Xanthatin and xanthinosin from the burs ofXanthium strumarium L. as potential anticanceragents1

Irving Ramırez-Erosa, Yaoge Huang, Robert A. Hickie, Ronald G. Sutherland, andBranka Barl

Abstract: Xanthatin and xanthinosin, 2 sesquiterpene lactones isolated from the burs of Xanthiun strumarium L.(cocklebur), showed moderate to high in vitro cytotoxic activity in the human cancer cell lines WiDr ATCC (colon),MDA-MB-231 ATCC (breast), and NCI-417 (lung). Xanthatin and xanthinosin were purified as the result of a multi-screening bioassay-guided study of wild plant species of the family Asteraceae, collected from various sites in Saskatche-wan, Canada. Seventy-five extracts at a single concentration of 100 mg/mL were evaluated for in vitro cytotoxicity to thehuman cancer cell lines used. The chloroform extract of Carduus nutans L. (nodding thistle) aerial parts (IC50, 9.3 mg/mL)and the hexane extract of Echinacea angustifolia DC. (narrow-leaved purple coneflower) root (IC50, 4.0 mg/mL) were mod-erately to highly cytotoxic to the lung cancer cell line. The chloroform extracts of X. strumarium L. burs and Tanacetumvulgare L. (tansy) aerial parts exhibited the highest cytotoxicity for all cell lines tested; their IC50 values, obtained frommultidose testing, ranged from 0.1 to 6.2 mg/mL (X. strumarium) and from 2.4 to 9.1 mg/mL (T. vulgare). Further purifica-tion of the chloroform fraction of X. strumarium yielded xanthatin and xanthinosin in high yields. This is the first timethat these compounds have been reported in the burs of X. strumarium. Their IC50 values are also reported herein.

Key words: xanthatin, xanthinosin, Asteraceae, cytotoxic activity, human cancer cell lines, Xanthium strumarium L.,Tanacetum vulgare L., Carduus nutans L., Echinacea angustifolia DC.

Resume : La xanthatine et la xanthinosine, deux lactones sesquiterpeniques isolees des capsules de Xanthium strumariumL. (lampourde glouteron), ont montre une activite cytotoxique moderee a elevee dans les lignees cellulaires cancereuseshumaines WiDr ATCC (colon), MDA-MB-231 ATCC (sein) et NCI-417 (poumon), respectivement. On a purifie la xantha-tine et la xanthinosine a partir des resultats d’une etude bioguidee de plusieurs plantes sauvages de la famille des Astera-ceae, prelevees dans divers sites de la Saskatchewan. On a evalue l’activite cytotoxique in vitro de soixante-quinze extraitsde 100 mg/mL contre les lignees de cellules cancereuses humaines. L’extrait au chloroforme des parties aeriennes (IC50

9,3 mg/mL) de Carduus nutans L. (chardon penche) et l’extrait a l’hexane de la racine de Echinacea angustifolia DC.(echinacee pourpre a feuilles etroites) (IC50 4,0 mg/mL) ont montre une activite cytotoxique moderee a elevee contre la li-gnee de cellules cancereuses pulmonaires. Les extraits au chloroforme des capsules de X. strumarium L. et des parties ae-riennes de Tanacetum vulgare L. (tanaisie vulgaire) ont demontre la plus haute cytotoxicite contre toutes les lignees decellules examinees; leurs valeurs d’IC50, obtenues a la suite d’une epreuve multidose, ont ete comprises entre 0,1 et6,2 mg/mL (X. strumarium) et entre 2,4 et 9,1 mg/mL (T. vulgare). Une purification additionnelle de la fraction chlorofor-mee de X. strumarium a produit de la xanthatine et de la xanthinosine en grandes quantites. On rend compte pour la pre-miere fois de l’activite de ces composes dans les capsules de X. strumarium. Leurs valeurs d’IC50 sont aussi indiquees.

Mots-cles : xanthatine, xanthinosine, Asteraceae, activite cytotoxique, lignees de cellules cancereuses humaines, Xanthiumstrumarium L., Tanacetum vulgare L., Carduus nutans L., Echinacea angustifolia DC.

[Traduit par la Redaction]

Introduction

For thousands of years, cultures throughout the world

have used plants or plant extracts for medicinal purposes.Many drugs commonly used today were developed fromplants used in traditional medicinal systems. Several drugs

Received 16 February 2007. Published on the NRC Research Press Web site at cjpp.nrc.ca on 21 November 2007.

I. Ramırez-Erosa, Y. Huang, and B. Barl.2,3 Department of Plant Sciences, University of Saskatchewan, Saskatoon, SK S7N 5A8,Canada.R.A. Hickie. Department of Pharmacology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada.R.G. Sutherland. Department of Chemistry, University of Saskatchewan, Saskatoon, SK S7N 5C9, Canada.

1This article is one of a selection of papers published in this special issue (part 2 of 2) on the Safety and Efficacy of Natural HealthProducts.

2Corresponding author (e-mail: branka.barl@shaw.ca).3Present address: Barl and Associates, 18-10235, 111 Street, Edmonton, AB T5K 2V5, Canada.

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have been developed following ethnobotanical leads, and alarge proportion of commercial drugs used today to treat avariety of diseases, including cancer, were originally discov-ered through research on plants (Farnsworth and Morris1976; Balandrin et al. 1985; Cragg and Newman 1999;Newman et al. 2003). Research dedicated to the isolationand identification of anticancer plant principles began in arational manner by collecting a variety of samples fromaround the world, or by relying on folklore. The folkloreand phytochemical literature of virtually every country hasnumerous references to the medicinal use of local plants, in-cluding the use of selected species in the treatment of cancer(Sneden 1984). For example, Huang (1999) describes 79plant species used in traditional Chinese medicine, as wellas their active principles, and the mechanisms of action ofthe compounds believed to exert anticancer effects.Although many of these plants are not mentioned in oldChinese traditional medical texts or in the Chinese pharma-copoeias, their properties have been studied at the NationalCancer Institute (NCI) in the United States. As a result,some plant extracts and compounds are currently beingtested in clinical trials.

