Biochemical Systematics and Ecology 37 (2009) 349–353

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Biochemical Systematics and Ecology

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Potential allelopathic effect of neo-clerodane diterpenes from Teucriumchamaedrys (L.) on stenomediterranean and weed cosmopolitan species

Antonio Fiorentino, Brigida D’Abrosca*, Assunta Esposito, Angelina Izzo, Maria TeresaPascarella, Grazia D’Angelo, Pietro MonacoDipartimento di Scienze della Vita, Laboratorio di Fitochimica, Seconda Universita degli Studi di Napoli, Via Vivaldi 43, 81100 Caserta, Italy

a r t i c l e i n f o

Article history:Received 9 March 2009Accepted 15 June 2009

Keywords:Teucrium chamaedrysLamiaceaeNeo-clerodaneDiterpenesAllelopathyMediterranean macchia

* Corresponding author. Tel.: þ39 0823 274564; fE-mail address: brigida.dabrosca@unina2.it (B. D

0305-1978/$ – see front matter � 2009 Elsevier Ltddoi:10.1016/j.bse.2009.06.006

a b s t r a c t

The allelopathic effects of neo-clerodane diterpenes, isolated from Teucrium chamaedrys(L.), have been evaluated on the seed germination and seedling growth of four coexistingMediterranean species (Dactylis hispanica, Petrorhagia velutina, Phleum subulatum andPetrorhagia saxifraga) and two cosmopolitan weeds (Amaranthus retroflexus and Avenafatua). All of the structures have been elucidated on the basis of their spectroscopicfeatures. The bioassays data, analyzed by principal component analysis, showed morenegative effects on weeds respect to coexisting species. Moreover D. hispanica, P. velutina, P.subulatum showed both stimulating or inhibiting effects depending on the type ofmetabolite and the concentration used in the test.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Mediterranean vegetation is one of the world’s biomes with high biodiversity, depending mainly on its environmentalcharacteristics and disturbance history (Naveh, 1989). It is well known that plants, on account of their static condition, to facebiotic and abiotic environmental pressures, synthesize bioactive compounds that can enhance the soil heterogeneity. In fact,these compounds, when released in the environment, can change the soil microbial and chemical components (Castaldi et al.,2009), influencing the neighbour plants and, consequently, the biodiversity level (Bouwmeester et al., 2003). Severalevidences have been recently reported, showing interactions between plants by means the production and release ofsecondary metabolites that can regulate the germination and/or growth of coexisting species (Fernandez et al., 2006). Thisphenomenon is mostly evident in several stenomediterranean species in which the massive production of different secondarymetabolites produces stimulating or inhibiting effects on herbaceous coexisting species (Esposito et al., 2008; Fiorentinoet al., 2008, 2009).

In this context we report the allelopathic potential of neo-clerodane diterpenes from Teucrium chamaedrys, a eur-imediterranean shrub species, typical of woodland and macchia vegetation ranging from 0 to 1500 m a.s.l.

Teucrium (Lamiaceae) is a large widely distributed genus of approximately 260 species of perennial herbs, shrubs, orsubshrub native to Mediterranean region and western Asia. The genus Teucrium is the most abundant source of furanic neo-clerodane diterpenes that have attracted interest on account of their activity against some economically important lep-idopteron, coleopteran and orthopteran pests (Ortego et al., 1995). Several neo-clerodane diterpenes and phenylethanoidglycosides have been isolated from T. chamaedrys (Bedir et al., 2003; Rodriguez et al., 1984).

ax: þ39 0823 274571.’Abrosca).

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A. Fiorentino et al. / Biochemical Systematics and Ecology 37 (2009) 349–353350

In this paper we report the isolation and structural characterization of nine neo-clerodanes, from T. chamaedrys, as well astheir effects on seed germination and seedling growth of four stenomediterranean coexisting species (Dactylis hispanica,Petrorhagia velutina, Petrorhagia saxifraga, and Phleum subulatum) and on two weeds (Amaranthus retroflexus and Avena fatua)frequently founded on disturbed site. The aim of this work is to verify the hypothesis that T. chamaedrys produce secondarymetabolites with selected modulated effects on coexisting and weed species.

