Flavonoids isolated from Medicago littoralis Rhode - Caryologia

CARYOLOGIA

Vol. 63, no. 1: 106-114, 2010

Flavonoids isolated from Medicago littoralis Rhode (Fabaceae): their ecological and chemosystematic significance Bertoli1 Alessandra, Daniela Ciccarelli2*, Fabio Garbari2 and Luisa Pistelli1 1

Alessandra Bertoli, Luisa Pistelli, Department of Bioorganic Chemistry and Biopharmaceutics, University of Pisa , via Bonanno 33, I-56126 Pisa, Italy 2 Daniela Ciccarelli, Fabio Garbari, Department of Biology, University of Pisa, via Luca Ghini 5, I-56126 Pisa, Italy

Dedicated to the memory of Prof. Ivano Morelli.

Abstract — Three flavonoids, laricitrin 3,5’-di-O-β-glucopyranoside (1), genistein 7-O-β-glucopyranoside (2), and biochanin A 7-O-β-glucopyranoside (= sissotrin) (3), were isolated for the first time from the aerial parts of Medicago littoralis Rhode. The structures of the isolated compounds were established by NMR and LCDAD-ESI-MS analyses. The chemosystematic of flavones and isoflavones identified was useful for discriminating different taxa inside the section Spirocarpos. In addition, the phytochemical profile of M. littoralis showed a behaviour due to the ecological conditions of sandy dunes, where this plant normally grows. Key words: Chemosystematic, Fabaceae, Flavones, Flavonoids, Isoflavones, Medicago.

INTRODUCTION The genus Medicago L. (Fabaceae) belongs to the subfamily Papilionoideae, tribe Trifolieae. It consists of 50 species of annual or perennial herbs, rarely shrubs, of which Medicago sativa L. (alfalfa) is well-known since it is used as a forage crop widely. Several taxonomical analyses have been carried out to establish which characters could be useful to delimit the genus, expecially regarding the generic circumscription of Medicago and Trigonella L., both belonging to the tribe Trifolieae (see SMALL et al. 1987). Investigations involving pollen morphology (SMALL 1981), floral characters (SMALL et al. 1981a), asymmetries in Trifolieae leaves (SMALL et al. 1981b), haemolytic

*Corresponding author: phone: +39(0)502211327; fax: +39(0)502211309; e-mail: [email protected]

saponins in the seeds (JURZYSTA et al. 1992), phenolic variation (CLASSEN et al. 1982; SALEH et al. 1982), stigma morphology (SMALL and BROOKES 1983), pollen-ovule pattern (SMALL 1986) or seed characters (SMALL and BROOKES 1990; DJEMEL et al. 2005) were performed in order to solve these taxonomical problems. Recently, molecular phylogeny analyses (BENA et al. 1998; BENA 2001) supported the transfer of 23 Trigonella species previously known as “medicagoids” to the genus Medicago, as proposed by SMALL et al. (1987) on the base of morphological data. The infrageneric classification of Medicago is reported in the Table 1 following mainly the classifications of LESINS and LESINS (1979), SMALL and JOMPHE (1989), SMALL (1990a-b), GILLESPIE and MCCOMB (1991). In the same table is also reported the geographical distribution of each species obtained from the main Floras (TUTIN 1968; HEYN and DAVIS 1970; PIGNATTI 1982; GREUTER et al. 1989; SALES and HEDGE 2000; GRIN Taxonomy for plants, 2006) in order to compare these taxa from an ecological point of view.

RUNNING TITLE : FLAVONOIDS IN MEDICAGO LITTORALIS RHODE

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TABLE 1 — The infrageneric classification and the geographical distribution of Medicago L. from the Mediterranean region, Middle East and Asia. Evidenced in bold the species living on maritime sands or extending their area of occupancy up to the seashore. Section

Subsection Species

Geographical distribution

Dendrotelis

M. arborea L. M. strasseri Greuter, Matthäs & Risse

Rocky places. Mediterranean region, Middle East and Asia. Rocky places. Europe.

