Primula spectabilis Tratt. aerial parts: Morphology, volatile compounds and flavonoids

Phytochemistry 72 (2011) 1371–1378

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Primula spectabilis Tratt. aerial parts: Morphology, volatile compounds and flavonoids Sara Vitalini a, Guido Flamini b, Aurora Valaguzza c, Graziella Rodondi d, Marcello Iriti a, Gelsomina Fico c,⇑ a

Dipartimento di Produzione Vegetale, Facoltà di Agraria, Università degli Studi di Milano, via Celoria 2, 20133 Milano, Italy Dipartimento di Scienze Farmaceutiche, Sede di Chimica Bioorganica e Biofarmacia, Università degli Studi di Pisa, via Bonanno 33, 56126 Pisa, Italy c Dipartimento di Biologia, Facoltà di Farmacia, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italy d Dipartimento di Biologia, Sezione di Botanica Sistematica e Geobotanica, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italy b

a r t i c l e

i n f o

Article history: Received 9 December 2010 Received in revised form 14 April 2011 Available online 24 May 2011 Keywords: Primula spectabilis Primulaceae Phytochemistry Morphology Flavonoids Apigenin C-glycoside Luteolin C-glycoside Glandular trichomes Volatile compounds

a b s t r a c t The vacuolar and epicuticular flavonoids and the volatiles of the leaves and parts of flower of P. spectabilis Tratt., an endemic species in the Italian Oriental Alps, were investigated. From a MeOH extract of the leaves two flavone glycosides, 8-C-b-glucopyranosylluteolin 7-O-a-arabinofuranoside (1) and 6-C-a-arabinofuranosylapigenin (2) were isolated, in addition to a flavone and three flavonols already known from species of Primula. From an EtOH extract of leaf exudates, 7,30 ,40 -tri-O-methylquercetin was obtained. The structures were elucidated on the basis of their 1D 1H- and 13C NMR data and 2D NMR techniques, as well as of HPLC–MS. The volatiles emitted by the leaves were mainly constituted by non-terpene derivatives, followed by comparable proportions of hemiterpens, oxygenated monoterpenes and sesquiterpene hydrocarbons. In flowers, monoterpene hydrocarbons were the most represented chemical class followed by non-terpene derivatives. Different proportions of compounds were found when individual parts of flowers were examined separately; calyx produced a greater proportion (approx. 49.5%) of non-terpenes as its volatile metabolites. P. spectabilis has glandular trichomes in the hyaline margins of the epidermal depressions, distributed on the adaxial leaf blade. Glandular hairs were also present on the corolla. Correlations of phytochemical data with the morphological features of leaf, flower and glandular hair are discussed, and a hypothesis is proposed on the ecological roles of the flavonoids and volatile compounds on the general fitness of the species and cross-pollination strategies. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Primula L. genus (Primulaceae) comprises 426 species distributed in temperate and cold regions of the Northern hemisphere and in mountains at tropical latitudes. Primula spectabilis Tratt. is an endemic species of the Eastern Alps (from Monte Grappa to the Brescia Pre-Alps), protected by the L.R. 27/09/77 n. 33 for the spontaneous flora. It only lives between 600 and 2500 m above the sea level, on humid and shaded rocks or stony and pebbly slopes (Ravazzi, 1999; Richards, 2003). This species belongs to sect. Auricula Duby and is characterized by involute leaf vernation and stomata on the adaxial surface of the leaf, as reported by Smith and Forrest (1929). The leaves are coriaceous, rhombic-ovate and pressed to the ground, and display glandular hairs on the upper surface, embedded in the parenchyma and in the cartilaginous margin. The scape ⇑ Corresponding author. Tel.: +39 0250314770; fax: +39 0250314764. E-mail addresses: [email protected] (S. Vitalini), fl[email protected] (G. Flamini), [email protected] (G. Rodondi), [email protected] (M. Iriti), gelsomina.fi[email protected] (G. Fico). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.04.010

is 2–15 cm in height, bearing 2–5 pinkish-red to lilac flowers with pedicels of 1–2 cm and a calyx of 7–12 mm; the bracts are triangular-lanceolate to linear (Pignatti, 1982; Tutin et al., 1993). The glands of Primula species produce farinas (formed as rod or needle-like crystals) and/or exudates (sticky secretions); in particular, the leaves of P. spectabilis produce an exudate deposition. Although the morphology of glandular hairs of some Italian Primula species has already been reported (Higuchi et al., 1999; Fico et al., 2007), no specific study on P. spectabilis has been published so far. The secondary metabolite content of Primula genus has not been completely characterized yet. Earlier studies reported the presence of saponins, especially in rhizome and roots (Calis, 1992; Morozowska and Wesoowska, 2004), responsible for its medicinal use as expectorant and antimicrobial drug (Della Loggia, 1993). Many works deal with the flavonoid composition of some Primula species: P. vulgaris (Harborne, 1968), P. elatior (PetitijeanFreytet, 1993), P. veris (Huck et al., 2000), P. denticulata (Tokalov et al., 2004), P. hirsuta, P. auricula and P. daonensis (Fico et al., 2007). Harborne (1968) used data on flavonoids in order to determine the relationship between flavonoid content and taxonomy in the Primulaceae. Among flavonols, mono- and diglycosilated

