2 (2.0 mg) in CCl4 (0.2 mL). After stir-

ring at room temperature for 12 h, the mixture waspoured into water (10 mL) and extracted with EtOAc(10 mL × 2). The EtOAc extract was concentrated invacuo and purified by preparative thin layer chromato-graphy (hexane:EtOAc, 2:1) to give the (S)-MTPAester (2b) as an oil; 1H-NMR (CDCl3): δ 7.62 (1H, d,J = 16.2 Hz, H-8′), 7.33 (2H, d, J = 8.6 Hz, H-4,8), 7.11(2H, d, J = 8.6 Hz, H-5,7), 6.33 (1H, d, J = 16.2 Hz,H-7′), 4.48 (2H, m, H-2).

Khellactone (3). Colorless needles; mp 173–175 °C; [α]D:110.5° (c = 0.91, MeOH); ESI-MS m/z: 263 [M+H]+.

Discophoridin (4). Colorless needles; mp 173–175 °C;[α]D: 110.5° (c = 0.91, MeOH); ESI-MS m/z: 263 [M+H]+.

Angelinol (5). Colorless plates; mp 180–182 °C; [α]D:−110.5° (c = 0.1, pyridine); ESI-MS m/z: 247 [M+H]+.

Tumor cells. LLC (mouse lung carcinoma) and NCI-H460 (human lung carcinoma) were purchased fromthe National Cancer Institute (NCI), NIH, USA. Thecells were maintained as monolayer cultures in RPMI1640 medium supplemented with 5% FBS, sodiumbicarbonate (2 g), penicillin G (100,000 units) and strep-tomycin (100 mg) in a humidified 5% CO2 atmosphereat 37 °C.

Animals. Specific pathogen-free female BDF-1 mice,purchased from Daehan-Biolink (Eomsung, Korea),were used at 4 weeks of age, weighing 14–16 g. Specificpathogen-free female Balb/c-nu/nu mice (nude mice),used for all the human tumor xenograft experiments,obtained from Daehan-Biolink (Eomsung, Korea),were used at 6 weeks and weighed between 19–21 g.The animals were fed with a commercial pellet chow ina temperature-controlled room at 25 ± 2 °C and allowedwater ad libitum.

Evaluation of activity in murine tumor models. Toevaluate the sensitivity of transplantable murine tumorsto the BDF-1 mouse, khellactone (3) was completelysuspended in 2% acacia gum. Adriamycin was used asa reference compound at a dosage of 2 mg/kg and dis-solved in 2% acacia gum. For the subcutaneous tumorassay (Kato et al., 1994), a suspension of 5 × 105 LLC cellsin 0.1 mL of 0.9% NaCl solution was carefully inocu-lated intradermally into the left axilla of mice. Then, thetumor-bearing mice were randomly assigned to treatmentexperimental groups (five or six mice in each group).Twenty-four hours after the tumor inoculation, micewere intraperitoneally given khellactone (5 mg or 10 mg/kg) and adriamycin once daily for 14 consecutive days.On the final day, tumors were excised and weighed.Mean tumor weights were determined, and used tocalculate the TGI expressed as a percentage

%TGI = 1 − (mean final tumor weight of treatedgroup (T )/mean final tumor weight of control

group (C)) × 100.

Evaluation of activity in human tumor models. To evalu-ate the sensitivity of human xenografts to khellactone,NCI-H460 cells (2 × 106/mouse) were injected s.c. intothe right flank of the nude mice. The experiment wasconducted as above.

Copyright © 2007 John Wiley & Sons, Ltd. Phytother. Res. 21, 406–409 (2007)DOI: 10.1002/ptr

408 H-K. LEE ET AL.

Statistical analysis. The significance of differencesbetween the experimental groups and control groupwas calculated by the Student’s t-test.

RESULTS AND DISCUSSION

Repeat column chromatography of the CHCl3-solublefraction of the MeOH extract of A. purpuraefoliaon silica gel and reverse phase silica gel columns ledto the isolation of five compounds (1–5). The knowncoumarin compounds were identified as khellactone (3)(Takata et al., 1988), discophoridin (4) and angelinol(5) (Chatterjee et al., 1978), which have been isolatedfrom Angelica species, based on their spectral andphysical data in comparison with those reported inthe literature.