Medicago

M. cancellata M. Bieb. M. daghestanica Rupr. M. hybrida Trautv. M. marina L. M. papillosa Boiss. M. pironae Vis. M. prostrata Jacq. M. rhodopaea Velen. M. rupestris M. Bieb. M. sativa L. M. saxatilis M. Bieb. M. suffruticosa Raymond

Steppe. Russian Federation. Steppe. Russian Federation. Woods and mountain slopes. Corbiéres and Pyrenees. Maritime sands. Shores of the Mediterranean, Black Sea and Atlantic. Middle East and Asia. Rocky places. Italy and Balcans. Dry grassland. From Italy and Austria to the Black Sea. Rocky places. Bulgaria. Rocky places. Krym. Cosmopolitan. Rocky places. Bulgaria and Krym. Rocky places. Pyrenees and Morocco.

Carstienses

M. carstiensis Jacq.

Bushy places. From Italy to Bulgaria.

Spirocarpos Pachyspirae M. constricta Durieu Grasslands. Europe and Asia. M. doliata Carmign. Dry lands. Mediterranean region and Middle East. M. italica Mill. (= M. tornata (L.) Miller) Dry grasslands. Mediterranean region extending to Portugal. M. lesinsii E. Small Mediterranean region. M. littoralis Rohde Mediterranean region extending to Portugal and W. France. M. murex Willd Mediterranean region extending to Portugal. M. rigidula L. Dry grasslands. South Europe. M. rigiduloides E. Small Middle East, Caucasus and Asia. M. sinskiae Uljanova Asia. M. soleirolii Duby Dry grasslands. Krym, probably introduced in Italy and France. M. syriaca E. Small Syria. M. truncatula Gaertn. Mediterranean region. M. turbinata L. Mediterranean region. Rotatae M. blancheana Boiss. Mediterranean region. M. noeana Boiss. Iraq and Turkey. M. rotata Boiss. Eastern Mediterranean region. M. rugosa Desr. Mediterranean region living also on maritime sands. M. scutellata L. Grasslands. South Europe. M. shepardii Post Turkey. Intertextae M. ciliaris L. Mediterranean region. M. granadensis Willd. Mediterranean region. M. intertexta L. Grasslands, expecially near the sea. Mediterranean region and Portugal. M. muricoleptis Tineo Grasslands, expecially near the sea. Mediterranean region. Leptospirae M. arabica L. Cosmopolitan. Mediterranean region. M. coronata L. Stenomediterranean. M. disciformis DC. Stenomediterranean. M. laciniata L. Naturalized locally in the Mediterranean region. M. lanigera C. Winkl. Afghanistan, Tajikistan and Turkmenistan. M. laxispira Heyn Iraq. M. minima L. Living on sandy dunes. Most of Europe. M. polymorpha L. Subcosmopolitan. South Europe. M. praecox DC. Dry lands and garrigues. Mediterranean region. M. sauvagei Nègre Morocco. M. tenoreana Ser. Mediterranean region.

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BERTOLI , CICCARELLI , GARBARI

and PISTELLI

Table 1 Contd. Section

Species

Geographical distribution

Geocarpa

M. hypogaea E. Small

Eastern Mediterranean region.

Lupularia

M. lupulina L. M. secundiflora Durieu

Throughout Europe. Mediterranean region.

Heynianae

M. heyniana Greuter

Europe and Middle East.

Orbiculares

M. orbicularis L.

South Europe.

Hymenocarpos

M. radiata L.

Afghanistan, Iran, Iraq, Israel, Jordan, Lebanon, Syria, Turkey and Caucasus.

Platycarpae

M. archiducis-nicolai Širj. M. cretacea M. Bieb. M. edgeworthii Širj. M. ovalis Širj. M. platycarpa L. M. plicata Širj. M. popovii Širj. M. ruthenica (L.) Ledebour

China. Ukraine, Krym and Asia. Asia. Spain, Algeria and Morocco. Russian Federation and Asia. Turkey. Soviet Middle Asia. Russian Federation and Asia.

Lunatae

M. biflora E. Small M. brachycarpa M. Bieb. M. huberi E. Small M. rostrata E. Small

Turkey and Caucasus. Iraq, Lebanon, Turkey and Caucasus. Turkey. Turkey.

M. arenicola E. Small M. astroites Trautv. M. carica E. Small M. crassipes E. Small M. fischeriana Trautv. M. halophila E. Small M. heldreichii E. Small M. medicaginoides E. Small M. monantha Trautv.