2. Material and methods

2.1. Plant material

Plant materials of T. chamaedrys (L.) were collected at vegetative state, in July 2006 at ‘‘Castel Volturno’’ Nature Reserve,a flat coastal area in the north of Naples (Southern Italy). The field samples were transported to the laboratory, separated inbelow and above biomass, dried at 40 �C in a ventilated oven and stored in paper bag until the extraction process. The area islocated on stabilized dunes of alluvial deposits and loose siliceous–calcareous sand, with a maximum elevation of 9 m abovethe sea level. The climate is typically Mediterranean with precipitations mostly occurring in autumn and winter witha drought period in summer. The soil is characterized by homogeneous sand with 97.1% sand, 1.25% loam, 1.6% clay, poor inorganic matter and nutrients (Rutigliano et al., 2004). Seeds of test species P. subulatum, D. hispanica, P. velutina and P. saxifragawere randomly collected in the field in July 2007, and stored in the laboratory at room temperature and darkness. Voucherspecimens (T. chamaedrys CE0037, P. subulatum CE0078, D. hispanica CE0089, P. velutina CE0115, P. saxifraga CE0112) have beendeposited at the Herbarium of the Dipartimento di Scienze della Vita of Second University of Naples. Seeds of the weeds A.retroflexus and A. fatua were furnished by Herbiseed (Reading, UK).

2.2. Phytochemical study

2.2.1. General experiment proceduresThe preparative HPLC apparatus consisted of a pump LC-10AD (Shimadzu, Japan), a refractive index detector (Shimadzu

RID-10A) and a Shimadzu Chromatopac C-R6A recorder. Preparative HPLC was performed using Luna RP-8 10 mm,250�10.0 mm i.d., (Phenomenex Torrance, Canada, CA) and Luna RP-18 10 mm, 250�10.0 mm i.d., (Phenomenex) columns.Analytical TLC was performed on Merck Kieselgel (Darmstadt, Germany) 60 F254 or RP-8 F254 plates with 0.2 mm layerthickness. Spots were visualized by UV light or by spraying with H2SO4�AcOH�H2O (1:20:4). The plates were then heatedfor 5 min at 110 �C. Flash column chromatography (FCC) was performed on Merck Kieselgel 60 (230–400 mesh) at mediumpressure. Column chromatography (CC) was performed on Fluka Amberlite XAD-4. NMR spectra were recorded at 300 MHzfor 1H and 75 MHz for 13C on a Varian Mercury 300 FT-NMR spectrometer Fourier transform NMR, in CD3OD or CDCl3solutions at 25 �C. Proton-detected heteronuclear correlations were measured using HSQC (optimised for 1JHC¼ 140 Hz) andHMBC (optimised for nJHC¼ 8 Hz). Optical rotations were measured on a Perkin-Elmer 343 polarimeter. Electronic ionizationmass spectra (EI-MS) were obtained with a HP 6890 instrument equipped with a MS 5973 N detector.

2.2.2. Isolation of compoundsDried epigeal part of T. chamaedrys (761.3 g) was powdered and extracted by maceration in ethyl acetate (EtOAc, 7 L) at 4 �C

in a dark refrigerated room. After five days the solution was filtered on Whatman paper and concentrated under vacuum,yielding 20.4 g of crude residue, which was stored at�80 �C until purification. The crude EtOAc extract was chromatographedby flash chromatography (FCC) on SiO2, eluting with CHCl3 and collecting fraction of 20 mL. The fractions 7–10 were purifiedon Sephadex LH-20 eluting with hexane/CHCl3/MeOH (2:1:1) to give three fractions A–C.

Fraction A was purified by RP-18 HPLC with H2O/MeOH/CH3CN (4:5:1) to give pure 1 (5.2 mg). Fraction B, re-chromato-graphed by FCC on SiO2 with CHCl3/iPrOH (19:1) solutions, furnished three fractions (B1-B3): B1the first one, resolved by RP-8HPLC with H2O/MeOH/CH3CN (4:5:1) led, to compounds 9 (6.0 mg), 4 (4.6 mg) and 1 (3.0 mg); B2 the second fractionsimilarly purified by RP-8 HPLC using as mobile phase H2O/MeOH/CH3CN (3:6:1) furnished pure metabolites 7 (4.6 mg) and 4(3.0 mg); B3 the third fraction was resolved by RP-8 HPLC with H2O/MeOH/CH3CN (11:7:2) to give pure compounds 5 (4.6 mg)and 8 (2.0 mg). Finally, fraction C was purified by RP-8 HPLC using as mobile phase H2O/MeOH/CH3CN (3:6:1) to have purecompounds 3 (5.0 mg), 7 (23.0 mg) and 2 (15.0 mg).