Most of the collections leading to the discovery of plant-derived anticancer agents have been from tropical rainforestareas, and less attention has being given to North Americanflora. In fact, only a few studies of medicinal plants havebeen undertaken, despite the well documented use of someplant species by Native Americans (Shemluck 1982;McCutcheon et al. 1992). The most common examples ofNorth American plant-derived drugs used today in cancertreatment include podophyllotoxin (and its less toxic semi-synthetic lignans, etoposide and teniposide) and paclitaxel.Podophyllotoxin is isolated from the rhizomes and roots ofthe American mayapple (Podophyllum peltatum L., Berberi-daceae) (Pettit 1995), indigenous to the eastern UnitedStates (Eastern being east of Oklahoma), and in woodlandsin Canada. Paclitaxel is isolated from the bark of the Pacificor western yew tree (Taxus brevifolia Nutt., Taxaceae)(Kingston 1993; Cragg and Newman 1999), found through-out the western region of northern California, Oregon,Washington, British Columbia, and Alaska (Pojar andMacKinnon 1994).

The plants in the family Asteraceae, also known as Com-positae (Robbers and Tyler 1999), are herbs, shrubs, vines,and trees. They are widely spread throughout the world, butare especially well adapted to fairly dry, temperate, andcooler climates (Shemluck 1982; Johnson et al. 1995; Hein-rich et al. 1998). Asteraceae has considerable economic im-portance through food plants (lettuce, globe artichoke,chicory, sunflower), but losses of livestock intoxicated byingesting plant species of this family is well known. Manyspecies are noxious weeds (dandelion, ragweed, thistle),while others are used in medicinal preparations or herbalteas (German chamomile) or as ornamentals (chrysan-themum, dahlia, zinnia, marigold) (Meyer and van Staden1989; Johnson et al. 1995). Recently, Echinacea purpureaL. Moench. (broad-leaved purple coneflower) and Echinaceaangustifolia DC. (narrow-leaved purple coneflower) have at-tracted much interest because they produce compounds thatstimulate the immune system (Foster 1991; Newall et al.1996; Jurgens 2000; Ross 2001). Plants in this family have

also been used in nutritional, culinary, and ceremonial appli-cations (Heinrich et al. 1998). Members of the family Aster-aceae are known to provide many interesting compounds.

As a result of the well documented toxicity exerted byseveral plants species of the Asteraceae, plant extract cyto-toxicity to human cancer cell lines has been a research topicin several laboratories throughout the world. The cytotoxic-ity of several compounds isolated from the flower extract ofChrysanthemum morifolium (Ukiya et al. 2002) and the cy-totoxic and anti-tumor effects of the methanol extract ofEmilia sonchiflora in mice (Shylesh and Padikkala 2000)are just a few examples of the potential use of these plantsin the development of anticancer drugs.

Given the similarity in flora between Saskatchewan (inwestern Canada) and northern China, some plant speciesused for cancer therapy in traditional Chinese medicine arereadily available in Saskatchewan. In this study, we usedtraditional Chinese medicine as a tool to screen wild plantsfrom Saskatchewan for potential cytotoxicity leading us toexamine 15 plant species from 12 genera of the family As-teraceae (Table 1). These were collected from various sitesand regions of Saskatchewan. The research described hereinaddresses the testing of these plant extracts for cytotoxic ac-tivity in vitro using the human cancer cell lines WiDr ATCC(colon), MDA-MB-231 ATCC (breast), and NCI-417 (lung),as well as the purification procedures and in vitro biologicalevaluation of xanthatin and xanthinosin as potential thera-peutic anticancer agents.

Materials and methods

Plant collectionPlant parts of interest from 15 plant species of the family

Asteraceae were collected from the end of May to the mid-dle of October in 1999 and 2000 from various sites in Sas-katchewan. The botanical identity of plants was verified byP.A. Ryan (Fraser Herbarium, University of Saskatchewan,Saskatoon, Saskatchewan), and a collection number wasgiven to each plant (Table 1). The plants were sorted,washed, air dried (until the moisture content was <10%),and kept in paper bags in a storage chamber at a controlledtemperature (10 8C) and humidity (<40%). Before extrac-tion, approximately 200 g of plant materials were ground ina laboratory mill (Model 4 Thomas-Wiley) by using a 2-millimetre sieve (20 mesh).

Plant extractionAll solvents were purchased from BDH Inc. (Toronto,

Ont.) or E. Merck (Darmstadt, Germany). A protocol forplant extraction using solvents of increasing polarity is pre-sented in Fig. 1. Approximately 100 g of the ground plantmaterial was extracted in 500 mL of methanol (MeOH),with 3 washes of 500 mL, over 72 h. The methanol extractswere pooled, filtered, and MeOH was evaporated in vacuo.The crude MeOH extract was resuspended in water (H2O,200 mL) and partitioned with hexane (Hex, 3 times, 2:1,1:1, and 1:1) to yield a hexane extract (extract 1). The re-maining aqueous phase was extracted 3 times (2:1, 1:1, and1:1) with chloroform (CHCl3) to yield a chloroform extract(extract 2). The remaining aqueous phase was evaporated todryness in vacuo. Methanol was then added (3 times,

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100 mL) to resuspend the water-soluble residue and after fil-tration, a methanol extract was obtained (extract 3). The re-maining residue was labeled as an aqueous extract (extract4). The plant residue obtained upon filtration of the firstMeOH extract was extracted under reflux with water for 1 h(2 times, 100 mL). Upon filtration, a boiling-water-solubleextract was collected (extract 5) and lyophilized, while theresidue was discarded. Each extract was dried under vac-uum for 10–24 h to completely remove any solvent residuebefore cytotoxic testing.

Evaluation of plant extract cytotoxicityThree human tumor cell lines were used to screen plant

extracts for anticancer effects: WiDr ATCC (colon), MDA-MB-231 ATCC (breast), and NCI-417 (lung). Cells weregrown in RPMI medium 1640 with L-glutamine (GIBCOBRL, Life Technologies, Burlington, Ont.), containing 10%FBS (Gibco BRL) and antibiotics (penicillin 100 U/mL andstreptomycin 100 mg/mL) (Gibco BRL), in either T-75 cm2

or T-25 cm2 tissue culture flasks and cultured at 37 8C inhumidified air and 5% CO2. The medium was changed twicea week. Generally, the cells were stored in liquid nitrogen.

A stock solution of 2 mg/mL of each test extract was pre-pared by completely dissolving 10 mg of extract in 0.1 mLDMSO, 20 mL Tween 80, and 4.9 mL RPMI medium 1640with L-glutamine (without FBS and antibiotics). The finalconcentration of the plant extract tested was 100 mg/mL, asrecommended by the NCI’s Developmental TherapeuticsProgram protocol for prescreening natural product extracts(microorganism, marine plant, marine animal, or terrestrialplant) (Boyd 1997). Each extract was prepared in triplicate.