Hydroxylomatin (1) was obtained as a white amor-phous powder with a positive optical rotation ([α]+30.3°). Its molecular formula C14H14O5 was confirmedfrom the molecular ion peak at m/z 263.1 [M+H]+ inthe ESI-MS of 1 as well as by high-resolution EIMSmeasurements. Hydroxylomatin (1) exhibited fourdoublet signals at δ 7.83 (J = 9.6 Hz), 7.38 (J = 8.4 Hz),6.77 (J = 8.4 Hz) and 6.16 (J = 9.6 Hz), which wereassigned to a coumarin skeleton in the 1H-NMR spec-trum, compared with that of badycoumarin A isolatedfrom A. flaccida (Seong et al., 1991). This observationwas further supported by the 13C-NMR spectral assign-ments (a carbonyl carbon at δ 163.2, two olefiniccarbons at δ 112.2 and 146.3, two aromatic carbons atδ 108.2 and 130.3, and four aromatic quaternarycarbons at δ 114.3, 115.1, 152.5 and 165.7) as well asby DEPT and 2D NMR (COSY, HMQC and HMBC)experiments. In addition, the 1H-NMR spectrum showedtwo oxygenated methylene protons at δ 3.53 (d, J =11.0 Hz) and 3.72 (d, J = 11.0 Hz), which correlatedwith an oxygenated quaternary carbon at δ 74.7 (C-2′)and a methyl carbon at δ 20.4 in the HMBC spectrum,indicating the presence of pyranocoumarin in 1, whichcompared favorably with that of the khellactoneskeleton. In addition, the 1H-NMR spectrum showedan oxygenated methine proton at δ 5.02 (t, J = 4.7 Hz),which correlated with the oxygenated carbon at δ 74.7and a methylene carbon at δ 27.5 (C-4′) in the HMBCspectrum. The β-orientation of the hydroxy group atC-3′ was deduced from the 1H-NMR coupling patternof H-3′ signal (δ 5.02, triplet), compared with thatof decanoyllomatin isolated from Seseli devenyense(Widelski et al., 2005). An equatorial methyl group(β-orientation, C-6′) at C-2′ was also deduced from acorrelation peak observed between H-3′ (δ 5.02) andH3-6′ (δ 1.21) in the NOESY spectrum. Furthermore,the carbonyl carbon at δ 163.2 (C-2) correlated withtwo olefinic protons at δ 7.83 (H-4) and 6.16 (H-3)in the HMBC spectrum. Therefore, the structure ofhydroxylomatin (1) was determined to be 3′β,5′-dihydroxy-3′,4′-dihydroseselin (Matsuda et al., 2000)(Fig. 1).

2-O-(4-Hydroxy-3-methoxy-E-cinnamoyl) (2) was firstisolated from Peucedanum decursivum by Kong andYao. However, they did not determine the absoluteconfiguration of C-1 in compound 2. In order todetermine the absolute stereochemistry at C-1, com-pound 2 was subjected to derivatization using the

Figure 1. Structures of compounds 1–5 isolated from A.purpuraefolia.

Figure 2. Chemical shift difference for the (S)-MTPA ester (2b)and (R)-MTPA ester (2a) in ppm at 400 MHz.

modified Mosher’s method (Ohtani et al., 1991). Com-pound 2 was treated with (+)- and (−)-α-methoxyl-α-trifluoromethylphenylacetyl chloride (MTPA-Cl) inthe presence of 4-(dimethylamino)-pyridine (DMAP)and gave the (R)- and (S)-MTPA esters (2a and 2b). Inthe 1H-NMR spectrum of the (S)-MTPA ester (2b), theproton signals assigned to H2-2, H-7′, and H-8′ wereobserved at a lower field than those of the (R)-MTPAester (2a), while signals due to H-4 and H-5 in theformer ester were shifted to a higher field than thosein the latter ester (Fig. 2). Therefore, the absolute con-figuration at C-1 was concluded to be 1S.

The compounds isolated from the roots of A.purpuraefolia (1–5) were tested for their cytotoxicactivity against LLC tumor cell line. However, com-pounds 1–5 were inactive (data not shown) againstthe test tumor cell line. The antitumor activity ofkhellactone (3), the major compound of the roots ofA. purpuraefolia, was estimated. This was tested usingan LLC mouse lung carcinoma in the BDF-1 mice andNCI-H460 human lung carcinoma in a human tumorxenograft model in nude mice. Khellactone (3) in 2%acacia gum was administered intraperitoneally at a doseof 5 or 10 mg/kg for 14 days. The growth of LLC can-cer cells was inhibited by khellactone (3). Compound 3reduced the tumor weights by 12.9% and 33.2% at adose of 5 and 10 mg/kg in BDF-1 mice, respectively,and the positive control, adriamycin, showed a 79.4%reduction at a dose of 2 mg/kg (Fig. 3). Furthermore,this compound (3) showed antitumor activity with a

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Copyright © 2007 John Wiley & Sons, Ltd. Phytother. Res. 21, 406–409 (2007)DOI: 10.1002/ptr

Figure 3. Effects of khellactone (3, KL) on tumor weight onintradermally inoculated LLC cells in mice by intraperitonealadministration. Data are expressed as mean ± SE (n = 5–6).Statistical significance: * p < 0.05 versus control.

Figure 4. Effects of khellactone (3) on tumor weight on intra-dermally inoculated NCI-H460 cells in nude mice by intraperi-toneal administration. Data are expressed as mean ± SE (n = 5).Statistical significance: * p < 0.05 versus control.

T/C value of 81.4% at a dose of 10 mg/kg with NCI-H460 tumor cells in nude mice, compared with that ofadriamycin (87.3% at a dose of 2 mg/kg) (Fig. 4). For-tunately, no loss of body weight was observed in theBDF-1 and nude mice administered with khellactone(3) at doses of 5 and 10 mg/kg and no significant differ-ence in histologic findings of kidney, lung, liver or uterusfrom both control and khellactone-treated mice wasfound (data not shown). Although further studiesare needed to clarify the mechanism of the antitumor

activities of khellactone, the present study suggests thatsuch coumarins could be good candidates as antitumoragents.

Acknowledgements

This research was supported by the grants (PF0300401-00) from thePlant Diversity Research Center of 21st Frontier Research Programfunded by the Ministry of Science and Technology of the KoreanGovernment.

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