Dried T. chamaedrys roots (300.2 g) were powdered and extracted in MeOH (2.4 L) for 5 days at 4 �C in a dark refrigeratedroom. After the removal of the solvents by Rotavapor�, the crude extract was dissolved in water and shaken in a separatorfunnel using EtOAc as extracting solvent. The aqueous fraction was chromatographed on Amberlite XAD-4 eluting first withH2O and then with MeOH. The MeOH eluate, was chromatographed by FCC on SiO2 eluting with the organic lower phase ofa CHCl3/MeOH/H2O (13:9:5) solution, to obtain a fraction which was purified by RP-18 HPLC using as mobile phase H2O/MeOH/CH3CN (4:5:1) to give pure metabolite 6 (3.5 mg).

2.3. Phytotoxicity test

A preliminary phase has been carried out to select seeds for uniformity. To this end a considerable number of seeds wereobserved under a binocular microscope to discard all undersized or damaged ones. A preliminary test to evaluate the

A. Fiorentino et al. / Biochemical Systematics and Ecology 37 (2009) 349–353 351

germinability of the three selected species was carried out on these seeds in a growth chamber KBW Binder 240 at 27 �C in thedark. The observed percentage of germinability for P. subulatum, D. hispanica, P. velutina and P. saxifraga was of 98.7%, 88.7%,98.3% and 95.8%, respectively. The bioassay experimental design included the use of three blocks of 100 seeds for each speciesand each treatment was conducted in ten replicates. Ten seeds of each species were poured onto a sheet of Whatman No 1filter paper in a Petri dish (50 mm of diameter). For each metabolite bioassay, three different concentrations have been tested:10�4 M, 10�6 M and 10�8 M pure metabolite solutions (defined as High, Medium and Low, respectively). The test solution ofpure metabolites (10�4 M) was prepared using (2-[N-morpholino]ethanesulfonic acid (MES; 10 mM, pH 6), while the others(10�6–10�8 M) were obtained by dilution. The metabolites were dissolved in DMSO (5 mL for each mL of MES). For each puremetabolite bioassay, parallel controls were carried out. After adding 10 seeds and 1.0 mL test solution, the Petri dishes weresealed with Parafilm� to ensure closed-system models. The dishes were placed in a growth chamber KBW Binder 240 at 27 �Cin the dark. Four days later (no more germination occurred after this time), germination percentage was analyzed. Succes-sively, seedlings were frozen at –20 �C to avoid subsequent growth until the root and shoot elongation measurement.Germination rate, root and shoot length were recorded by using a Fitomed� system (Castellano et al., 2001), that allowedautomatic data acquisition and statistical analysis by its associated software. Data are reported as percentage differences withrespect to control. Therefore, zero represents the control, positive values show stimulation while negative values representinhibition of the studied parameters.

The statistical significance of differences between groups was determined by Student’s t-test, calculating mean values forevery parameter (germination average, shoot and root elongation) and their population variance within a Petri dish. The levelof significance was set at P< 0.05 (Castellano et al., 2001).

Moreover a numerical clustering of data bioassay was made on the basis of percentage differences from control. Anaverage linkage agglomeration criterion and the Euclidean distance as dissimilarity coefficient were applied by the software‘‘SYN-TAX’’ version 2000 for Windows (Podani, 2000).

3. Results and discussion

The phytochemical investigation of Teucrium chamaedrys leaf and root extracts led to the isolation and spectroscopicelucidation of nine neo-clerodane diterpenes 1–9 (Fig. 1).

All off the isolated neo-clerodane, with the exception of compound 8, have been previously reported as T. chamaedrysmetabolites. Compound 1 was isolated from this plant for the first time by Reinbol’d and Popa (1974) and was also reported asconstituent of Teucrium scordium extract (Jakupovic et al., 1985). Compounds 2, 3, 4, 7, and 9 named, respectively, teucrin G,chamaedroxide, teucrin F, teucrin A and teuflidin were already isolated from T. chamaedrys (Rodriguez et al., 1984; Egurenet al., 1982; Savona et al., 1982). NMR data of compound 5 were in good accordance with those reported by Bedir et al. (2003).

Compound 6, named teuflin, identified in the aerial part of T. chamaedrys (Fernandez-Gadea et al., 1983), was isolated forthe first time from Teucrium viscidum var miquelianum (Node et al., 1981). Compound 8, named 2b-hydroxyteucvidin hasbeen isolated from plants of Teucrium webbianum (Savona et al., 1986).