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derivatives characterize this genus. The most common sugars are glucose, rhamnose, xylose and galactose, while the main aglycons are quercetin, kaempferol and isorhamnetin; on average, sugar moiety linkage favours the 3-position. This is the first study on the hair morphology of the Italian endemic species P. spectabilis and its vacuolar and epicuticular flavonoids. Moreover, the volatile compounds emitted by the leaves, the whole flower and its parts are also investigated.

2. Results 2.1. Aerial part structure, trichome types, morphology and distribution The leaves of P. spectabilis show a rhombic-ovate outline and are coriaceous and thickly imbricate, forming a basal rosette (Fig. 1). They are distinguished by a white (hyaline), cartilaginous margin which folds up to the upper epidermis. The adaxial lamina is densely covered with glandular trichomes producing sticky secretions with a strong aromatic smell. They are embedded and randomly interposed among stomata (Fig. 2A and B). Hairs can also be found on the abaxial surface and along the margins.

Fig. 1. P. spectabilis whole plant.

Fig. 2. LM micrograph of epistomatic leaf of P. spectabilis: both stomata and glandular trichomes are in the upper epidermis (A); LM micrograph of a single big-headed leaf glandular trichome (B); LM micrograph of transverse section of an epistomatic leaf of P. spectabilis: the glandular trichomes are located in the epidermal crypts; numerous glandular hairs can also bee found on the involute leaf margin (C); LM micrograph of glandular hairs of P. spectabilis petals with long conical stalks and round glands (D, E). Abbreviations: gtr, glandular trichome; gh, glandular head; st, stomata; uep, upper epidermis; lep, lower epidermis; ma, margin; me, mesophyll; n, neck; s, stalk.

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S. Vitalini et al. / Phytochemistry 72 (2011) 1371–1378 Table 1 Flavonoid profile of Primula spectabilis.

R1 R2 R6 O

R 5O

Fig. 3. Fluorescence micrograph of a cross-section of P. spectabilis leaf: distribution of epicuticular flavonoids (yellow autofluorescence) in hand section of fresh leaf, at UV light; note the blue autofluorescence of cuticle on the whole upper leaf surface (365 nm excitation filter, 400 nm barrier filter). Abbreviations: uep, upper epidermis; me, mesophyll; ef, epicuticular flavonoids; c, cuticle.

At light microscopy (LM) analysis, fully-developed capitate trichomes consist of a basal epidermal cell and a short, rectangular stalk supporting a wide, globose, unicellular glandular head. The leaves display a dorsoventral structure, showing a thickwalled epidermis with a well developed cuticle. The upper surface is characterized by crypts exhibiting a trichome at the bottom. Some glandular hairs are also visible on the leaf margin, where the mesophyll gradually disappears, replaced by the thick-walled cells of mechanical tissue (Fig. 2C). Thricomes composed of a large, cylindrical stalk, a small, rectangular neck and a globose head are randomly spread on the upper surface of petals (Fig. 2D and E). Fluorescence microscopy observations of the fresh leaves evidenced that epicuticular flavonoids are not uniformly distributed on the entire surface: they are rather stored in the epidermis crypts. Their location can be detected through the specific yellow autofluorescence, easily distinguishable from the blue fluorescence of the cuticle (Fig. 3).