Turkey. Iran, Iraq, Israel, Jordan, Lebanon, Syria, Turkey, Caucasus and Soviet Middle Asia. East Aegean islands and Asiatic Turkey. Iran, Iraq, Lebanon, Syria and Turkey. Bulgaria. Turkey. Turkey. Europe, Middle East and Asia. Afghanistan, Iran, Iraq, Israel, Jordan, Lebanon, Syria, Turkey, Caucasus and Soviet Middle Asia. Iran, Iraq, Turkey, Caucasus and Soviet Middle Asia. Turkey. Iran. Iran, Iraq, Syria and Turkey. France, Portugal, Spain, Algeria, Morocco and Tunisia. Turkey. Afghanistan. Mediterranean region. Turkey. Turkey.

Buceras

Subsection

Erectae

Deflexae Reflexae Isthmocarpae

M. orthoceras Trautv. M. pamphylica E. Small M. persica E. Small M. phrygia E. Small M. polyceratia Trautv. M. rigida E. Small M. retrorsa E. Small M. monspeliaca Trautv. M. isthmocarpa E. Small M. rhytidiocarpa E. Small

The present report represents the first result of a phytochemical study of Italian populations of Medicago littoralis Rhode (strand medic). This species is a therophyte growing on sandy soils near the sea and distributed in the Mediterranean region extending to Portugal and West France. Whole plant is covered by hair;

the leaves are ternate with obovate or cordate, cuneate, dentate towards the apex leaflets. The yellow flowers are grouped in a terminal raceme; flowering in March-May. The legumes are in a very close spiral of 3-6 turns, discoid to cilindrical, glabrous, spiny or not (TUTIN 1968). There are few photochemical studies on the

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TABLE 2 — Distribution of flavonoids in the genus Medicago. The species that living on maritime sands or extending their area up to the seashore are evidenced in bold. Species Sec. Dendrotelis M. arborea a,b Sec. Medicago M. cancellata a,b M. hybrida a,b M. marina a,b M. sativa a,b,d,e,f Sec. Spirocarpos Subsec. Pachyspirae M. doliata a,b M. littoralis a,b M. murex a,b M. rigidula a,b M. truncatula a,b,c M. turbinata a,b Subsec. Rotatae M. blancheana a,b M. rotata a,b M. scutellata a,b Subsec. Intertextae M. ciliaris a,b M. intertexta a,b Subsec. Leptospirae M. arabica a,b M. coronata a,b M. laciniata a,b M. minima a,b M. polymorpha a,b Sec. Geocarpa M. hypogaea a,b Sec. Lupularia M. lupulina a,b Sec. Orbiculares M. orbicularis a,b Sec. Hymenocarpos M. radiata a,b Sec. Platycarpae M. cretacea a,b M. platycarpa a,b Sec. Buceras Subsec. Erectae M. medicaginoides a,b M. monantha a,b M. polyceratia a,b Subsec. Reflexae M. monspeliaca a,b

Flavonols

Isoflavones

Flavones

x

Isoflavans

Pterocarpans Coumestans

x

x

x x x

x x x

x x

x x

x

x

x

x

x x

x x

x

x x

x x

x

x

x x

x x

x

x

x x x x x x x

x x

x

x x x x x

x x

x x x x x

x x x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x x x x

x

x

Phytochemical data source: a = Southon (1994); b = Hegnauer and Hegnauer (2001); c = Kowalska et al. (2007); d = Stochmal et al. (2001a); e = Stochmal et al. (2001b); f = Stochmal et al. (2001c).

