Biochemical Systematics and Ecology 38 (2010) 232235

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Biochemical Systematics and Ecology

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Terpenoids and phenolics from Inula ensifolia

Anna Stojakowska a, *, Janusz Malarz a, Szymon Zubek b, Katarzyna Turnau c, Wanda Kisiel a
a b c

w, Poland Department of Phytochemistry, Institute of Pharmacology, Polish Academy of Sciences, 12 Sm etna Street, 31-343 Krako w, Poland Mycology Unit, Institute of Botany, Jagiellonian University, 46 Lubicz Street, 31-512 Krako w, Poland Institute of Environmental Sciences, Faculty of Biology and Earth Sciences, Jagiellonian University, 7 Gronostajowa Street, 30-387 Krako

a r t i c l e i n f o
Article history: Received 18 May 2009 Accepted 12 December 2009 Keywords: Inula ensifolia Asteraceae Thymol derivatives Norisoprenoids Flavonoids Dicaffeoylquinic acids

1. Subject and source Inula ensifolia L. (Asteraceae, Inuleae) is a herbaceous perennial, about 45 cm high, with grey-green, lance-shaped leaves and anthodia composed of bright-yellow, narrow ray orets surrounding darker disc orets. Whole plants of I. ensifolia were w Upland, Poland; coordinates: 50 330 9400 N, 20170 9700 collected from xerothermic grasslands nearby Kalina-Lisiniec (Miecho E), in June 2008, and identied by Teresa Anielska M.Sc. from the Institute of Environmental Sciences, Jagiellonian University w. A voucher specimen (08/08) has been deposited at the Garden of Medicinal Plants, Institute of Pharmacology, in Krako w. Polish Academy of Sciences, Krako

2. Previous work The species lacks detailed phytochemical investigation. Literature data are sparse. Wollenweber et al. (1997) examined ve Inula species (Inula britannica L., Inula germanica L., Inula salicina L., Inula helenium L., I. ensifolia L.) with respect to exudate avonoid production. However, no exudate avonoids were found in I. ensifolia aerial parts. Thin-layer chromatography of methanolic extracts from disc and ray owers of I. ensifolia revealed the presence of phenolic acids (caffeic and chlorogenic) ter and Do sa, 2002). Secondary metabolites of plants from the genus Inula, and avonoids (apigenin and hyperin) (Pe including monoterpenoids, sesquiterpenoids and avonoids, have been reviewed by Konovalov and Khubieva (1997) and recently by Zhao et al. (2006). Although the plant has no any documented medicinal use, an antiproliferative activity of thy et al., 2007). methanolic extract from I. ensifolia against human cancer cell lines in vitro has been reported (Re

* Corresponding author. Tel.: 48 12 6623217; fax: 48 12 6374500. E-mail address: stoja@if-pan.krakow.pl (A. Stojakowska). 0305-1978/$ see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2009.12.011