O

OOO

HO

O

OH

O

R

O

OR3

OO

R2

R=R1=H R2=H R3= HR=R1=H R2=OH R3= HR=OH R1=R2=HR3= HR=R2=H R1=OH R3= H

O

OO

O

OH

HOOH

O

OO

O

OH

OH

O

OO

O

OH

HOOH

R1

O

O

O

OH

OO OH

6

7

8

9

1 2 3

4

5

Fig. 1. Chemical structures of compounds 1–9.

-50

-25

0

25

5 7 2 4 3 1 9 6

%F

RO

M C

ON

TR

OL

Germination

-0,5

-0,25

0

0,25

0,5

-0,5 -0,25 0 0,25 0,5

7

5

2

3

1

4

6

9

-60

-40

-20

0

20

40

60

7 5 2 3 4 1 6 9

-40

-15

10

35

D. hispanica P. subulatum P. velutina

P. saxifraga A. retroflexus A. fatua

COMPOUNDS

7 5 2 6 9 3 1 4

Root elongation

-0,4

-0,2

0

0,2

0,4

-0,4 -0,2 0 0,2 0,4

-- A

XIS

F

2 -->

7

5

2

3

4

6

1

9

Shoot elongation

-0,6

-0,3

0

0,3

0,6

-0,6 -0,3 0 0,3 0,6

-- AXIS F1 -->

7

5

2

6

9

1

3

4

High concentration Medium concentration Low concentration

High concentration Medium concentration Low concentration

A B

Fig. 2. Bioassay results of the effects of neo-clerodane diterpenes from T. chamaedrys on coexisting and weed species. (A) Principal component analyses of thebioassay data of germination, root and shoot elongation; (B) effects of the metabolites on germination, root elongation, and shoot elongation of test speciesreported in accordance with the PCAs of Fig. 2A. H¼ 10�4 M, M¼ 10�6 M, L¼ 10�8 M.

A. Fiorentino et al. / Biochemical Systematics and Ecology 37 (2009) 349–353352

A. Fiorentino et al. / Biochemical Systematics and Ecology 37 (2009) 349–353 353

All of the compounds, with the exception of metabolite 8, have been tested on four coexisting herbs, D. hispanica, P.subulatum, P. velutina and P. saxifraga, and on two weeds A. retroflexus and A. fatua.

To evaluate the effects at three different concentration levels (high, medium, low) of the eight pure compounds on thetested species, the data matrix of 24 bioassay values (mean germination and root and shoot elongation percentage differencesfrom control)� 6 species was analyzed by Principal Component Analysis from the package ‘‘SYN-TAX’’ version 2000 forWindows (Podani, 2000). The choice of PCA as an ordination method was based on its recognized usefulness as interpretationtool when applied to short compositional gradient. The ordination of the effects of the three concentration levels of the eightpure compounds on the germination and shoot and root elongation on tested species, according to the first and secondprincipal component is shown in Fig. 2A. A clear gradient could be detected along the first axis where the most phytotoxiccompounds had negative scores, whereas the less phytotoxic compounds were progressively recognized towards positivescores. Moreover a separation, according to the effects of the concentration levels, was evident on the second axis with stronginhibition effects due to the highest concentration of terpenoids grouped towards negative scores and prevailing stimulatingeffects distributed on the positive scores. In particular a similar pattern was observed for germination and root elongationwith compounds 7, 5, and 2 that are the most phytotoxic compounds at all the tested concentrations; compounds 3, 4 and 1showed high toxicity level at the highest concentration and a wide differentiation of effects between the three level ofconcentrations; compounds 6 and 9 showed the less phytotoxic tested metabolites. The bioassay results on shoot elongationevidenced a slightly similar pattern, confirming compounds 7 and 5 as the most phytotoxic, but compound 4 showed thelowest phytotoxic effects. The pattern of phytotoxicity, in relation to the responses of each species and to the concentrationlevels, is evidenced along the phytotoxicity gradient produced by PCA as reported in Fig. 2A. It is clearly evidenced that thetested species showed different behaviors in relation to the type of compound and to the levels of concentration used in thetests. These results confirm our hypothesis that T. chamaedrys produce a wide range of metabolites with different chemicalproprieties responsible of the observed modulating effects.

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