R3

R4 OH

1 2 3 4 5 6 7

O

R1

R2

R3

R4

R5

R6

OH H H OH OH H OCH3

OH OH OH OH OH OH OCH3

H H H Gent Soph OH OH

H Araf Glc H H H H

Araf H H H H H CH3

Glc H Glc H H Glc H

Araf =

HO O HO

OH Glc =

HO O OH

2.2. Flavonoid isolation and identification OH

From the MeOH leaf extract, two new flavone glycosides, luteolin 7-O-a-arabinofuranosyl-8-C-b-glucopiranoside (1), apigenin 6-C-a-arabinofuranoside (2) were isolated, besides a flavone and three flavonols already known from Primula species: apigenin 6,8-di-C-glucopyranoside (3), quercetin 3-O-gentiobioside (4), quercetin 3-O-sophoroside (5), and kaempferol 8-C-glucoside (6). From an EtOH extract of leaf exudates, 7,30 ,40 -tri-O-methylquercetin (7) was obtained. The structures were elucidated on the basis of their 1H- and 13C NMR data and 2D-NMR techniques, as well as of HPLC–MS. Compound 1 was obtained as a yellowish powder that appears on TLC as a yellow spot after treatment with Naturstoffreagenz A-PEG. The negative ESI–MS spectrum returned a quasimolecular peak [M–H]– at 579 m/z, corresponding to the molecular formula C26H28O15, supported also by elemental analysis (see Section 4). The 13C NMR spectrum showed 26 signals, sorted by DEPT experiments into 14 CH, 2 CH2 and 10 quaternary C. In the 1H NMR spectrum obtained in DMSO-d6, a three-proton ABM system was present (d 7.47, dd, J = 8.8, 2.0 Hz; 7.40, d, J = 2.0 Hz; 6.73, d, J = 8.8 Hz), typical of a 30 ,40 -disubstituted ring B of a flavonoid nucleus. Ring A showed only a resonance as one-proton singlet at d 6.31. Moreover, another one-proton singlet was present at d 6.42. These data are consistent with a luteolin skeleton bearing a further substituent on ring A. Furthermore, the signals of two anomeric sugar protons were clearly visible as a broad singlet at d 4.94 and as a doublet (J = 10.1) at d 4.81. Analysis of the 13C NMR spectrum identified the two sugar units as b-glucopyranose and a-arabinofuranose. The uncommon high field shift of the glucose

OH Gent =

HO O O OH

O O OH

OH OH

OH OH

Soph =

HO O OH HO

OH O O OH

OH OH .

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Table 2 Volatiles of different plant parts of Primula spectabilis sampled by SPME. Constituents

l.r.i.

Isopentyl alcohol Pentyl alcohol Ethyl isovalerate 2-Heptanone Anisole a-Thujene a-Pinene b-Pinene 6-Methyl-5-hepten-2-one Myrcene 6-Methyl-5-hepten-2-ol Octanal p-Methyl anisole p-Cymene Limonene 1,8-Cineole Benzyl alcohol c-Terpinene cis-Linalool oxide (furanoid) trans-Linalool oxide (furanoid) ortho-Guaiacol 2-Nonanone 1-Undecene Methyl benzoate n-Undecane Linalool Nonanal 2,6-Dimethylphenol Phenylethyl alcohol p-Ethyl anisole cis-Verbenol Veratrole (1,2-dimethoxybenzene) 1-Nonanol cis-Linalool oxide (pyranoid) 4-Terpineol a-Terpineol Methyl salicylate n-Dodecane Decanal trans-Carveol Isobornyl formate Isobornyl acetate Dihydroedulan IA 2-Undecanone n-Tridecane Undecanal Myrtenyl acetate Methyl 2,6-dihydroxybenzoate b-Elemene Sativene n-Tetradecane Methyl eugenol Dodecanal Longifolene b-Caryophyllene b-Gurjunene a-Guaieme Aromadendrene (E)-b-farnesene a-Humulene (E)-geranylacetone (E)-b-ionone b-Selinene Valencene a-Selinene n-Pentadecane (E,E)-a-farnesene 7-epi-a-Selinene d-Cadinene Caryophyllene oxide 1-Hexadecene Ethyl laurate n-Hexadecane

735 756 856 891 919 932 937 982 987 992 994 1003 1022 1027 1032 1035 1036 1062 1075 1090 1091 1092 1094 1095 1100 1101 1104 1106 1110 1120 1143 1150 1173 1175 1179 1193 1194 1200 1206 1218 1233 1287 1290 1293 1300 1308 1328 1386 1390 1392 1400 1402 1409 1410 1420 1433 1438 1442 1454 1455 1457 1486 1488 1493 1495 1500 1505 1515 1523 1583 1591 1596 1600

Primula spectabilis Leaves

Whole flowers

Corolla

Calyx

14.5 0.4 0.9 0.3 0.3 4.4 – – 1.1 0.5 0.4 – 0.4 – 0.6 0.9 – – 2.2 2.7 – 1.1 – – – 8.7 – – – 23.7 – 17.7 – 0.3 – 0.3 – – – – – – 0.2 0.5 – – – – 1.1 0.4 – 0.4 – 0.7 6.7 1.3 0.3 – 0.5 0.6 – – 0.3 – 0.5 – 0.4 – – – – 0.2 –

– – – – – – 46.0 0.9 1.8 1.0 – 0.7 – 0.1 2.6 0.1 0.9 1.3 – tr 2.7 – 0.1 0.8 – 1.3 2.5 – 0.8 – – – tr – tr – 16.3 0.9 2.2 – – 0.9 – – 0.5 tr 0.4 1.0 0.5 – 1.5 – tr – 2.5 – – 0.2 – – 1.0 0.9 – 1.4 – 0.8 0.5 – – – – – 1.4