The cytotoxic activity of the plant extracts was evaluatedusing the CellTiter 96 nonradioactive cell proliferation as-say. The CellTiter 96 assay is based on the cellular conver-sion of a tetrazolium salt into a formazan product that iseasily detected using a 96-well plate reader, which is the in-tensity of the color absorbance directly proportional to thelevel of cell proliferation or viability (Promega Corporation1999; Page 1997; Sieuwerts et al. 1995). The cell growth ra-

tio of cancer cells in different concentrations of each plantextract was calculated using the following formula:

Y ¼ 1 � T

C

� �� 100

where Y is the cell growth ratio of cancer cell mortality (%) ateach sample concentration, T is the mean absorbance of trea-ted cells, and C is the mean absorbance of negative control.

The IC50 values of selected plant extracts (those that pro-duced the highest mortality rates) were determined from adose–response curve by using 4 different concentrations(12.5, 25, 50, and 100 mg/mL). Analyses were done in tripli-cate for each concentration.

Analysis of extracts of Xanthiun strumarium bychromatographic methods

Analytical thin-layer chromatographyAnalytical thin-layer chromatography (TLC) was carried

out on aluminum-backed plates of silica gel 60 F254 (5 �10 cm, 0.25 mm layer thickness; E. Merck, Darmstadt, Ger-many), and after elution with a suitable solvent system, theplates were examined under UV light (254/366 nm). Theplates were sprayed with 1% (w/v) vanillin–H2SO4 stainingreagent (Wagner and Bladt 1984). Compounds were visual-ized after color development upon heating at 110 8C for2 min. The developing solvent system for CHCl3 extractionof the cocklebur burs was Hex–EtOAc (ethyl acetate) (2:3).

Analytical high-performance liquid chromatographyAnalytical high-performance liquid chromatography

(HPLC) was performed using a Nova-Pak C18 column(150 � 3.9 mm i.d., 4 mm) from Waters Corporation (Mil-ford, Mass.), and a Waters 996 photodiode array (PDA) de-tector.

The system was equipped with an autosampler, the PDAdetector was set to scan in the region of 192–400 nm, andthe column effluent was monitored at 210 and 275 nm. Theextracts (fractions) were prepared at a concentration of

Table 1. Botanical and common names of plants screened.

Species (common name) Plant part collected Collection site

Artemisia campestris L. (plains wormwood) Whole plant LR-009Artemisia ludoviciana Nutt. (prairie sage) Whole plant SP2–021Artemisia vulgaris L. (common wormwood) Aerial parts US-043Aster brachyactis Blake (rayless aster) Aerial parts WS-072Aster ciliolatus Lindl. (Lindley’s aster) Root LR-059Carduus nutans L. (nodding thistle) Aerial parts SWS-035Cirsium arvense (L.) Scop. (Canada thistle) Aerial parts US-033Echinacea angustifolia DC. (narrow-leaved purple coneflower) Root US-074Helianthus petiolaris Nutt. (prairie sunflower) Whole plant SWS-036Matricaria matricarioides (Less.) Port. (pineappleweed) Whole plant SWS-082Solidago mollis Bartl. (velvety goldenrod) Whole plant SS-064Sonchus arvensis L. (perennial sow-thistle) Whole plant SWS-065Tanacetum vulgare L. (tansy) Aerial parts SS-070Taraxacum officinale Webb. (dandelion) Root OL-017Xanthium strumarium L. (cocklebur) Fruit SR-047

Note: Key to collection sites: LR, La Ronge, Sask.; SP-2, Saskatoon plot No. 2; US, University of Saskatchewan Hor-ticulture plot; WS, Saskatoon west side; SWS, Saskatoon south-west side; SS, Saskatoon Silverspring area; OL, Outlook,Sask.; SR, South Saskatchewan River bank.

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1 mg/mL, the injection volume was 5 mL, and fractions wereeluted using a solvent system with a gradient (80%H2O : 20% MeCN (methyl cyanide) to 40% H2O : 60%MeCN) over 35 min at a flow rate of 1 mL/min. Data werecollected and analyzed using Millennium32 chromatographysoftware (Waters Corporation).

Fractionation and isolation of target compounds from achloroform extract of X. strumarium burs

Fractionation by vacuum liquid chromatography usinggradient elution

Vacuum liquid chromatography (VLC) of a chloroform

extract was performed according to Pelletier et al. (1986).The adsorbent silica gel (Type H, size 10–40 mm), pur-chased from Sigma Chemical Co. (St. Louis, Mo.) was firstloaded into a glass funnel and allowed to settle by gentletapping. A vacuum was then applied, and the adsorbent wascompressed to a hard layer by pressing with a rubber stopperand tapping. Once uniform and tight packing of the adsorb-ent was achieved, the vacuum was released, the solvent oflow polarity (Hex) was poured quickly onto the adsorbentsurface, and the vacuum was reapplied. The column wasthen sucked dry and the CHCl3 extract preadsorbed on silicagel (1:3 ratio) was carefully introduced onto the surface ofthe packing. Enough solvent was used to completely cover

Fig. 1 Selected protocol for plant extraction using solvents of increasing polarity.

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the top surface of the packing and the vacuum was appliedgently to draw the sample into the packing. The solvent gra-dient used consisted of 100% Hex to 100% EtOAc to100% MeOH. Ten 100-millilitre fractions were collectedand fractions with similar TLC profiles were pooled.

Fractionation by flash-column chromatographyFlash-column chromatography (FCC) of the chloroform

extract and of selected fractions from the VLC fractionswas performed on silica gel (EM grade 60, mesh size 230–400, 60 A) (1 A = 0.1 nm). The column was packed with aslurry of the silica gel – solvent system (2:1, column bedlength, 16 cm); target fractions were dissolved in the mini-mum amount of the solvent system and applied with a pip-ette to the top of the silica bed. The column wassubsequently eluted with the solvent system using a flow ofnitrogen adjusted to 5.08 cm/min (2 inches/min) and 5-millilitre fractions were collected for further analysis.

Fraction VLC-4 was purified by FCC using a 2 cm (i.d.)glass column and a mixture of Hex–EtOAc (2:3) as the sol-vent system. Fraction VLC-5 was purified by FCC by usinga 2 cm (i.d.) glass column and a mixture of Hex–EtOAc(3:7). Fractions VLC-6 and VLC-7 were purified by FCCusing a 2.5 cm (i.d.) glass column and a mixture ofCH2Cl2–MeOH (9.6:0.4).