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3. Present study The present report deals with the isolation of ve thymol derivatives (15) from the roots of I. ensifolia, as well as atocopherol (6), two norisoprenoids (7, 8), three quercetin derivatives (911) and four caffeoylquinic acids (1215) from its aerial parts (Fig. 1). The air-dried roots of I. ensifolia (10.8 g) were powdered and exhaustively extracted with CHCl3 at room temperature with shaking. The obtained extract was concentrated under reduced pressure to give 0.162 g of an oily residue. This residue, after fractionation by preparative TLC (Merck, Art. 1.05553; hexane-EtOAc, 9:1) followed by semipreparative HPLC on a Delta-Pak C-18 column (particle size 15 mm, 25 100 mm) coupled to a dual wavelength UV/vis detector operating at 205 and 270 nm, using MeOHH2O mixture (13:7) at a ow rate of 6 ml min1, yielded: 1 (21.9 mg), 2 (2.1 mg), a mixture (2.8 mg) of 3 and 4 (by 1 H NMR), and a fraction (1.5 mg) containing 5 as the main component (by 1H NMR and ESIMS). The aerial parts (59.6 g) of I. ensifolia were powdered and extracted successively with CHCl3, followed by pure MeOH at room temperature with shaking and the solvents were evaporated under reduced pressure which furnished crude CHCl3 (2.98 g) and MeOH (5.50 g) extracts. The crude CHCl3 extract was fractionated by column chromatography on a silica gel (Merck, Art 7754) using gradients of EtOAc in hexane as a solvent system. Elution with hexane-EtOAc 9:1 followed by preparative TLC in the solvent system of the same composition led to the isolation of 6 (3.3 mg). From the more polar fractions (hexane-EtOAc, 1:1) a mixture (2 mg) of 7 and 8 (by 1H NMR) was isolated. The crude MeOH extract was subjected to column chromatography on Sephadex LH-20 (Pharmacia Biotech) eluted with MeOH/H2O. The eluted fractions were monitored by analytical RP-HPLC using an Agilent 1200 Series HPLC system (Agilent Technologies, USA) equipped with a Rheodyne manual sample injector, quaternary pump, degasser, column oven and a diode array detector. Chromatographic separations were carried out at 25  C, on a Zorbax Eclipse XDB-C18 column (4.6 150 mm, 5 mm particle size; Agilent Technologies, USA) with a mobile phase consisting of H2O/HCOOH/CH3COOH 99/0.9/0.1 (solvent A) and MeCN/MeOH/HCOOH/CH3COOH 89/10/0.9/0.1 (solvent B), at a ow rate of 1 ml/min, using 10 ml injections. The gradient elution conditions described by Spitaler et al. (2006) were used. This procedure yielded: 12 (100 mg), 13 (300 mg), a mixture (160 mg) of 9 and 10 (by 1H NMR and co-HPLC with authentic samples), a mixture (304 mg) of 13, 14 and 15 (by 1H NMR), and 11 (60 mg), in that order. The mixtures were not further separated, as the 1H NMR signals could be readily assigned to the respective compounds. Optical rotations were determined with a PolAAr31 automatic polarimeter (Optical Activity LTD). 1H and 13C NMR spectra were measured in CDCl3 or in CD3OD (phenolic compounds) on a Varian Mercury-VX 300 spectrophotometer operating at 300.08 MHz (1H) and 75.46 MHz (13C). COSY experiments were carried out using the same instrument. Chemical shifts (d in ppm) were referenced to TMS. Mass spectra (ESIMS) were recorded on a Bruker Esquire 3000 mass spectrometer. The isolated compounds were identied based on their physical ([a]D, whenever possible) and spectroscopic data, including NMR and MS, and their comparison with literature data for: 7-isobutyroyloxythymol methyl ether (1, Shtacher and Kasman, 1971; Anthonsen and Kjsen, 1971), 10-isobutyroyloxy-8,9-epoxythymol isobutyrate (2), 10-(2-methylbutyroyloxy)-8,9-epoxythymol isobutyrate (3), 10-isovaleroyloxy-8,9-epoxythymol isobutyrate (4) (Bohlmann et al., 1969; Zee et al., 1998), 7,10diisobutyroyloxy-8,9-epoxythymol isobutyrate (5, Bohlmann and Zdero, 1977), a-tocopherol (6, Baker and Myers, 1991; Malik vez et al., 1997; Xian et al., 2006), loliolide (8, et al., 1997), megastigmane aglycone - 3b-hydroxy-5b,6b-epoxy-b-ionone (7, Cha Kisiel, 1992; El Hattab et al., 2008), quercetin-3-O-b-glucopyranoside (isoquercitrin, 9, Shoeb et al., 2007), quercetin-3-O-bgalactopyranoside (hyperin, 10, An et al., 2008), quercetin-3-O-b-(600 -caffeoylgalactopyranoside) (11, Shigematsu et al., 1982), chlorogenic acid (12, Pauli et al., 1999), 1,5-, 3,4- and 3,5-dicaffeoylquinic acids (1315, Merfort, 1992; Basnet et al., 1996; Islam et al., 2002). 4. Chemotaxonomic signicance A total of 15 terpenoid and phenolic compounds have been isolated from the roots and aerial parts of I. ensifolia. Except for compounds 10 and 12, the other isolated constituents are reported for the rst time from I. ensifolia. Quinic acid derivatives (1215) appeared to be major secondary metabolites (over 1% yield) of the aerial parts. The paraphyletic genus Inula L., heterogenous with respect to morphology and chromosome numbers, comprises ca. 100 species native to Eurasia and Africa (Bremer, 1994). Within the genus, a group of resiniferous taxa, including I. helenium a well known medicinal plant, has been distinguished by Anderberg (1991). Roots of the resiniferous species usually contain essential oils with the eudesmane-type sesquiterpene lactones alantolactone and/or isoalantolactone as major constituents. s et al., 1998; Francisco-Ortega et al., Separate position of these species has been conrmed by ITS sequence analysis (Eldena 2001). Within Inula species lacking resin canals, a classication based on morphological data has been proposed (Anderberg, 1991). According to this proposal, I. ensifolia has been included in the I. salicina group together with I. germanica, Inula hirta, Inula helvetica and Inula viscidula. Of these, only I. salicina and I. germanica have been phytochemically investigated (Anthonsen and Kjsen, 1971; Konovalova et al., 1974; Bohlmann et al., 1978, 1985). Both plant species contained sesquiterpene lactones in their aerial parts (I. germanica germacranolides, I. salicina eudesmanolides). The compounds were absent from the plant material under study. This absence of sesquiterpene lactones situates I. ensifolia closer to some representatives of the postulated Inula decurrens group, i.e. Inula bifrons and Inula conyza. Moreover, I. germanica is rich in exudate avonoids, derivatives of luteolin, scutellarein and quercetagenin, whereas only one derivative of luteolin was found in I. salicina exudate and no avonoids in an exudate of I. ensifolia were found (Wollenweber et al., 1996). It seems that, neither exudate avonoids nor esteried thymol derivatives are helpful chemotaxonomical markers within Inula. The avonoids due

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O O OMe

7 6 5 4

CH2R1
1 2

O O OR2

3 8

10

1
2 3 4 5

R1 H H H OiBu

R2 iBu MeBu iVal iBu

CH3 H3C HO CH3 O

CH3

CH3

CH3

CH3 CH3

11 2 3 1 4 6 5

12 7

O O
13 8 9

11

HO
10

3 2

4 1

5 6

O
8 7 10

HO

7
OH OH HO
6

OR4
2 5 3 4

O
OR OH O

HOOC

OR3

OR1

OR2

9 10 11

R - Glc R - Gal R - (6''-caffeoyl)Gal

12 13 14 15

R1 H Caff H H

R2 H H Caff Caff

R3 H H Caff H

R4 Caff Caff H Caff

Fig. 1. Structures of thymol derivatives (15), a-tocopherol (6), norisoprenoids (7, 8), avonoids (911) and quinic acid derivatives (1215), isolated from Inula ensifolia (iBu, isobutyroyl; MeBu, 2-methylbutyroyl; iVal, isovaleroyl; Caff, caffeoyl; Glc, b-glucopyranosyl; Gal, b-galactopyranosyl).

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to limited data available, and the thymol derivatives due to their common occurrence in members of Inuleae and in plants of Helenieae and Eupatorieae tribes. Further phytochemical studies of Inula sp. are needed to support classication efforts, which nowadays are based mainly on morphological traits. References
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