– – – – – – 35.7 0.2 1.6 0.5 – – – – 2.4 0.2 1.1 1.0 – – 3.2 – – 1.6 – 1.7 2.2 – 1.5 – – – tr – 0.1 – 27.1 0.5 2.3 – – 0.7 – – 0.4 0.1 0.1 0.8 tr – 0.8 – tr – 1.4 – – – – – 0.7 1.4 – 1.6 – 0.6 0.7 – – – tr – 2.1

– – – – – – 28.6 1.2 – 0.4 – – – – 0.8 5.9 – – 0.6 2.4 – – 3.2 – 0.4 – 0.5 0.5 – – 0.5 – 3.1 – 2.6 – 0.3 0.7 0.4 0.2 0.5 – – – 1.0 – – – 0.9 – 2.8 – 0.7 – 9.0 – – 4.4 – 0.3 0.1 – – 0.1 – – – 0.5 0.7 1.3 1.0 – 0.2

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S. Vitalini et al. / Phytochemistry 72 (2011) 1371–1378 Table 2 (continued) Constituents

l.r.i.

Longiborneol Tetradecanal n-Heptadecane Methyl myristate n-Octadecane Hexadecanal Sandaracopimara-8(14),15-diene epi-13-Manoyl oxide Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Diterpenes Non-terpene derivatives Total identified

1601 1613 1700 1730 1800 1842 1962 2012

Primula spectabilis Leaves

Whole flowers

Corolla

Calyx

0.3 – – 0.4 – – – – 5.5 15.1 12.8 0.3 0 62.5 96.2

– – 0.9 – 0.2 tr – – 51.9 3.7 5.1 0.0 0.0 36.9 97.6

– 0.1 1.4 – 0.6 – – – 31.0 12.8 15.8 1.3 0.0 34.1 96.4

– 0.4 18.6 – 0.2 – 0.1 0.5 31.0 3.4 2.7 0.0 0.6 49.5 95.6

anomeric carbon (d 72.1) and the large coupling constant of its proton doublet (J = 10.1 Hz) supported the hypotesis of a C-glycosidic link. The position of this link was established at C-8 of luteolin through the downfield shift experienced by this carbon atom (about 11 ppm) and by HMBC experiments. The latter experiment also permitted the arabinose moiety to be placed on the 7-OH. Therefore, 1 was identified as luteolin 7-O-a-arabinofuranosyl-8C-glucopyranoside (Table 1). Compound 2 was obtained as a pale yellowish powder that appears on TLC as a yellow spot after treatment with Naturstoffreagenz A-PEG. The negative ESI–MS spectrum returned a quasimolecular peak [M–H]– at 401 m/z, corresponding to the molecular formula C20H18O9, supported also by elemental analysis (see Section 4). The 13C NMR spectrum showed 18 signals, sorted by DEPT experiments into 8 CH, 1 CH2 and 9 quaternary C. The 1 H NMR spectrum obtained in DMSO-d6 was very similar to that of compound 1, besides the presence of two 2-protons doublets (J = 8.8 Hz) centred at d 6.77 and 7.66, indicating a 40 -substitution on ring B of the flavonoid nucleus. The flavonoid unit was then recognized as a substituted apigenin. Further resonances were present between d 4.91 and the water signal that partially overlapped them. From the 13C NMR spectrum and the very small coupling constant of the anomeric proton, the sugar moiety was identified as a-arabinofuranose. Through HMBC experiments, a-arabinofuranose was found to be connected via a C-glycosidic linkage to C-6 of apigenin. Therefore, 2 was identified as apigenin 6-C-a-arabinofuranoside (Table 1). Besides 1 and 2, a flavone and three flavonols (3–6) already known in Primula genus were isolated (Gluchoff-Fiasson et al., 1997; Guanghou and Leong, 2004; Kamara et al., 2003; Siciliano et al., 2004). On the basis of these results, a characteristic flavonoid profile for this species was evidenced (Table 1). P. spectabilis stores 6-C-, 8-C- and 3-O-glycosides of apigenin, luteolin, kaempferol and quercetin in its vacuoles; the sugar moieties were glucose, arabinose and mono- and disaccharides based on these sugars. In addition 7,30 ,40 -tri-O-methylquercetin (7) was identified in the exudate.