Purification by preparative TLCPreparative TLC (PTLC) of target fractions was per-

formed on silica gel plates (EM, 60 F254, 20 � 20 cm,0.25 mm thickness). The fraction was dissolved in the mini-mum amount of the selected developing solvent system andapplied to the plate. The plate was developed, air dried, andobserved under UV light. The developed bands were scrapedfrom the plate with the aid of a scraping knife, and com-pounds were recovered from the silica by repeated washes

with CH2Cl2–MeOH (95:5). Two solvent systems were usedfor various preparative thin-layer chromatography purifica-tions: (i) Hex–EtOAc (2:1), developed twice; and(ii) CH2Cl2–MeOH (95:5), developed once.

Purification of xanthinosin by reverse-phase columnchromatography

Reverse-phase chromatography (RP-CC) was performedby using a Waters Sep-Pak Vac 20cc (5 g) C18 cartridge.The fraction containing xanthinosin (approximately 60 mg)was dissolved in 6 mL of H2O–MeCN (6:3), and the car-tridge was equilibrated with 50 mL of the same solvent mix-ture. Four 50-millilitre fractions were then eluted with agradient of H2O–MeCN (6:3), H2O–MeCN (6:4), 100%MeCN, and 100% MeOH.

Purification of xanthatin by semipreparative HPLCSemipreparative HPLC (SP-HPLC) was carried out by us-

ing a Waters Nova-Pak HR C18 (300 � 7.8 mm i.d., 6 mm,60 A) column, and a Waters 996 PDA detector. The injec-tion volume was 250 mL. Fractions were collected manuallyusing gradient elution of 80% H2O : 20% MeCN to 40%H2O : 60% MeCN over 55 min at a flow rate of 5 mL/min.

Spectroscopic studiesOptical rotations were determined using a Rudolph Instru-

ment, Inc. model DigiPol DP781 136–9805 polarimeter. Themelting point of xanthatin was determined in a Fisher melt-ing point apparatus. Infrared spectra were recorded on aBio-Rad Digilab Division FTS-48 spectrometer. Spectrawere measured by the diffuse reflectance method on samplesdispersed in KBr. 1H NMR spectra were obtained with aBruker AMX 300 or AMX 500 spectrometer. Chemicalshifts (d) are reported in parts per million (ppm) relative totetramethylsilane. The d values were referenced to CHCl3

Table 2. Quantity and percentage of fractions extracted from 15 plant species of the Asteraceae family collected in Saskatchewan.

Plant speciesCrude MeOHextract, gb

Fraction, g (%)a

Hexaneextractc

CHCl3extractc

MeOHextractc

Aqueousextract Recoveryd

Boiling waterextracte

Artemisia campestris 13.9 2.0 (14.4) 0.6 (4.3) 9.7 (69.8) 0.9 (6.5) 13.2 (95.0) 17.4 (20.2)Artemisia ludoviciana 18.1 2.0 (11.0) 0.8 (4.4) 13.8 (76.2) 0.7 (3.9) 17.3 (95.6) 12.6 (15.4)Artemisia vulgaris 10.2 2.6 (25.5) 0.2 (2.0) 5.8 (56.9) 0.3 (2.9) 8.9 (87.3) 7.9 (8.8)Aster brachyactis 18.7 5.3 (28.3) 0.1 (0.5) 12.3 (65.8) 0.6 (3.2) 18.3 (97.9) 8.2 (10.1)Aster ciliolatus 11.9 1.0 (8.4) 0.3 (2.5) 9.1 (76.5) 0.9 (7.6) 11.3 (95.0) 26.5 (30.1)Carduus nutans 15.1 2.1 (13.9) 0.2 (1.3) 9.9 (65.6) 1.5 (9.9) 13.7 (90.7) 17.0 (20.0)Cirsium arvense 15.9 2.3 (14.5) 0.3 (1.9) 8.8 (55.3) 3.4 (21.4) 14.8 (93.1) 16.5 (19.6)Echinacea angustifolia 18.0 2.8 (15.6) 0.1 (0.6) 11.2 (62.2) 1.3 (7.2) 15.4 (85.6) 24.2 (29.5)Helianthus petiolaris 16.2 4.8 (29.6) 0.6 (3.7) 8.9 (54.9) 1.1 (6.8) 15.4 (95.1) 19.2 (22.9)Matricaria matricarioides 15.7 3.4 (21.7) 0.2 (1.3) 9.2 (58.6) 2.3 (14.6) 15.1 (96.2) 26.5 (31.4)Solidago mollis 18.6 4.3 (23.1) 0.9 (4.8) 9.3 (50.0) 0.9 (4.8) 15.4 (82.8) 19.3 (23.7)Sonchus arvensis 22.5 3.3 (14.7) 0.3 (1.3) 14.5 (64.4) 2.6 (11.6) 20.7 (92.0) 6.1 (7.9)Tanacetum vulgare 22.8 3.2 (14.0) 0.7 (3.1) 17.1 (75.0) 1.6 (7.0) 22.6 (99.1) 12.4 (16.1)Taraxacum officinale 24.2 4.3 (17.8) 0.3 (1.2) 10.1 (41.7) 7.8 (32.2) 22.5 (93.0) 37.8 (49.9)Xanthium strumarium 9.4 3.8 (40.4) 0.5 (5.3) 4.3 (45.7) 0.4 (4.3) 9.0 (95.7) 11.0 (12.1)

aThe percentages of fractions extracted are indicated in parentheses.bThe amount of extract obtained from 100 g of dry plant material.cPercentage was calculated based on the crude MeOH extract.dRecovery of extract from the crude MeOH extract.ePercentage was expressed relative to the mass (g) of plant residue that remained upon MeOH extraction.

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(7.27 ppm). First-order behaviour was assumed in the analy-sis of 1H NMR spectra and multiplicities were indicated byone or more of the following: s, singlet; d, doublet; t, triplet;q, quartet; m, multiplet; and br, broad. Spin coupling con-stants (J values) are reported to the nearest 0.5 Hz.13C NMR data were collected on a Bruker AMX 300 spec-trometer at 75.5 MHz or a Bruker AMX 500 spectrometer at125.8 MHz. The 13C chemical shifts (d values) were refer-enced to CDCl3 (77.2 ppm). The multiplicity of 13C signalsrefers to the number of attached protons (i.e., s = C, d = CH,

t = CH2, q = CH3) and were determined based upon HMQCexperiments (xanthatin and xanthinosin), chemical shift, andconsistency within a series of similar structures. Electronimpact mass spectroscopy was performed in a VG 70SEmass spectrometer (solid probe; source temperature, 200 8C;electron energy, 70 eV).

