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

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 ensifoliaL. (Asteraceae, Inuleae) is a herbaceous perennial, about 45 cm high, with grey-green, lance-shaped leaves

and anthodia composed of bright-yellow, narrow ray florets surrounding darker disc florets. Whole plants ofI. ensifoliawerecollected from xerothermic grasslands nearby Kalina-Lisiniec (Miechow Upland, Poland; coordinates: 503309400 N,201709700

E), in June 2008, and identified by Teresa Anielska M.Sc. from the Institute of Environmental Sciences, Jagiellonian University

in Krakow. A voucher specimen (08/08) has been deposited at the Garden of Medicinal Plants, Institute of Pharmacology,

Polish Academy of Sciences, Krakow.

2. Previous work

The species lacks detailed phytochemical investigation. Literature data are sparse. Wollenweber et al. (1997) examined fiveInulaspecies (Inula britannicaL., Inula germanicaL., Inula salicinaL., Inula heleniumL., I. ensifoliaL.) with respect to exudate

flavonoid production. However, no exudate flavonoids were found in I. ensifoliaaerial parts. Thin-layer chromatography ofmethanolic extracts from disc and ray flowers ofI. ensifoliarevealed the presence of phenolic acids (caffeic and chlorogenic)

and flavonoids (apigenin and hyperin) (Peter and Dosa, 2002). Secondary metabolites of plants from the genus Inula,including monoterpenoids, sesquiterpenoids and flavonoids, have been reviewed by Konovalov and Khubieva (1997) and

recently byZhao et al. (2006). Although the plant has no any documented medicinal use, an antiproliferative activity of

methanolic extract fromI. ensifolia against human cancer cell lines in vitro has been reported (Rethy et al., 2007).

* Corresponding author. Tel.: 48 12 6623217; fax: 48 12 6374500.

E-mail address:stoja@if-pan.krakow.pl(A. Stojakowska).

Contents lists available atScienceDirect

Biochemical Systematics and Ecology

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0305-1978/$ see front matter

2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.bse.2009.12.011

Biochemical Systematics and Ecology 38 (2010) 232235

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3. Present study

The present report deals with the isolation of five thymol derivatives (15) from the roots of I. ensifolia, as well as a-

tocopherol (6), two norisoprenoids (7, 8), three quercetin derivatives (911) and four caffeoylquinic acids (1215) from its

aerial parts (Fig. 1).

The air-dried roots ofI. ensifolia(10.8 g) were powdered and exhaustively extracted with CHCl3at 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 15mm, 25 100 mm) coupled to a dual wavelength UV/vis detector operating at 205 and 270 nm,

using MeOHH2O mixture (13:7) at a flow rate of 6 ml min1, yielded: 1 (21.9 mg), 2 (2.1 mg), a mixture (2.8 mg) of3 and 4 (by

1H NMR), and a fraction (1.5 mg) containing 5 as the main component (by 1H NMR and ESIMS).

The aerial parts (59.6 g) ofI. ensifoliawere powdered and extracted successively with CHCl3, followed by pure MeOH atroom temperature with shaking and the solvents were evaporated under reduced pressure which furnished crude CHCl 3(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 of6 (3.3 mg). From the more polar fractions

(hexane-EtOAc, 1:1) a mixture (2 mg) of7and8(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, 5mm 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 flow rate of 1 ml/min, using 10ml 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) of9 and10(by 1H NMR and co-HPLC with

authentic samples), a mixture (304 mg) of13, 14and 15(by 1H NMR), and11(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 (din

ppm) were referenced to TMS. Mass spectra (ESIMS) were recorded on a Bruker Esquire 3000 mass spectrometer. The isolated

compounds were identified 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-epox-

ythymol isobutyrate (3), 10-isovaleroyloxy-8,9-epoxythymol isobutyrate (4) (Bohlmann et al., 1969; Zee et al., 1998), 7,10-

diisobutyroyloxy-8,9-epoxythymol isobutyrate (5, Bohlmann and Zdero, 1977), a-tocopherol (6, Baker and Myers, 1991; Malik

et al., 1997), megastigmane aglycone - 3b-hydroxy-5b,6b-epoxy-b-ionone (7, Chavez et al.,1997; Xian et al., 2006), loliolide (8,

Kisiel, 1992; El Hattab et al., 2008), quercetin-3-O-b-glucopyranoside (isoquercitrin, 9,Shoeb et al., 2007), quercetin-3-O-b-galactopyranoside (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 significance

A total of 15 terpenoid and phenolic compounds have been isolated from the roots and aerial parts ofI. ensifolia. Except for

compounds10and 12, the other isolated constituents are reported for the first time fromI. ensifolia. Quinic acid derivatives

(1215) appeared to be major secondary metabolites (over 1% yield) of the aerial parts.

The paraphyletic genusInulaL., 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 byAnderberg (1991). Roots of the resiniferous species usually contain

essential oils with the eudesmane-type sesquiterpene lactones alantolactone and/or isoalantolactone as major constituents.

Separate position of these species has been confirmed by ITS sequence analysis ( Eldenas et al., 1998; Francisco-Ortega et al.,

2001). WithinInulaspecies lacking resin canals, a classification based on morphological data has been proposed (Anderberg,

1991). According to this proposal,I. ensifoliahas been included in the I. salicinagroup together withI. 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 sesqui-

terpene lactones in their aerial parts (I. germanica germacranolides, I. salicina eudesmanolides). The compounds wereabsent from the plant material under study. This absence of sesquiterpene lactones situates I. ensifolia closer to some

representatives of the postulated Inula decurrensgroup, i.e. Inula bifronsand Inula conyza. Moreover, I. germanicais rich in

exudate flavonoids, derivatives of luteolin, scutellarein and quercetagenin, whereas only one derivative of luteolin was found

in I. salicina exudate and no flavonoids in an exudate ofI. ensifoliawere found (Wollenweber et al., 1996). It seems that, neitherexudate flavonoids nor esterified thymol derivatives are helpful chemotaxonomical markers withinInula.The flavonoids due

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1

2

3

4

5

R1 R2

H iBu

H MeBu

H iVal

OiBu iBu

6

7 8

9 R - Glc

10 R - Gal

11 R - (6''-caffeoyl)Gal

12

13

14

15

R1 R2 R3 R4

H H H Caff

Caff H H Caff

H Caff Caff H

H Caff H Caff

OMe

O

O

CH2R1

O

O

OR2

O

1

2

34

5

6

7

8

9

10

O

O

OH

12

34 5

6 7

8

9

10

11 12

13

O

O

OH

12

34

5

6

7

8

9 10

11

O

OR

OH

OH

OOH

OH1

OR2

OR3

OR4

OR1

HOOC

6

12

3

45

O

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

OH

Fig. 1. Structures of thymol derivatives (15), a-tocopherol (6), norisoprenoids (7, 8), flavonoids (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 ofInulasp. are needed to support classification efforts, which nowadays are based mainly

on morphological traits.

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