reached an important percentage (36.9%). These compounds were followed by comparable percentages of oxygenated monoterpenes (3.7, 12.8 and 3.4% in whole flowers, corolla and calyx, respectively), and sesquiterpene hydrocarbons (5.1, 15.8 and 2.7% in whole flowers, corolla and calyx, respectively). Monoterpene hydrocarbons constituted an important part of the volatiles emitted by the flowers (51.9, 31.0 and 31.0% in whole flowers, corolla and calyx, respectively). Oxygenated sesquiterpenes were scarcely represented (1.3% only in corolla). The large differences observed between the whole flower and its parts may account for the presence of the fertile parts in the whole flower: these are known to substantially contribute to the emission of volatiles (Flamini et al., 2007, 2003). Some studies have evidenced that in certain species floral structures other than perianth are the main producers of volatiles within a flower, i.e. stamens (Jullien et al., 2008) nectary disks (Custodio et al., 2006) and staminoids (Flamini et al., 2002). Even pollen may play an important role on the whole emission of a flower (Dobson and Bergström, 2000). From a qualitative point of view, sesquiterpene hydrocarbons were the most characterizing compounds (27 chemicals out 55) followed by non-terpene hydrocarbons (16 chemicals). Monoterpenes were less represented (11 and 6 compounds for oxygenated and hydrocarbon ones, respectively). Only one representative of oxygenated sesquiterpenes was detected, while seven occurrences of hydrocarbon were detected. Two diterpenes were identified, but only among the volatiles released by the calyx. The main compound was always a-pinene (28.6–46.0%), followed by methyl salicylate in the case of whole flowers and corolla (16.3% and 27.1%, respectively) and n-heptadecane for calyx (18.6%). On the contrary, methyl salicylate was only a minor constituent for the calyx (0.3%). Considering the chemical classes, the emission of the leaves is more similar to that of calyx than of corolla. In fact, non-terpene derivatives constituted 62.5% of the whole emission. However, the compounds forming this mixture are quite different from those of flowers: the main volatiles were identified as p-ethyl anisole (23.7%), veratrole (17.7%) and isopentyl alcohol (14.5%), all absent in the flower emissions.

3. Discussion 2.3. Volatile compounds The questions to be answered are: In the headspace around the flowers, 54 chemicals were identified, accounting for 95.6% to 97.6% of total volatiles (Table 2). Among them, with the exception of whole flowers, non-terpene derivatives were the most represented (34.1% and 49.5% for corolla and calyx, respectively); however, also in the whole flower they

(1) Why is the study of P. spectabilis inside Primula genus interesting? (2) What are the phytochemical and morphological elements of novelty in this study?

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P. spectabilis is an endemic primrose of Southern calcareous Pre-Alps and it is considered a patroendemic species (Kress, 1963). The aim was to verify whether flavonoid production and secretory tissue, together with the entire structure of leaf and flower, may characterize this entity. A careful description of leaf morphology, scarcely reported in the literature, and of flower and trichomes, not previously reported at all, was necessary. Therefore, the leaf and flower morphology, with particular care to the sites of flavonoid deposition, were investigated. Unlike other Italian Primula species (Fico et al., 2007 and our recent observations still not shown), very short trichomes were noted. They are typically located in epidermal depressions, creating dark spots on the adaxial surface, clearly visible with a magnifying glass. This condition could explain the recurring descriptions in the literature as ‘‘leaves with glandular points’’ (Pignatti, 1982), or ‘‘leaves with sunken dot-glands’’ (Zhang and Kadereit, 2004). The structure of flower hairs (trichomes are three times higher than in the leaf) and their localization on the epidermal surface (and not in crypts like in the leaf), may warrant their maximum exposure to the environment and pollinator attraction, i.e. the chief role of the flower. On the basis of previous phytochemical studies on Primula genus, only P. spectabilis presents apigenin and luteolin among flavonoid aglycons. Some of these aglycons (1–6) are C-6 and C-8 substituted (another element of novelty for the genus), and among the sugar moieties commonly described for Primula entities, sophorose is distinctive for this species. An epicuticular flavonoid was isolated as well (7): it can also be found in some species of different families, such as Asteraceae, Cistaceae, Geraniaceae, where it is described not only in the exudates of the aerial parts, but also in roots and fruits (ValantVetschera et al., 2010; Zhang et al., 2006; Ivancheva and Petrova, 2000; Hui et al., 1999; Wollenweber et al., 1995; Ivancheva and Wollenweber, 1989). Its lipophilicity and low molecular weight explain its production by trichomes (Valant-Vetschera and Brem, 2006). A UV-protective role is often claimed for flavonoids, and it could be of particular interest for a species, like P. spectabilis, exposed to severe irradiation in its habitat. In particular, the epicuticular flavonoids might protect the plant from solar exposure in addition to the preeminent action of the wall phenolic compounds (see the epidermal blue fluorescence in Fig. 3) (Burchard et al., 2000; Tattini et al., 2004). Furthermore, the flavonoid secretion in the crypts (see the epidermal yellow fluorescence in Fig. 3), characterized by the absence of stomata, may prevent the obstruction of stomatal pores, scattered in the adaxial epidermis, thus preserving their functionality. Only a few studies were previously carried out on volatiles surrounding the headspace of Primula species. Gaskett et al. (2005) reported that P. elatior and P. farinosa produce qualitatively different bouquets. The former species emits primarily limonene (about 94%) with small amounts of three other monoterpene hydrocarbons: myrcene, a-pinene and sabinene (about 2%, 0.7% and 2.7%, respectively). P. farinosa mainly produces non-terpene derivatives, such as benzaldehyde, 4-oxoisophorone, benzyl alcohol and benzyl acetate (about 35%, 27%, 29% and 8.4%, respectively). Interestingly, the authors did not find intraspecific variations in scent between the two floral morphs (pin and thrum) in either species. P. spectabilis is characterized by a completely different composition of the volatile emission in comparison with the two species mentioned above. Even if more studies are required, it seems that each species is characterized by a distinctive profile, probably to attract different pollinators (Gaskett et al., 2005). Actually, cross-pollination is a fundamental requisite for most Primula species, particularly in the case of heterostyly (such as the case of

