Biochemical Systematics and Ecology 39 (2011) 228–231

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Biochemical Systematics and Ecology

journal homepage: www.elsevier .com/locate/biochemsyseco

An unusual drimane sesquiterpene glucoside from roots ofPetrorhagia velutina

Antonio Fiorentino*, Brigida D’Abrosca, Monica Scognamiglio, Nikolaos Tsafantakis,Pietro MonacoDipartimento di Scienze della Vita, Laboratorio di Fitochimica, Seconda Università degli Studi di Napoli, via Vivaldi 43, 81100 Caserta, Italy

a r t i c l e i n f o

Article history:Received 20 September 2010Accepted 6 March 2011Available online 29 March 2011

Keywords:Petrorhagia velutinaCaryophyllaceaeDrimane sesquiterpene1D and 2D NMR data

* Corresponding author. Tel./fax: þ39 (0) 823274E-mail addresses: antonio.fiorentino@unina2.it, a

0305-1978/$ – see front matter � 2011 Elsevier Ltddoi:10.1016/j.bse.2011.03.003

1. Subject and source

Specimens of Petrorhagia velutina (Guss.) P. W. Ball. et Heywood (Caryophyllaceae) were collected at “Castel Volturno”Nature Reserve, a flat coastal area located in the north of Naples (Southern Italy). The area is situated on stabilized dunes ofalluvial deposits and loose siliceous-calcareous sand, with a maximum elevation of 9 m above sea level. The climate istypically Mediterranean with precipitation mostly occurring in autumn and winter with a drought period in summer. Thesoil is characterized by homogeneous sand with 97.1% sand, 1.25% loam, 1.6% clay, poor in organic matter and nutrients(Rutigliano et al., 2004). The plants were identified by Dr Assunta Esposito of the Second University of Naples and a voucherspecimen (CE0115) has been deposited at the Herbarium of the Dipartimento di Scienze della Vita of the Second Universityof Naples.

2. Previous work

In a previous work on the aerial parts of P. velutina the isolation of a dimeric p-coumaroyl glucose and monomericp-coumaroyl and feruloyl derivatives was reported (D’Abrosca et al., 2010a). Furthermore, from the apolar leaf extract, a newpheophorbide, as well as other nine chlorophyll derivatives, was isolated and characterized (D’Abrosca et al., 2010b). Finally,from leaf and root methanol extracts of this plant the antiproliferative activity against human hepatoblastoma cancer cell lineHepG2 of eight flavonoid C-glycosides was analyzed (Pacifico et al., 2010).

576.ntonio.fiorentino25@virgilio.it (A. Fiorentino).

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Table 11D and 2D spectroscopic NMR data of drimane sesquiterpene glucoside 1 in pyridine-d5.

d 1H DQF-COSY (H/H) d 13C DEPT HMBC (H/C) NOESY (H/H)

1 3.61 m 2 70.7 CH 2a, 2b, 152a 1.77 m 1, 3, 2b 26.3 CH2 12b 1.90 m 1, 3, 2a 1, 14, 153a 1.30 m 2a, 2b, 3b 29.7 CH2 3b, 53b 2.61 m 2a, 2b, 3a 3a,4 – – 37.6 C – –

5 2.43 t (5.1) 6 38.7 CH 4, 6, 9, 10, 13, 15 6a, 96a 2.01 m 5, 6b, 7 25.1 CH2 5. 76b 2.53 m 5, 6a, 7 77 6.81 m 6, 9 133.6 CH 6a, 6b8 – – 127.4 C – –

9 3.88 m 7, 11proS, 11proR 43.6 CH 5, 11proS10 – – 38.8 C – –

11proS 4.52 dd (9.3, 9.0) 9, 11proR 67.7 CH2 8, 9, 12 911proR 4.09 dd (9.3, 8.7) 9, 11proS 8, 9, 12 1512 – – 169.4 C – –

13 0.98 s – 17.9 CH3 3, 4, 5, 14 2b, 6b, 14a, 14b, 1514a 3.93 d (9.6) 14b 78.2 CH2 10 , 3, 4, 5, 13 10 , 13, 14b14b 3.29 d (9.6) 14a 10 , 3, 4, 5, 13 10 , 13, 14a15 0.74 s – 14.5 CH3 1, 10, 11 1, 2b, 6b, 11proR, 1310 4.84 d (7.8) 20 105.8 CH 20 , 30 , 14a, 14b 30 , 50 , 14a, 14b20 4.04 m 10 , 30 75.4 CH 40

30 4.25 ov 20 , 40 79.1 CH 10 , 50 10

40 4.24 ov 30 , 50 72.0 CH 20

50 4.00 m 40 , 6’a, 6’b 79.0 CH 10 , 60 10 , 60

6’a 4.61 dd (11.7, 2.2) 50 , 6’b 63.3 CH2 50 50

6’b 4.43 dd (11.7, 5.4) 50 , 6’a 50 50

Data acquired at 300 MHz for 1H and 75 MHz for 13C at 25 �C. TMS was used as internal reference; d ¼ doublet; dd ¼ doublet of doublet; m ¼ multiplet;ov ¼ overlapped; s ¼ singlet; t ¼ triplet.