Results

The yield of the 75 extracts generated from the selected

Table 3. Cytotoxicity of 75 extracts from selected plant species of the family As-teraceae, expressed as percentage mortality of WiDr human colon cancer cells.

No. Plant (extract) % Mortality No. Plant (extract) % Mortality

1 074 (Hex) 100 39 033 (BW) 55.62 070 (Hex) 100 40 017 (CHCl3) 53.33 047 (Hex) 99.1 41 082 (MeOH) 52.74 070 (CHCl3) 98.9 42 082 (BW) 51.25 047 (CHCl3) 95.7 43 021 (BW) 49.86 037 (Hex) 95.6 44 009 (BW) 49.57 082 (Hex) 95.5 45 074 (MeOH) 47.88 036 (Hex) 94.7 46 059 (MeOH) 44.69 033 (Hex) 94.4 47 047 (W) 40.6

10 065 (CHCl3) 93.5 48 070 (MeOH) 36.911 082 (CHCl3) 93.3 49 033 (W) 34.712 021 (CHCl3) 93.1 50 017 (BW) 30.113 009 (Hex) 92.8 51 035 (BW) 30.014 035 (Hex) 92.1 52 043 (Hex) 29.515 035 (CHCl3) 90.4 53 072 (MeOH) 27.316 074 (CHCl3) 88.7 54 082 (W) 25.817 021 (Hex) 87.8 55 043 (W) 19.218 036 (CHCl3) 87.2 56 043 (BW) 15.819 033 (MeOH) 85.8 57 035 (W) 14.920 033 (CHCl3) 84.9 58 043 (MeOH) 4.821 065 (MeOH) 82.8 59 072 (BW) 4.222 043 (CHCl3) 82.1 60 065 (W) 023 059 (CHCl3) 79.3 61 065 (BW) 024 059 (BW) 78.0 62 074 (BW) 025 059 (Hex) 77.2 63 070 (BW) 026 037 (CHCl3) 75.2 64 074 (W) 027 035 (MeOH) 74.5 65 017 (Hex) 028 047 (MeOH) 74.5 66 070 (W) 029 009 (CHCl3) 74.3 67 072 (W) 030 072 (Hex) 70.0 68 036 (BW) 031 009 (MeOH) 65.8 69 036 (W) 032 072 (CHCl3) 65.4 70 037 (BW) 033 021 (MeOH) 64.9 71 037 (W) 034 065 (Hex) 64.4 72 036 (MeOH) 035 009 (W) 63.6 73 017 (W) 036 021 (W) 63.6 74 037 (MeOH) 037 059 (W) 58.9 75 017 (MeOH) 038 047 (BW) 58.7

Note: 009, Artemisia campestris L.; 021, Artemisia ludoviciana Nutt.; 043, Artemisia vul-garis L.; 072, Aster brachyactis Blake; 059, Aster ciliolatus Lindl.; 035, Carduus nutans L.;033, Cirsium arvense (L.) Scop.; 074, Echinacea angustifolia DC.; 036, Helianthus petiolarisNutt.; 082, Matricaria matricarioides (Less.) Port.; 037, Solidago mollis Bartl.; 065, Sonchusarvensis L.; 070, Tanacetum vulgare L.; 017, Taraxacum officinale Web.; 047, Xanthiumstrumarium L.; Hex, hexane extract; CHCl3, chloroform extract; MeOH, methanol extract;W, aqueous extract; BW, boiling water extract.

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parts of 15 plant species is presented in Table 2. The yieldof crude MeOH extracts ranged from 9.4 g (X. strumarium)to 24.2 g (T. officinale), and upon partitioning of these ex-tracts with organic solvents, CHCl3 extracts gave the lowestyields, followed by Hex and MeOH extracts. High recov-eries of water-soluble extracts were obtained when plant ma-terials that remained upon MeOH extraction were refluxedin boiling water.

All 75 extracts were tested in triplicate and at a concen-tration of 100 mg/mL for cytotoxicity to 3 human cancer

cell lines, and resulted in cell mortality rates that rangedfrom 0 to 100%. These results are provided in Tables 3, 4,and 5. In general, only the Hex and CHCl3 extracts causedsignificant mortality rates, whereas the more polar extracts(MeOH, aqueous, and boiling water) were less toxic or nottoxic at all. The Hex extracts of E. angustifolia, T. vulgare,X. strumarium, Solidago mollis, Matricaria matricarioides,Helianthus petiolaris, Artemisia campestris, and Carduusnutans; and the CHCl3 extracts of T. vulgare, X. strumarium,M. matricarioides, Artemisia ludoviciana, and C. nutans, re-

Table 4. Cytotoxicity of 75 extracts from selected plant species of the family As-teraceae, expressed as percentage mortality of MDA-MB-231 human breast cancercells.

No. Plant (extract) % Mortality No. Plant (extract) % Mortality

1 047 (Hex) 99.1 39 033 (Hex) 68.92 082 (Hex) 97.1 40 009 (BW) 67.23 065 (Hex) 96.6 41 072 (CHCl3) 61.94. 037 (Hex) 96.6 42 021 (MeOH) 60.35 047 (CHCl3) 95.8 43 059 (MeOH) 58.96 074 (Hex) 95.7 44 059 (W) 58.97 082 (CHCl3) 95.5 45 035 (Hex) 56.48 021 (CHCl3) 94.7 46 033 (W) 52.19 035 (CHCl3) 93.2 47 009 (MeOH) 50.8