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(1) Laricitrin 3,5 -O-β-diglucopyranoside

(2) Genistein 7-O-β-glucopyranoside R=H (3) Biochanin A7-O-β-glucopyranoside R=Me Figure 1 — Chemical structures of the flavonoids (1-3) isolated from the aerial parts of Medicago littoralis.

aerial green parts of M. littoralis collected out of Italy (INGHAM 1979; SALEH et al. 1982), which show the presence of several typical compounds such as daidzein, luteolin, medicarpin, sativan, 3’,4’,7-trihydroxyisoflavone and vestitol. Previous phytochemical studies on the genus Medicago showed the presence of flavones, flavonols, isoflavones, isoflavans, coumestans and pterocarpans (SOUTHON 1994; HEGNAUER and HEGNAUER 2001; STOCHMAL et al. 2001a-c; KOWALSKA et al. 2007). The aim of this paper was the isolation and the characterization of some typical flavonoids of M. littoralis aerial parts collected in sandy dunes to suggest chemosystematic considerations. MATERIALS AND METHODS Plant material - The plant grows on the back of sandy dunes of Marina di Vecchiano (Pisa, Italy) within the Natural Park of Migliarino - San Rossore - Massacciuccoli. Aerial parts of Medicago littoralis were randomly collected from the same population during the flowering phase at the end of April 2004. A voucher specimen (3688/4)

and PISTELLI

was deposited in the Herbarium Horti Botanici Pisani (PI), Italy. Chemicals and equipments - Acetonitrile, HCOOH and methanol were HPLC grade solvents by Baker (Netherlands). HPLC-Water was purified by a Milli-Q Plus system (Millipore Milford, MA, USA). Reference chemical material including luteolin, daidzein, and the isolated compounds (laricitrin 3,5’-di-O-β-glucopyranoside, genistein 7-O-β-glucopyranoside and biochanin A 7-O- β-glucopyranoside) were part of an home-made database where each compound was used as standard with an HPLC grade purity of 98-99%. The analysis were performed by a Surveyor Thermofinnigan liquid chromatograph pump equipped with a Thermofinnigan Photodiode Array Detector and a LCQ Advantage mass detector. The semipreparative HPLC system consisted of a Waters 600E (Millipore) pump and 990 photodiode array detector (PDA) and a column Merck LiChroCART 250RP-18 column (30 x 1 cm, 10 µm) was used. Extraction and LC-DAD-MS screening - The aerial parts (184 g) of Medicago littoralis, air-dried at room temperature and powdered, were extracted in a Soxhlet apparatus with n-hexane, CHCl3 and MeOH in turn. After removal of solvent under vacuum, the following residues were obtained: RH (5.49 g), RC (6.53 g), and RM (25.81 g). RM (3.73 g) was chromatographed on a Merck Lichroprep RP-8 column (40-63 mm) eluted with MeOH-H2O (7:3) to obtain 19 crude fractions (M1-M19). Fractions were checked by TLC [RP-18, MeOH-H2O (7:3)], Merck Kieselgel 60 F254. TLC chromatograms were visualized under UV light at 254 and 366 nm and sprayed with Naturstoffreagents-PEG. The most interesting purified fractions were screened by LC-DADESI-MS using an analytical Lichrosorb RP-18 column (250 x 4.6 mm i.d., 5μm, Merck). The analyses (20 μl, 2 mg/ml methanolic extract solution) were carried out by a linear gradient using water with 0.1% HCOOH (solvent A), CHCN (solvent B) and MeOH (solvent C) at flow 3 rate of 1.0 ml/min in the following conditions: from 20:80 v/v (B-A) to 40:60 (B-A) in 20 min, then to 40:60 v/v (B-A) (20 min), and then conditioning to the initial condition (20:80 v/v B-A) for 10 min. The total analytical run time was 50 min for each sample. The spectral data from the PDA detector were collected during the whole run in the range 210-500 nm and the peaks were detected at 330 nm for all analysed compounds. LC-ESI-MS analyses were performed in the same chromatographic conditions using these specific