P. spectabilis); many of them release a complex bouquet capable of attracting different insects. It has been observed that generalist adaptation to insect pollination is often revealed by scent blends dominated by benzenoids and/or linalool and linalool oxides (Andersson et al., 2002). These compounds seem to be imperative to attract butterflies. At the same time, the presence of single compounds in large relative amounts has been shown to have a food potential signalling for bees (Borg-Karlson et al., 1996). In the case of P. spectabilis, it seems that both strategies have been adopted, with a large emission of benzenoids (22.5% and 35.3% in the whole flowers and corolla, respectively), and high percentages of single compounds such as a-pinene (46% and 35.7% in the whole flower and corolla, respectively) and methyl salicylate (16.3% and 27.1%). Such behaviour may be a strategy to overcome the selfincompatibility fertilization due to heterostyly. This hypothesis may be confirmed by Crema et al. (2009), who reported some observations on the related species P. apennina. Also in this species, the heterostyly requires pollen exchange among individuals of different morphs. The long-range pollen transfer was attributed to the hawk moth Macroglossum stellatarum, while the fertilization between spatially close individuals is performed by Staphylinidae coleopterans, characterized by a very low mobility. In the calyx the percentage of benzenoids dropped to 1.1%, probably because of its lesser importance as attractant organ. On the contrary, similarly to the leaf, it emitted appreciable amounts of linalool oxides. Considering the preeminent protective role of this whorl, these chemicals may be involved in the defence against herbivores, as observed for Osmanthus fragrans (Mura et al., 2000). These different patterns and gradients of emission of the diverse floral parts may play an important role in the attraction of pollinators and also in their optimal orientation for foraging purposes within the flower (Dobson et al., 1990; Knudsen and Tollsten, 1991). A synergy between olfactory (by means of volatile compounds) and visual (by means of epicuticular flavonoids) attraction may well be an acceptable hypothesis. As expected, volatiles emitted by the leaves were quite different from those of the flowers. The attractive role of the vegetative parts is normally less important than in the flowers, even if it cannot be completely neglected (a synergistic action leaf-flower may be ascribed to the common emission of attractant volatiles such as b-caryophyllene or linalool) (Raguso et al., 2003; Raguso and Pichersky, 1999). However, in the leaf a defensive role against herbivores, pathogens and competing organisms, seems to be the most likely and useful function for the plant. The elevated production of benzenoids with a different structure (alkoxybenzenes) with respect to those synthesized by the flower is noteworthy. They may be involved in the chemical defence of leaves, both as repellent against herbivorous and as attractant for parasitoids, as well as allelopathic agents (Akhtar et al., 2007; Paduraru et al., 2008; Pettersson and Boland, 2003). In conclusion, morphological and chemical data seem to univocally characterize P. spectabilis. An ecological role of the secondary metabolites in correlation with the aerial part structure was proposed.

4. Experimental 4.1. Plant material The aerial parts of P. spectabilis L. were sampled from randomly selected individuals collected in Valvestino Alps in Lombardy (Italy), in July 2007, and were determined according to Pignatti (1982). The voucher specimen (Ps-101) was deposited at the Dipartimento di Biologia, Università degli Studi di Milano (Italy).