10 070 (CHCl3) 92.5 48 072 (MeOH) 41.811 036 (Hex) 92.0 49 021 (W) 40.112 074 (CHCl3) 91.3 50 009 (W) 38.913 070 (Hex) 91.3 51 074 (W) 36.214 043 (Hex) 91.2 52 017 (CHCl3) 34.715 036 (CHCl3) 90.8 53 059 (BW) 31.016 009 (Hex) 89.7 54 070 (BW) 25.117 065 (CHCl3) 86.4 55 047 (BW) 19.118 033 (MeOH) 86.1 56 035 (BW) 17.719 072 (Hex) 85.6 57 074 (BW) 11.620 021 (Hex) 85.5 58 017 (BW) 10.721 072 (BW) 85.5 59 082 (MeOH) 4.322 043 (CHCl3) 85.0 60 033 (BW) 4.123 065 (MeOH) 84.3 61 036 (BW) 3.824 070 (W) 81.9 62 047 (W) 2.725 043 (BW) 80.2 63 082 (BW) 026 037 (CHCl3) 78.6 64 035 (MeOH) 027 021 (BW) 76.7 65 065 (W) 028 047 (MeOH) 75.5 66 035 (W) 029 009 (CHCl3) 74.4 67 082 (W) 030 070 (MeOH) 74.4 68 017 (Hex) 031 072 (W) 74.1 69 036 (MeOH) 032 074 (MeOH) 73.9 70 037 (BW) 033 043 (W) 73.7 71 036 (W) 034 043 (MeOH) 73.4 72 037 (W) 035 033 (CHCl3) 71.8 73 037 (MeOH) 036 059 (Hex) 70.2 74 017 (MeOH) 037 065 (BW) 70.1 75 017 (W) 038 059 (CHCl3) 68.9

Note: 009, Artemisia campestris L.; 021, Artemisia ludoviciana Nutt.; 043, Artemisia vul-garis L.; 072, Aster brachyactis Blake; 059, Aster ciliolatus Lindl.; 035, Carduus nutans L.;033, Cirsium arvense (L.) Scop.; 074, Echinacea angustifolia DC.; 036, Helianthus petiolarisNutt.; 082, Matricaria matricarioides (Less.) Port.; 037, Solidago mollis Bartl.; 065, Sonchusarvensis L.; 070, Tanacetum vulgare L.; 017, Taraxacum officinale Web.; 047, Xanthiumstrumarium L.; Hex, hexane extract; CHCl3, chloroform extract; MeOH, methanol extract;W, aqueous extract; BW, boiling water extract.

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sulted in mortality rates greater than 90% for all 3 cell linesused. In contrast, Hex extracts from Sonchus arvensis werehighly toxic (96.6%) only to the human breast cancer cellline, and Cirsium arvense (94%) was toxic to the colon andlung cell lines.

In addition to the above extracts, all plant extracts thatcaused greater than 80% cell mortality were further sub-jected to determination of IC50 values using multidose test-ing at 4 different concentrations. In total, 22, 25, and 36extracts were tested against WiDr, MDA-MB-231, andNCI-417 cell lines, respectively. A plant extract is regarded

as cytotoxic if its IC50 value is less than 10 mg/mL (Jacksonet al. 2000), and a pure compound is defined as cytotoxic ifits IC50 value is less than 4 mg/mL (Fiebig et al. 1985).

The CHCl3 extracts from X. strumarium and T. vulgareexhibited significant cytotoxic effects in all 3 cell linestested, and their IC50 values were, respectively, 3.3 and9.1 mg/mL for WiDr; 6.2 and 5.6 mg/mL for MDA-MB-231;and 0.1 and 2.4 mg/mL for NCI-417. While the previous ex-tracts demonstrated no discernable cell-type selectivity, theHex extract from E. angustifolia and the CHCl3 extractfrom C. nutans showed cytotoxic effects only on the lung

Table 5. Cytotoxicity of 75 extracts from selected plant species of the family As-teraceae, expressed as percentage mortality of NCI-417 human lung cancer cells.

No. Plant (extract) % Mortality No. Plant (extract) % Mortality

1 047 (Hex) 99.2 39 047 (W) 76.72 082 (Hex) 96.8 40 035 (BW) 76.43 074 (Hex) 96.1 41 074 (MeOH) 75.24 047 (CHCl3) 95.0 42 082 (W) 70.85 037 (Hex) 94.9 43 070 (W) 70.56 033 (Hex) 94.6 44 047 (MeOH) 66.97 021 (CHCl3) 94.3 45 072 (BW) 66.18 082 (CHCl3) 94.1 46 043 (MeOH) 64.89 035 (Hex) 93.5 47 021 (BW) 63.7

10 065 (Hex) 93.3 48 059 (W) 57.911 070 (CHCl3) 92.8 49 009 (BW) 56.412 037 (CHCl3) 92.7 50 043 (BW) 56.213 035 (CHCl3) 92.4 51 072 (W) 55.714 036 (CHCl3) 91.9 52 043 (W) 54.015 043 (Hex) 90.9 53 072 (CHCl3) 48.616 070 (Hex) 90.4 54 009 (MeOH) 45.717 036 (Hex) 90.3 55 035 (W) 44.818 009 (Hex) 88.9 56 009 (W) 41.919 033 (W) 88.7 57 021 (MeOH) 41.920 059 (BW) 88.6 58 059 (MeOH) 41.721 043 (CHCl3) 88.4 59 017 (BW) 41.522 021 (Hex) 88.0 60 065 (BW) 36.423 059 (Hex) 87.6 61 021 (W) 36.024 074 (CHCl3) 85.7 62 074 (W) 11.125 017 (CHCl3) 85.6 63 074 (BW) 8.826 033 (CHCl3) 85.6 64 017 (Hex) 5.727 082 (MeOH) 85.2 65 070 (BW) 4.728 035 (MeOH) 85.2 66 036 (W) 3.029 082 (BW) 84.9 67 072 (MeOH) 030 059 (CHCl3) 84.2 68 037 (BW) 031 047 (BW) 83.7 69 036 (BW) 032 072 (Hex) 83.6 70 065 (W) 033 009 (CHCl3) 82.9 71 036 (MeOH) 034 033 (BW) 82.5 72 037 (W) 035 065 (MeOH) 82.0 73 037 (MeOH) 036 033 (MeOH) 80.5 74 017 (MeOH) 037 065 (CHCl3) 79.5 75 017 (W) 038 070 (MeOH) 78.5

Note: 009, Artemisia campestris L.; 021, Artemisia ludoviciana Nutt.; 043, Artemisia vul-garis L.; 072, Aster brachyactis Blake; 059, Aster ciliolatus Lindl.; 035, Carduus nutans L.;033, Cirsium arvense (L.) Scop.; 074, Echinacea angustifolia DC.; 036, Helianthus petiolarisNutt.; 082, Matricaria matricarioides (Less.) Port.; 037, Solidago mollis Bartl.; 065, Sonchusarvensis L.; 070, Tanacetum vulgare L.; 017, Taraxacum officinale Web.; 047, Xanthiumstrumarium L.; Hex, hexane extract; CHCl3, chloroform extract; MeOH, methanol extract;W, aqueous extract; BW, boiling water extract.

