Food Chemistry 136 (2013) 595–603 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Comprehensive analysis of Phyteuma orbiculare L., a wild Alpine food plant ⇑ Christian Abbet a, Ivan Slacanin b, Matthias Hamburger a, Olivier Potterat a, a Division of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland b Ilis Institute and Laboratory, Chemin de la Passerelle 17, CH-2503 Bienne, Switzerland article info abstract Article history: Plants which have been traditionally eaten by the alpine population may provide new opportunities for Received 16 February 2012 agricultural development in mountain regions. In this context we have investigated the chemical compo- Received in revised form 8 August 2012 sition of Phyteuma orbiculare (Campanulaceae), a perennial herb whose leaves have been eaten as salad Accepted 10 August 2012 by rural populations in Valais (Switzerland). Extracts of different polarities were subjected to comprehen- Available online 21 August 2012 sive metabolite profiling using a dereplication platform combining HPLC–PDA-MS, and offline NMR anal- ysis. Twenty-three compounds, including various phenolic glycosides, a new dimeric phenylpropanoid Keywords: glucoside, saponins, and fatty acids were identified online, or after targeted isolation. Selected phenolic Phyteuma orbiculare constituents were quantitatively assessed by HPLC–PDA analysis. In addition, substances relevant for P. spicatum P. hemisphaericum nutrition, such as b-carotene, fatty acids, ascorbic acid and minerals were quantified in leaves and flow- P. ovatum ers. The antioxidant capacity was determined with an ORAC assay, and total phenolic compounds were Alpine plants quantified. Finally, the phytochemical profile was compared to that of the related species P. spicatum, P. Salad hemisphaericum and P. ovatum. Saponins Ó 2012 Elsevier Ltd. All rights reserved. Fatty acids Dimeric phenylpropanoid glucoside 1. Introduction characterised by a pleasant nutty taste, and have been used until re- cently as salad by the rural Alpine population. In addition, the flow- The alpine regions of Europe have a rich ancestral tradition with ers were eaten as sweets by young shepherds. Despite a broad regard to the consumption of wild plants as medicines or food (Pie- geographic distribution, no information was available on the chem- roni & Giusti, 2009). However, knowledge about these plants and ical constituents of the entire genus Phyteuma when we began our their uses tends to become lost as much of it has been transmitted investigations. In a first contribution, we reported the isolation and through oral traditions. Ancient food plants with interesting gusta- the structure elucidation of two triterpene saponins with structur- tory properties may represent interesting opportunities for devel- ally unique aglycons from the aerial parts of P. orbiculare (Abbet opment of mountain agriculture if they can be suitably taken et al., 2011). In a continuation of our investigation, we now report into cultivation. Rocket salad (Eruca sativa) is an example of the a comprehensive phytochemical profiling of the aerial parts of this successful development from a locally used food plant into a main- plant. In addition, we provide data from a quantitative analysis of stream salad (Sastry, 2003). However, prior to any considerations nutritionally valuable substances, such as ascorbic acid, b-carotene, regarding possible cultivation and development, a comprehensive minerals, and fatty acids. The antioxidant capacity was measured chemical analysis of such plants is required as the basis for an with an ORAC (oxygen radical absorbance capacity) assay and the assessment of their nutritional value and safety. phenolic compounds were quantified in the leaves and flowers. Fi- We recently carried out an ethnobotanical survey in Lower and nally, the phytochemical profiles of leaves and flowers of P. orbicu- Central Valais (Switzerland) of traditionally used fruits, vegetables lare were separately investigated with the aid of HPLC–PDA-ESIMS and spices. During semi-structured interviews with informants, analyses, and compared to those of three further Phyteuma species, we established that various species from the genus Phyteuma (Cam- P. spicatum L., P. ovatum L., and P. hemisphaericum L. panulaceae) have been traditionally used as edible plants. The genus Phyteuma, commonly known as rampion, is native to Europe 2. Material and methods and Western Asia and contains about 40 perennial herbs (Shulkina, Gaskin, & Eddie, 2003) including the commonly occurring species P. 2.1. Plant material orbiculare L. and P. spicatum L. The leaf rosettes of both species are The aerial parts of Phyteuma orbiculare L. (Voucher nr. 532) used ⇑ Corresponding author. Tel.: +41 61 267 15 34; fax: +41 61 267 14 74. for metabolite profiling and compound isolation were collected by E-mail address: [email protected] (O. Potterat). C. Abbet on 24th June 2009 in l’Amônaz, near Orsières, Valais, 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.08.018 596 C. Abbet et al. / Food Chemistry 136 (2013) 595–603 Switzerland. The leaves and flowers of P. spicatum L. (Nr. 794), P. (3 Â 2 L, each 24 h). The extracts were evaporated to dryness under ovatum L. (Nr. 795), and a second batch of P. orbiculare (Nr. 799) reduced pressure to obtain 11.9 g of dichloromethane extract, and with leaves and flowers separately harvested and used for compar- 42.3 g of methanol extract. ative studies were collected on 11th June 2011 in Ferret, near Ors- A portion (10.3 g) of the dichloromethane extract was loaded ières, Valais, Switzerland. P.hemisphaericum L. (Nr. 796) was onto a silica gel column (100 Â 6 cm i.d.) eluted with a gradient collected on 11th June 2011 in la Breya, near Orsières. The plants of n-hexane–ethyl acetate–methanol to give 16 fractions (Fr. 1– were identified by C. Rey, Senior Scientist at the Agroscope Chan- 16). After liquid/liquid partition of Fr. 13 (600 mg) between n-hex- gins-Wädenswil ACW research station in Conthey, Switzerland. ane and methanol, a portion (20 mg) of the n-hexane fraction Voucher specimens are kept at the Division of Pharmaceutical Biol- (324 mg) was purified by semi-preparative HPLC on RP-18 with a ogy, University of Basel, Switzerland. gradient of 10 to 100% acetonitrile in 30 min, to afford compounds 18 (1 mg) and 19 (1 mg). 2.2. Chemicals and reference compounds A portion (42.3 g) of the methanol extract (43.7 g) was dissolved in 100 mL water and then loaded onto a Diaion HP-20 column Solvents were from Scharlau. Technical grade solvents were (70 Â 400 mm i.d.) eluted with water (7 L), followed by methanol used after redistillation for extraction and column chromatography (15 L). A portion (5.0 g) of the methanol fraction (6.4 g) was sepa- (CC). HPLC grade solvents were employed for HPLC. HPLC grade rated on a Sephadex LH-20 column (7 Â 100 cm i.d.) eluted with water was obtained by an EASY-pure II (Barnstead, Dubuque IA, methanol. 15 fractions (Fr. 1–15) were collected. Fr. 3 (263 mg) USA) water purification system. Deuterated solvents were pur- and 4 (539 mg) were submitted to flash chromatography on RP- chased from Armar Chemicals (Döttingen, Switzerland). Other 18 with methanol–water (20–100%) as eluent to give 14 (40 mg), chemicals used were analytical grade. Linoleic acid (20), a-linole- and 15 (51 mg), respectively. Frs. 11, 12, 14 and 15 were purified nic acid (22), palmitic acid (23), adenosine (1), chlorogenic acid by semi-preparative HPLC on RP18 with methanol–water gradients (2) and quercetin-3-O-b-glucoside (9) were purchased from Sig- (see Supplementary Information Data D1). 250 mg of Fr. 11 ma–Aldrich (Buchs, Switzerland). Ursolic acid (21) was obtained (849 mg) gave compounds 2 (1 mg), 3 (3 mg), and 4 (5 mg). Like- from Extrasynthese (Genay, France). wise, compounds 3 (3 mg) and 7 (2 mg) were isolated from 51 mg of Fr. 12 (101 mg). Compounds 6 (5 mg), 12 (2 mg), and 13 (2 mg) 2.3. General experimental procedures were obtained after purification of 40 mg of Fr. 14 (96 mg). Finally, 42 mg of Fr. 15 (63 mg) provided compounds 1 (6 mg), 6 (3 mg), 9 Sephadex LH-20 was purchased from GE Healthcare. Diaion HP- (4 mg), and a mixture of two compounds (14 mg). They were sepa- 20 resin was obtained from Sigma–Aldrich. Silica gel (0.063– rated by Sephadex LH-20 CC (40 Â 1 cm i.d.) with methanol, to ob- 0.200 mm) was purchased from Merck. Flash chromatography tain 10 (6 mg), and 12 (5 mg). Compound 11 (6 mg) was isolated was performed on a SepacoreÒ chromatography system (Büchi from 100 mg of Fr. 6 (213 mg), after separation by silica gel CC with Labortechnik, Flawil, Switzerland) equipped with a pre-packed a gradient of n-hexane–ethyl acetate–methanol. Fraction 10 RP-18 cartridge (40 Â 150 mm, 40–63 lm, Büchi Labortechnik). (418 mg) was separated by silica gel CC eluted with a gradient of Semi-preparative HPLC was performed on an Agilent 1100 series ethyl acetate–methanol. Semi-preparative HPLC on RP18 with a instrument equipped with a PDA detector. Separations were car- gradient of 10 to 50% acetonitrile in 30 min afforded compounds ried out at 25 °C on a SunFire C18 column (5 lm, 150 Â 10 mm 16 (2 mg), and 17 (4 mg). 20 i.d., Waters, Milford, MA, USA) equipped with a precolumn Tangshenoside VII (8): White amorphous powder. [a] D À19.4 (c (10 Â 10 mm i.d.). Gradients of acetonitrile and water were used; 0.05, MeOH). UV (MeOH) kmax (log e): 220 (4.03), 268 (3.72).