electrospray ionization values: sheath gas flowrate 72 psi, auxilary gas flow 10 psi, capillary voltage -16 V and capillary temperature 280°C . Full scan spectra from m/z 200 to 500 amu in the negative ion mode were obtained. The identification of each constituents was carried out by the comparison of each peak in the extracts with the retention times, UV and MS spectra of an home-made database of flavonoid compounds. Isolation and purification of flavonoids - The filtered solution of fraction M2 (1 g) was subjected to column chromatography over a Merck Lichroprep RP-8 column (40-63 µm) eluted with MeOH-H2O (3:7) to obtain 11 subfractions (E1-E11). Subfraction E7 (30 mg) was purified by semipreparative HPLC-PDA. The gradient profiles were based on two solvents, denoted A (CH3CN) and B (H2O), were employed. Initial conditions were 20% A, 80% B with a linear gradient reaching 40% A, 60% B; this was followed by isocratic elution, after which the programme returned to the initial solvent composition (t = 40 min). Column was mantained at room temperature, and the flow rate was 1.3 ml/min. Injection volumes were 100 μl, and UV detection was at 330 nm. This semipreparative HPLC/UV gave 4 subfractions (E7.1-E7.4). Fraction M4 was subjected to column chromatography under the same conditions of fraction M2 to obtain 2 subfractions (M4.1-M4.2). The nuclear magnetic resonance (NMR) spectra of the isolated flavonoids were registered on a Bruker AC-200 spectrometer and a Bruker Avance 400 spectrometer using CD3OD or DMSO-d6 as solvent. TMS was used as internal standard. All the 1D and 2D NMR experiments were carried on using the standard Bruker library of microprograms. MS spectra of the isolated compounds were registered by direct infusion in LCQ Advantage ion trap mass spectrometer using a methanolic solution (1 μg/ ml, 5 μl/min) with the following ESI parameters: sheath gas flow-rate 72 arbitrary units, auxilary gas flow 10 arbitrary units, capillary voltage -16 V and capillary temperature 280°C. RESULTS AND DISCUSSION The preliminary screening by LC-DAD-ESIMS analyses of Medicago littoralis (aerial parts) collected during the flowering phase on the sandy dunes of Marina di Vecchiano (Pisa, Italy) showed the presence of some characteristic fla-

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vonoids such as lutein and daidzein previously reported in the literature (SOUTHON 1994; HEGNAUER and HEGNAUER 2001). However, our phytochemical investigation on this species showed also laricitrin 3,5’-diO-β-glucopyranoside (1), genistein 7-O-βglucopyranoside (2), and biochanin A 7-O-β -glucopyranoside (= sissotrin) (3) as characteristic compounds (Fig. 1). These flavonoids were isolated for the first time in Medicago littoralis. After the extraction, the methanolic extract was chromatographed by semi-preparative HPLC to isolate the constituents. Subfractions E7.2 (2 mg) and E7.4 (2 mg) gave pure compounds (1) and (2), respectively. Compound (3) was identified as pure constituent of subfraction M4.1 (2.8 mg). The structural elucidation of the isolated compounds were performed on the basis of NMR and ESI-MS experiments. The signals in the 1H and 13C NMR spectra of the isolates were superimposable on those reported in the literature for laricitrin 3,5’-di-O-βglucopyranoside (1) (TORCK et al. 1983; KOWALSKA et al. 2007), genistein 7-O-β-glucopyranoside (2) (AGRAVAL 1989), and biochanin A 7-O-βglucopyranoside (= sissotrin) (3) (GUGGOLZ et al. 1961). Except for laricitrin 3,5’-di-O-β-glucopyranoside, isolated in the floral parts of M. arborea L. (TORCK et al. 1983), M. lupulina L. (BURDA and JURZYSTA 1988) and M. truncatula Gaertn. (KOWALSKA et al. 2007), the other flavonoids had never been recorded in the genus Medicago. Previous phytochemical studies showed the presence of luteolin, daidzein and 3’,4’,7-trihydroxyisoflavone in the hydrolized plant of M. littoralis (SALEH et al. 1982), while sativan, vestitol and medicarpin were isolated from the leaves of the same species (INGHAM 1979). Flavonoids are well accepted as chemical markers in plant taxonomy as a useful tool for the characterization and classification of higher plants, because of their widespread occurrence and chemical stability (HARBORNE and TURNER 1984; HEGNAUER and HEGNAUER 2001). Regarding a significant connection between the distribution of secondary metabolites and the infrageneric classification of Medicago (Table 2), it seems clear that flavones and isoflavones may be particularly useful for discriminating different taxa inside the section Spirocarpos Ser. Isoflavones, in fact, occur in the subsections Intertextae (Urb.) Heyn and Leptospirae (Urb.) Heyn, but they are absent in Pachyspirae (Urb.) Heyn (except for M. littoralis). In the same way as re-