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cancer cell line, with IC50 values of 4 and 9.3 mg/mL, re-spectively. The IC50 values of all remaining extracts listedin Tables 6, 7, and 8 were above 10 mg/mL, which is con-sidered to be a low cytotoxic effect. The bioassays for Hexand CHCl3 extracts of T. vulgare, X. strumarium, and E. an-gustifolia were repeated 3 times more and the results werereproducible. HPLC analysis (220 nm) of the CHCl3 extractof the burs of X. strumarium showed 4 major peaks with re-tention times between 15 and 19 min. Bioassay-guided frac-tionation of the CHCl3 extract of the burs of X. strumariumallowed the purification of sesquiterpene lactones xanthatin(16.2 min) and xanthinosin (18.8 min). Spectroscopic data(from 1H NMR and 13C NMR) and electron impact – massspectrometry of these compounds were in accordance withthose reported in the literature for xanthatin and xanthinosin(Ginesta-Peris et al. 1994; Marco et al. 1993; Bohlmann etal. 1978; McMillan et al. 1975b). HPLC chromatograms of

xanthatin and xanthinosin are shown in Figs. 2 and 3. Onlyxanthatin showed a strong absorbance at 275 nm. Becausethe retention times and Rf values for these compounds weresimilar, their purification was accomplished through a seriesof successive chromatographic techniques described in Ma-terials and methods. Techniques employed included VLC(Hex–EtOAc), FCC (Hex–EtOAc, 3:7), preparative thin-layer chromatography (Hex–EtOAc, 2:1, 2 times), and semi-preparative reverse-phase HPLC (H2O–MeCN gradient).Spectroscopic data of xanthatin and xanthinosin are pre-sented in Appendix A (Fig. A1 and Table A1) and AppendixB (Fig. B1 and Table B1).

We also determined the IC50 values for xanthatin and xan-thinosin at 8 different concentrations (100, 50, 25, 12.5,6.25, 3.1, 1.5, and 0.8 mg/mL) for WiDr (colon) and MDA-MB-231 (breast) human tumor cell lines in vitro. Xanthatinexhibited IC50 values of 6.15 ± 0.07 mg/mL and 13.9 ± 1.13,

Fig. 2 HPLC chromatogram of xanthatin (210 nm) with a UV spectrum of the corresponding peak (retention time, 16.2 min). An HPLCchromatogram at 275 nm is also shown (inset).

Fig. 3 HPLC chromatogram of xanthinosin (210 nm) with a UV spectrum of the corresponding peak (retention time, 18.8 min).

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respectively, while those for xanthinosin were 2.65 ± 0.07and 4.8 ± 0.56 mg/mL, respectively. These results are sum-marized in Table 9.

Discussion

Some members of the family Asteraceae have been pri-marily classified as weeds in Saskatchewan, but they are at-tractive for study because they grow abundantly inSaskatchewan’s climate and are reputed to have anticancerproperties according to traditional Chinese medicine.

Of the 15 species addressed in this study, 8 have been re-

ported to be used against cancer (Hartwell 1968), and 9 havebeen previously assayed for anticancer properties by theNCI Database, but were found to be ineffective. Unfortu-nately, the plant parts of the 9 species tested by NCI werenot specified, thus it has been assumed that whole plantswere used in the studies. No record in the NCI studies oncytotoxicity for Artemisia campestris (plains wormwood),Aster brachyactis (rayless aster), Aster ciliolatus (Lindley’saster), Carduus nutans (nodding thistle), Matricaria matri-carioides (pineapple weed), or Solidago mollis (velvetygoldenrod) was found. It is noteworthy that at least one spe-cies from each genus selected for this study has been used in

Table 6. IC50 values for the 5 extracts that were most toxic to the WiDrhuman colon cancer cells. Values represent the means of 3 replications.

No. Species, extracta IC50

1 Xanthium strumarium (cocklebur; 047), chloroform 3.32 Tanacetum vulgare (tansy; 070), chloroform 9.13 Xanthium strumarium (cocklebur; 047), hexane 12.24 Sonchus arvensis (perennial sow thistle; 065), hexane 15.15 Tanacetum vulgare (tansy; 070), hexane 15.2

aCommon names followed by the code assigned to each species are given inparentheses.

Table 7. IC50 values for the 5 extracts that were most toxic to the MDA-MB-231 human breast cancer cells. Values represent the means of 3 re-plications.

No. Species, extracta IC50

1 Tanacetum vulgare (tansy; 070), chloroform 5.62 Xanthium strumarium (cocklebur; 047), chloroform 6.23 Aster brachyactis (rayless aster; 072), boiling water 11.44 Artemisia campestris (plains wormwood; 009), hexane 11.45 Artemisia ludoviciana (prairie sage; 021), chloroform 18.9

aCommon names followed by the code assigned to each species are given inparentheses.

Table 8. IC50 values of the 5 extracts that were most toxic to the NCI-417 human lungcancer cells. Values represent the means of 3 replications.

No. Species, extracta IC50

1 Xanthium strumarium (cocklebur; 047), chloroform 0.12 Tanacetum vulgare (tansy; 070), chloroform 2.43 Echinacea angustifolia (narrow-leaved purple coneflower; 074), hexane 4.04 Carduus nutans (nodding thistle; 035), chloroform 9.35 Artemisia ludoviciana (prairie sage; 021), chloroform 11.8

aCommon names followed by the code assigned to each species are given in parentheses.

Table 9. IC50 values for xanthatin, xanthinosin, and the chloroform extract of Xanthiumstrumarium L. burs. Values represent the means ± SD of 3 replications.

IC50, mg/mL ± SD

WiDr cells MDA-MB-231 cells

047-CHCl3 chloroform extract 4.4±1.56 10.3±5.80Xanthatin 6.15±0.07 13.9±1.13Xanthinosin 2.65±0.07 4.8±0.56

Note: WiDr cells, human colon cancer cells; MDA-MB-231 cells, human breast cancer cells.

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the treatment of cancer (Hartwell 1968; Tang and Eisen-brand 1992; Huang 1999; Graham et al. 2000).

Partitioning of the crude MeOH extracts with solvents ofincreasing polarities was shown to be a good screeningmethod for cytotoxic compounds in plants. When the plantextracts were tested on the human cancer cell lines, NCI-417 (lung) and WiDr (colon) were the most susceptible,whereas MDA-MB-231 (breast) was the least susceptible.These cancer cell lines were selected for this study becausethey are among the most prevalent human cancers and alsothe most difficult to treat.