A. Fiorentino et al. / Biochemical Systematics and Ecology 39 (2011) 228–231 229

3. Present study

Fresh plants were transported to the laboratory, separated into aerial parts and roots, dried at 40 �C in a ventilated ovenand stored in paper bag until the extraction process. Dried roots (163 g) were extracted at 4 �C in methanol for three days toobtain a crude extract (9.6 g) which was chromatographed on Amberlite XAD-4 with water and then with methanol. Thealcoholic eluate was chromatographed on Sephadex LH-20 and eluted with pure water and fractions of 20 mL each werecollected. Fraction 4 was chromatographed by RP-18 HPLC with MeOH-H2O (3:1) to give a fraction which, when purified byTLC with the lower phase of CHCl3/MeOH/H2O (13:7:3) solution, gave pure compound 1 (4 mg).

Fig. 1. Diagnostic Nuclear Overhauser Effects evidenced in the aglycone by the NOESY experiment.

A. Fiorentino et al. / Biochemical Systematics and Ecology 39 (2011) 228–231230

Themolecular formula of this newcompoundwas C21H32O9, as suggested by the presence of a pseudomolecular peak atm/z 451.1939 [M þ Na]þ in the positive HR-MS spectrum (calculated 451.1939). The complete structure was elucidated on thebasis of its 1D and 2D NMR spectroscopic features. Its 13C NMR spectrum showed 21 signals identified, on the basis of a DEPTexperiment, as twomethyls, sixmethylenes, ninemethines and four quaternary carbons. The base peak atm/z 267.4543 in theHR-MS spectrum, due to the protonated aglycone, indicated the loss a hexose moiety. Accordingly, the carbon at d 105.8,correlated in the HSQC experiment to the proton at d 4.84, as well as the presence of four methine and a methylene carbinolcarbons, indicated the presence of a sugar moiety whose chemical shift values at d 79.1, 79.0, 75.4, 72.0 and 63.3 were in goodaccordance with those of the glucopyranose. Among the remaining 15 carbons, one carboxyl carbon at d 169.4, two carbinolmethylenes at d 78.2 and 67.7, one carbinol methine at d 70.7, and two olefin carbons at d 127.4 and 133.6, were evident.

The 1H-NMR spectrum showed two methyl singlets at d 0.74 and 0.98, a methine proton geminal to oxygen at d 3.61, twodiastereotopic oxygenated methylenes at d 3.29/3.93 and at d 4.09/4.52, an olefin multiplet at d 6.81, two methines at d 3.88

O

O

OHO

OHHO OH

H

HO

OH1

3

1'6'

9

10

7

1211

1

and 2.43 and three aliphatic methylenes ranging from 2.65 to 1.20 ppm. The proton at d 3.61 showed, in the COSYexperiment,cross peaks with both the methylene protons at d 1.77 and 1.90, which correlated with the protons at d 1.30 and 2.61.Furthermore, the TOCSY experiment allowed a further spin system constituted by the olefinic proton, the remaining meth-ylenes at d 4.09/4.52 and 2.01/2.53 and the methines at d 2.45 and 3.88, to be identified.

The HMBC experiment indicated the correlation between the C-12 carboxyl carbon and the H-11ProS proton at d 4.52whichwas also correlated, together with the H-11ProR proton at d 4.09, with the C-9 methine at d 43.6 and the C-8 and C-10quaternary carbons at d 127.4 and 38.8, respectively. These and all the other information derived from the 2D NMR experi-ments (Table 1) were in good accordance with a glucoside having 1,13-dihydroxy-7-drimene-12,11-lactone as aglycone. Theheterocorrelation, in the HMBC experiment, between the anomeric proton and the C-13 carbon at d 78.2, as well as thatobserved between the H-13 protons and the anomeric carbon allowed the glycosylation site to be defined at the C-13 carbon.Furthermore, the coupling constant value of the anomeric protonwas in good agreement with the presence of a b-anomer forthe glucose moiety. The belonging of the sugar to D-series is tentative from biogenetic considerations.

The NOESY experiment (Fig. 1) enabled the relative configuration at the chiral carbons to be defined. In fact, the NOEbetween H-15 and H-13methyls and those between the H-15methyl with the H-1 proton agreed with an a-orientation of thehydroxyl at C-1 carbon. On the other hand, the NOE evidenced between the H-5 proton and the H-14 and H-9 protons andbetween the H-15 methyl and the �11ProR proton accorded with S and R configurations for the C-5 and C-9 carbons,respectively. These data allowed compound 1 to be identified as (1S,4R,5S,9R,10S) 1,14-dihydroxy-7-drimene-12,11-olide14-O-b-D-glucopyranoside.

4. Chemotaxonomic remarks

P. velutina is an eurimediterranean herbaceous species, belonging to the Caryophyllaceae family, typical of woodland andmacchia vegetation. The genus Petrorhagia includes a few annual and perennial species and, to the best of our knowledge, theonly phytochemical investigations of this genus are by D’Abrosca et al. (2010ab) and Pacifico et al. (2010).

Drimane sesquiterpenoids are restricted in their distribution among plant families and are therefore useful as chemo-systematic markers (Jansen and de Groot, 2004). In fact, they are mainly reported from species belonging to the Canellaceae(Manguro et al., 2003), Winteraceae (Rodríguez et al., 2005), and Apiaceae (Zhou et al., 2000) families. Although the presentwork reports the isolation of a drimane-type sesquiterpene, for the first time, from a species of Caryophyllaceae, differentsesquiterpenes have been reported as flower scent component in Dianthus, Saponaria (Jürgens et al., 2003) and Silene species(Jürgens et al., 2002; Jürgens, 2004).

Acknowledgments

The authors are very grateful to dr. Angela Chambery of Dipartimento di Scienze della Vita (Seconda Università di Napoli)for the ESI Q-TOF HR-MS spectra.

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References

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