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4.2. Morphological analysis For morphological observations, leaves were fixed in a mixture of 37% formaldehyde, glacial acetic acid, 50% ethanol (5:5:90 by volume) for several days. Samples were then washed in distilled water for 20 min, cut by razor blade into pieces of approximately 5 mm, and peeled or embedded in methacrylate resin. The epidermal tissues, peeled from the superior leaf blade by microscopy tweezers, were cleared by using sodium hypochlorite 5%, washed in distilled water and mounted in a 90% solution of glycerin for semipermanent slides. Samples were then dehydrated through a graded ethanol series and embedded in methacrylate resin (Technovit 7100; Heraeus Kulzer, Werhrheim, Germany). Specimens were sectioned with a rotary microtome (Leica, Wetzlar, Germany) and the leaf sections (5–8 lm) were routinely stained with 1% toluidine O in sodium tetraborate. The sections were then mounted in Eukitt (O. Knidler GmbH & Co.; Freiburg, Germany) for permanent slides. Permanent and semipermanent slides were examined and photographed with a LEICA DM light microscope. Fluorescence microscopy studies were carried out by preparing thin hand sections of the leaf, mounted in a 90% solution of glycerin for semipermanent slides. Semipermanent slides were examined and photographed with a LEICA DM-RD light/ epifluorescence microscope equipped with a Ploe mopack 1 System and a mercury lamp (OSRAM HBO 100 W) with a UV-1A filter (365 nm excitation filter, 400 nm barrier filter). 4.3. Phytochemical analysis The dried and powdered leaves (62 g) of P. spectabilis were defatted with n-hexane and then extracted with CH2Cl2, CH2Cl2–MeOH (9:1) and MeOH, three times for each solvent. HPLC analyses were performed with a Merck-Hitachi L 6200 system equipped with a photo Diode Array Detector Hewlett Packard 1040, controlled by HP-Chemstation (Hewlett Packard) software, using a Merck LiChrospher 100 RP-18 column (5 lm, 250  4 mm, flow rate 1.3 ml/min) with binary gradient elution [A: H2O (pH 3.5 with HCl); B: ACN; gradient: 0–10 min 88% A, 10–15 min 82% A, 30 min 55% A, 35–42 min 100% B, minimum re-equilibration time between two injections: 10 min]. The detection range was 250–360 nm. The concentration of samples was 100 mg/ml and the injection volume was 100 ll. After removal of the solvent, the extracts were separately chromatographed on a Sephadex LH-20 column, using MeOH as eluent, to obtain 19 fractions, combined together according to TLC analyses [Silica 60 F254 gel-coated aluminium sheets; eluent: n-BuOH-CH3COOH–H2O (60:15:25)]. The flavonoid-containing fractions, selected using NTS–PEG (Naturstoffe Reagenz A-Polyethylenglycol) as spray reagent, were submitted to RP–HPLC on a C18 l-Bondapak column (300  7.8 mm, flow rate 2.5 mL min1) with MeOH/H2O (40:60) to yield compounds 1–6. The epicuticular flavonoids were extracted by immersion of fresh leaves in EtOH 95% for 1 min. The extract was then chromatographed on a Sephadex LH-20 column, using MeOH as eluent, in order to obtain 23 fractions, combined together according to TLC analyses [Silica 60 F254 gel-coated aluminium sheets; eluent: CH3COOH–HCOOH–CH3COOC2H5–H2O (11:11:100:27)]. This technique allowed the isolation of compound 7. All the isolated compounds were submitted to NMR spectroscopic measurements with Bruker AC 400 (400 MHz) apparatus using CD3OD or DMSO-d6 as solvents, and the chemical shifts were expressed in d (ppm) referring to solvent peaks: dH 3.31 or 2.49 and dC 49.0 or 39.5, respectively. The HPLC–MS and UV–VIS spectra were performed on a HP 1090L instrument equipped with a Diode Array Detector, managed by a HP 9000 workstation interfaced with