While several plant extracts tested showed a high level ofcytotoxic activity at a single concentration of 100 mg/mL,dose–response experiments showed that the CHCl3 extractsof X. strumarium, T. vulgare, and C. nutans, and the Hexextract of E. angustifolia, were the most active.

The presence in plants of biologically active sesquiterpenelactones containing the a-methylene-g-lactone ring is welldocumented. When this ring is absent, (e.g., in santonin) noinhibitory effect is found (Hehner et al. 1998).

Roussakis et al. (1994) demonstrated that xanthatin iso-lated from the bioassay-directed fractionation of the activeCH2Cl2 soluble residue from the leaves of X. strumarium ex-hibited IC50 values of 0.018 and 0.009 mg/mL when testedon 2 murine lymphocytic leukemia cell lines, P-388 and L-1210, and against the human bronchial epidermoid carci-noma NSCLC-N6 (non-small cell lung cancer). In a similarfashion 8-epi-xanthatin and 8-epi-xanthatin epoxide, isolatedfrom the leaves of X. strumarium, demonstrated a significantinhibition of the proliferation of cultured A549 (human non-small cell lung cancer), SK-OV-3 (ovarian cancer), SK-MEL-2 (melanoma), XF498 (central nervous system cancer),and HCT-15 (colon cancer) cells in vitro (Kim et al. 2003).Ross et al. (1999) also demonstrated that parthenolide, ex-tracted from the leaves of T. vulgare and related species, in-hibited cell growth in an irreversible fashion when tested ontumor cell lines at concentrations above 5.0 mmol/L.

Xanthinosin has been reported in the leaves of X. struma-rium (McMillan et al. 1975a; McMillan et al. 1975b) and inthe aerial parts of X. indicum Koen. Ex. Roxb. (McMillan etal. 1975b), and Inula helenium (Asteraceae) (Bohlmann etal. 1978). Xanthinosin has also been reported as the onlysesquiterpene lactone in leaves of indigenous Xanthiumplants of India and northern Europe (McMillan et al. 1975a;McMillan et al. 1975b). Xanthatin and xanthinosin havebeen reported to be present in the fruit (burs) of X. macro-carpum (Lavault et al. 2005), but there is no evidence thatthese compounds are present in the burs of X. strumariumIn this study, the high content of xanthinosin in the burs ofX. strumarium, as well as the cytotoxic activity observed forxanthinosin, xanthatin and related fractions on the selectedhuman cancer cell lines suggest that further examination ofthe cytotoxic potential of these compounds should be pur-sued. In addition, it is worthwhile to examine the anticancerproperties of sesquiterpene lactones with xanthanolide-typeskeleton, which are simple plant metabolites.

AcknowledgementsThe authors are grateful to Saskatchewan Department of

Agriculture and Food, Agriculture Development Fund, for

funding this work. Thanks to Dr. Svein Carlsen, ConniePercy and the Saskatoon Cancer Centre for making their tis-sue culture facility available for cytotoxic activity testing.I.R.-E. thanks the Consejo Nacional de Ciencia y Tecnolo-gıa (CONACyT) from Mexico for a graduate student schol-arship. Thanks are also extended to Peggy Ann Ryan,Gerald Ivanochko and Donna Dunlop for assistance withcollection and verification of the plant material.

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Appendix A. XanthatinValues found for xanthatin (C15H18O3) were the follow-

ing: white powder; mp = 113–117 8C; [a)20D = –198 (c =1.0, CHCl3). �

MeCNmax nm: 277.1, 206.6

IR �max (KBr) cm–1: 1727 (>C = O), 1765 (methylene lac-tone), 1663, 1609, 1589. MS m/z (rel. int.): [M+] 246.125(37%) [C15H18O3]; [M+–Me] 231 (21); [M+–H2O] 228 (4.5);[M+–C3H7

+] 203 (28); [203–CO] 175 (51); [C9H13+] 121

(57); 109 (100).

Appendix B. XanthinosinValues found for xanthinosin (C15H20O3) were the follow-

ing:Colorless oil; [a]30D = –428 (c = 0.45, CHCl3). �MeCN

max

nm: 206.6. IR �max (KBr) cm–1: 1720 (>C=O), 1778, 1754(methylene lactone). MS m/z (rel. int.): [M+] 248.141 (62%)[C15H20O3]; [M+–H2O] 230 (37); [230–Me] 215 (12); [M+–C3H7

+] 205 (14); [M+–CH3COCH3] 190 (100); [M+–C3H7CO+] 177 (24); [123–C = O] 95 (51).

Fig. B1. Chemical structure of xanthinosin.

Table B1. 1H NMR and 13C NMR data for xanthinosin.

Position dC (ppm) dH (ppm)

1 146.7 —2 34.9 2.27, 2H, m3 43.2 2.5, 2H, m4 208.4 —5 122.1 5.51, 1H, dd, J = 8.5, 2 Hz6 26.4 2.5, 1H, m

1.99, 1H, bt (ddd), J = 14 Hz7 48.6 2.5, 1H, m8.b 82.5 4.22, 1H, ddd, J = 12, 10, 2.5 Hz9 37.5 1.75, 1H, ddd, J = 12, 12, 4 Hz

2.27, 1H, m10 34.5 2.5, 1H, m11 140.2 —12 170.5 —13 118.9 5.42, 1H, d, J = 3 Hz

6.13, 1H, d, J = 3 Hz14 18.9 1.12, 3H, d, J = 7.5 Hz15 30.4 2.14, 3H, s

Table A1. 1H NMR and 13C NMR data for xanthatin.

Position dC (ppm) dH (ppm)

1 145.2 —2 148.9 7.09, 1H, d, J = 16 Hz3 125.1 6.23, 1H, d, J = 16 Hz4 198.9 —5 138.5 6.31, 1H, dd, J = 9, 3.5 Hz6 27.6 2.82, 1H, dddd J = 16.5, 9,

2.5 Hz2.27, 1H, m

7 47.6 2.58, 1H, m8.b 81.9 4.32, 1H, dtd, J = 12, 10, 2.5 Hz9 37.1 1.87, 1H, ddd, J = 13, 12, 4 Hz

2.41, 1H, dt, J = 13, 4, 2.510 29.5 2.29, 1H, m11 139.6 —12 170.1 —13 119.4 5.51, 1H, d, J = 3 Hz

6.23, 1H, d, J = 3 Hz14 19.3 1.19, 3H, d, J = 7.5 Hz15 28.3 2.33, 3H, s

Fig. A1. Chemical structure of xanthatin.

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