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TABLE 3 — Flavonoids from M. littoralis and their occurrence in the genus Medicago. The species that living on maritime sands or extending their area up to the seashore are evidenced in bold. Species Sec. Dendrotelis M. arborea Sec. Medicago M. cancellata M. hybrida M. marina M. sativa Sec. Spirocarpos Subsec. Pachyspirae M. doliata M. littoralis M. murex M. rigidula M. truncatula M. turbinata Subsec. Rotatae M. blancheana M. rotata M. scutellata Subsec. Intertextae M. ciliaris M. intertexta Subsec. Leptospirae M. arabica M. coronata M. laciniata M. minima M. polymorpha Sec. Geocarpa M. hypogaea Sec. Lupularia M. lupulina Sec. Orbiculares M. orbicularis Sec. Hymenocarpos M. radiata Sec. Platycarpae M. cretacea M. platycarpa Sec. Buceras Subsec. Erectae M. medicaginoides M. monantha M. polyceratia Subsec. Reflexae M. monspeliaca

Flavonols 1

2

Isoflavones 3 4

Flavones 5 6

Isoflavans 7 8

x

x

x

x

Pterocarpans 9

x

x

x x

x

x x

x

x

x

x

x

x

x x

x x

x x

x

x x

x x

x

x x x

x x x x x x

x x

x x

x x

x

x

x x x

x

x

x

x

x x

x

x

x

x

x

x

x

x

x

x

x

x

1 = Laricitrin 3,5’-O-glc; 2 = Genistein 7-O-glc; 3 = Sissotrin; 4 = Daidzein; 5 = 3’,4’,7-trihydroxyisoflavone; 6 = Luteolin; 7 = Sativan; 8 = Vestitol; 9 = Medicarpin.


gards flavones, it is interesting that these compounds occur in the subsection Leptospirae, but they are absent in Pachyspirae except again for M. littoralis. Our results are partially in accordance with molecular phylogenetic studies (BENA et al. 1998) and biochemical analyses of seed composition that show M. truncatula and M. littoralis as very closely related species (DJEMEL et al. 2005). M. truncatula, in fact, shows the same phytochemical profile of M. littoralis except for isoflavones (KOWALSKA et al. 2007). Moreover, this study was carried out in order to find relationships between the phytochemical profile of M. littoralis and the ecological conditions of sandy dunes, where the plants grows. M. littoralis is able to synthesise flavonols, isoflavones, flavones, isoflavans and pterocarpans (SOUTHON 1994; HEGNAUER and HEGNAUER 2001). This behaviour is also found in M. sativa, M. arabica, M. orbicularis and M. radiata (Table 2), which belong to different sections or subsections and live in different habitats. Anyway, if we consider the specific flavonoid and not the chemical class (see Table 3), the mentioned species produce different flavonoids. All the plants that live on maritime sands were able to synthesize flavonols (except for M. marina L., M. coronata L. and M. minima L.) and most of them produced isoflavones and flavones depending on the subsection considered. On the other hand, considering flavonoids isolated from M. littoralis samples and their occurrence in the genus Medicago (see Table 2-3), a so large variety in the flavonoidic composition has never been observed for plants living near the sea yet. In conclusion, this study showed laricitrin 3,5’-di-O-β-glucopyranoside (1), genistein 7-O-β-glucopyranoside (2), and sissotrin (3) as characteristic flavonoids of the aerial parts of M. littoralis collected on the sandy dunes during the flowering period. Therefore, the list of chemotaxonomic markers of M. littoralis can be added by these flavonoids (1-3). To evaluate more deeply the flavonoidic production in this species, the authors plan to study its phytochemical profile also during seasonal and growth-phase variations. REFERENCES AGRAVAL P.K., 1989 — Carbon-13 NMR of Flavonoids. Elsevier, Amsterdam. BENA G., 2001 — Molecular Phylogeny supports the morphologically based taxonomic transfer of the “medicagoid” Trigonella species to the genus Medi-

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Published May 30th 2010 Editore: Università degli Studi di Firenze Registrazione Tribunale di Firenze n. 478 del 13/7/1951 Redazione: Dipartimento di Biologia Vegetale Via La Pira, 4 - 50121 FIRENZE Direttore Responsabile: Dr. ALESSIO PAPINI Stampato a Firenze da Edizioni Tassinari - Firenze - May 2010