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a HP 1100 MSD API-electrospray unit. Melting points (uncorrected) were determined with the aid of a Kofler apparatus. 4.4. New compounds Luteolin 7-O-a-arabinofuranosyl-8-C-glucopyranoside (1): yellowish powder; 1H NMR (400 MHz, DMSO-d6): d 2.58 (1H, m, H4000 ), 3.01 (1H, br d, J = 8.8 Hz, H-600 a), 3.24 (overlapped with water signal, H-500 ), 3.35 (overlapped with water signal, H-400 ), 3.44 (overlapped with water signal, H-300 ), 3.51 (overlapped with water signal, H-5000 a), 3.54 (overlapped with water signal, H-300 0 ), 3.59 (overlapped with water signal, H-2000 ), 3.61 (overlapped with water signal, H-600 ), 3.79 (1H, m, H-5000 b), 4.02 (1H, t, J = 9.08 Hz, H-200 ), 4.81 (1H, d, J = 10.1 Hz, H-100 ), 4.94 (1H, brs, H-100 0 ), 6.31 (1H, s, H-6), 6.42 (1H, s, H-3), 6.73 (1H, d, J = 8.8 Hz, H-50 ), 7.40 (1H, d, J = 2.0 Hz, H-20 ), 7.47 (1H, dd, J = 8.8 and 2.0 Hz, H-60 ); 13C NMR (100 MHz, DMSO-d6, 65 °C): d 59.4 (t, C-600 ), 61.8 (t, C-5000 ), 71.1 (d, C-400 ), 72.1 (d, C-100 ), 74.9 (d, C-200 ), 76.0 (d, C-3000 ), 79.1 (d, C300 ), 81.8 (d, C-2000 + C-500 ), 83.1 (d, C-4000 ), 99.4 (d, C-6), 101.0 (d, C3), 104.3 (s, C-10), 104.6 (s, C-8), 107.4 (d, C-1000 ), 112.7 (d, C-20 ), 115.5 (d, C-50 ), 119.5 (d, C-60 ), 120.4 (s, C-10 ), 146.5 (s, C-30 ), 152.1 (s, C-40 ), 156.4 (s, C-2), 157.7 (s, C-5), 160.3 (s, C-9), 163.5 (s, C-7), 180.9 (s, C-4); ESIMS (negative mode) m/z 579 [M–H]–; elemental analysis: found: C 54.11%, H 4.69%, requires: C 53.80%, H 4.86%. 6-C-a-arabinofuranoside (2): pale yellowish powder; 1H NMR (400 MHz, DMSO-d6): d 3.19 (overlapped with water signal, H500 a), 3.22 (overlapped with water signal, H-400 ), 3.26 (overlapped with water signal, H-500 b), 3.32 (overlapped with water signal, H300 ), 3.81 (1H, m, H-200 ), 4.91 (1H, brs, H-100 ), 6.18 (1H, s, H-8), 6.38 (1H, s, H-3), 6.77 (2H, d, J = 8.8 Hz, H-30 + H-50 ), 7.66 (2H, d, J = 8.8 Hz, H-20 + H-60 ); 13C NMR (100 MHz, DMSO-d6, 65 °C): d 61.0 (t, C-500 ), 76.9 (d, C-100 ), 78.2 (d, C-300 ), 80.1 (d, C-200 ), 84.6 (d, C-400 ), 97.5 (d, C-8), 100.7 (d, C-3), 105.0 (s, C-10), 106.2 (s, C-6), 116.2 (d, C-30 + C-50 ), 119.9 (s, C10 ), 127.1 (d, C-20 + C-60 ), 157.4 (s, C-9), 160.1 (s, C-40 ), 160.6 (s, C-5), 163.6 (s, C-2), 163.8 (s, C-7), 178.6 (s, C-4); ESIMS (negative mode) m/z 401 [M–H]–; elemental analysis: found: C 59.98%, H 4.32%, requires: C 59.70%, H 4.51%. 4.5. Volatile compounds The solid phase microextraction (SPME) was carried out with Supelco SPME devices coated with polydimethylsiloxane (PDMS, 100 lm), used for sampling the headspace of Primula aerial parts placed into a 10 ml glass septum vial and allowed to equilibrate for 30 min. After equilibration time, the fibre was exposed to the headspace for 30 min at room temperature. At the end of sampling, the fibre was withdrawn into the needle and transferred to the injection port of the GC and GC/MS system, operating as follows. The GC analyses were accomplished with a HP-5890 Series II instrument equipped with HP-WAX and HP-5 capillary columns (30 m  0.25 mm, 0.25 lm film thickness), and with the following conditions: temperature programme 60 °C for 10 min, followed by an increase of 5 °C/min to 220 °C; injector and detector temperatures at 250 °C; carrier gas helium (2 mL/min); splitless injection; detector dual FID. The identification of the chemicals was performed for both the columns through comparison of their retention times with those of pure authentic samples and by means of their Linear Retention Indices (LRI) relative to the series of n-hydrocarbons. GC/EIMS analyses were performed with a Varian CP-3800 gaschromatograph equipped with a HP-5 ms capillary column (30 m  0.25 mm; coating thickness 0.25 lm) and a Varian Saturn 2000 ion trap mass detector. The analytical conditions were the following: injector and transfer line temperatures 250 °C and 240 °C respectively; oven temperature from 60 °C to 240 °C at

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3 °C/min; carrier gas helium at 1 mL/min; splitless injection. The identification of the constituents was based on a comparison of their retention times with those of authentic samples, comparing their linear retention indices relative to the series of n-hydrocarbons, and on computer matching against commercial mass spectra (NIST 98 and ADAMS 95) and those of our library, built up from pure substances and components of known essential oils and MS literature data [346 (Stenhagen et al., 1974); 347 (Massada, 1976); 348 (Jennings and Shibamoto, 1980); 364 (Swigar and Silverstein, 1981); 349 (Davies, 1990); 350 (Adams, 1995)]. Moreover, the molecular weights of all the identified substances were confirmed by GC/CIMS, using MeOH as CI ionizing gas.

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