A new ferulol derivative isolated from the aerial parts of Ferulago nodosa (L.) Boiss. growing in Sicily (Italy)

Abstract Ferulago nodosa (L.) Boiss. (Apiaceae) is a species occurring in the Balkan-Tyrrhenian area being present in Crete, Greece, Albania, and probably in Macedonia. Although the western disjointed population of Sicily has been classified as an endemic sub-species, F. nodosa subsp. geniculata (Guss.) Troia & Raimondo, it is not officially accepted. From the aerial parts of the Sicilian accession of this species four known metabolites (1–4), and a new ferulol derivative (5), were isolated and characterized. The structure of the new compounds was determined by mean of extensive NMR spectroscopic experiments. Graphical Abstract


Introduction
The genus Ferulago W.D.J. Koch (Apiaceae) includes about 50 taxa mainly growing in the Mediterranean area, from Portugal to Iran, and from Russia to north-western Africa belonging to the Apiaceae family. Recently, a comprehensive review of Ferulago genus including traditional uses, chemical composition of volatile and non-volatile metabolites, and in vitro and in vivo biological properties, have been published . Furthermore, the chemical composition, hypoglycemic, and antilipidemic effects of the essential oil of the aerial parts of F. nodosa have been reported .

Results and discussion
Powdered dry aerial parts (600 g) were extracted with acetone. The extract was chromatographed over silica gel column using a gradient solvent system (P.Et.-AcOEt). Further purification of the main fractions yielded compounds 1-5 ( Figure 1). Compound 1 was identified as 2,3,4-trimethylbenzaldehyde (Kubeczka and Ullmann 1981;Ruberto et al. 1999;Maggio et al. 2013) and its presence can be justified, as previously reported (Riela et al. 2011), by the loss of the ester moiety of the ferulol derivatives co-occurring in the same plant and subsequent rearrangement.
The ferulol ester 4, isolated in significant amount, was previously identified, and characterised in Ferulago nodosa (Ruberto et al. 1999). Its spectroscopical data agreed with dose reported in literature (Ruberto et al. 1999). The mass spectrum of the compound 5 indicated a molecular formula of C 20 H 26 O 6 with eight degrees of unsaturations. The 1 H and 13 C NMR spectra showed signals for two a,b-unsatured ester groups ( Hz, H-4) mutually coupling with a diagnostic coupling constant (J ¼ 5.85 Hz), and a carboxyl carbonyl at d C7 ¼ 171.43 ppm. The 2 D-NMR experiments ( 1 H-1 H COSY, HMBC, and HSQC) also clarified the exact skeleton, and the assignment to the individual protons and carbons. The connection between ester moiety, and the ring portion, was mainly based on the HMBC correlations. In fact, H 1 (d H1 ¼ 5.24) showed a correlation spot with C-1 0 (d C ¼ 166.19). Furthermore, the HMBC correlations between H-4 (d H4 ¼ 7.03), and the carbon C-7 (d C7 ¼ 171.43), indicated that the carboxylic group bound to carbon 5. Consequently, we assumed that our compound was an acid derivate of ferulol ester, not previously described in the literature.

General experimental procedures
Column chromatography was performed using silica gel (70-230 mesh ASTM, Merck No. 7734) deactivated with 15% deionized water. Lyophilisation was conducted using CoolSafe instrument (4-15 L Freeze Dryers). Optical rotations were measured on a JASCO P-1010 instrument. The NMR spectra were recorded on a Bruker Avance II instrument (400 MHz for 1 H-NMR and 100 MHz for 13 C-NMR). The thin-layer chromatography used aluminum oxide 60 F254 neutral (Merck). Mass spectrum of isolated compounds was obtained using a HPLC/ESI/Q-TOF HRMS apparatus. The conditions for the HPLC analysis were the following of the work proposed by : HPLC conditions were as follows: water, acetonitrile and formic acid were of HPLC/MS grade; the HPLC system was an Agilent 1260 Infinity; a reversed-phase C 18 column (ZORBAX Extended-C 18 2.1 Â 50 mm, 1.8 lm) with a Phenomenex C18 security guard column (4 mm Â 3 mm) were used; the flow-rate was 0.4 mL/min and the column temperature was set to 30 C. The mass spectra were recorded using an Agilent 6540 UHD accurate-mass Q-TOF spectrometer equipped with a Dual AJS ESI source 6.

Extraction and isolation
Dried and chopped aerial parts (600 g) of F. nodosa were extracted with acetone (3 litres, 7 days Â 3 times) at room temperature. The acetone extracts were concentred in vacuum to give two gums (10.5 g). The extract of the aerial parts was chromatographed over silica gel column using a gradient solvent system (P.Et.-AcOEt). The various fractions of the initial column were subsequently re-chromatographed over silica gel, and silica flash columns, using a gradient solvent system (P.Et.-AcOEt-1:1 ! 1:9) to yield in order of increasing polarity the following compounds: 1 (70 mg), 2 (344 mg), 3 (26 mg), 4 (140 mg), and the new compound 5 (18 mg).

Conclusions
Our chemical investigation of aerial parts of the Sicilian accession of Ferulago nodosa resulted in the isolation and identification of five compounds, including two derivatives 2,3,4-trimethylbenzenes (1 and 3), and three ferulol derivative (2, 4 and 5), one of which (5) has never been isolated from plant species. The different compounds were characterised by HPLC-MS, [a] D , 1 H-NMR, 13 C-NMR, and two dimensional (2 D)-NMR spectra. The 2 D-NMR experiments ( 1 H-1 H COSY, HMBC, and HSQC) clarified the exact skeleton of new compound (5). In fact, the connection between ester moiety and the ring portion (H 1 , d H1 ¼ 5.24 and C-1 0 , d C ¼ 166.19), and the HMBC correlations between H-4 (d H4 ¼ 7.03) and the carbon C-7 (d C7 ¼ 171.43), indicated that the carboxylic group bound to carbon 5. The present investigation suggested, once again, the chemical diversity of a genus extensively studied but still today full of potential novelties. From a chemotaxonomic point of view, further studies on the Balkan-Aegean population of this taxa, never previously investigated, would be necessary and desirable in order to confirm or not, the occurrence of two different subspecies.

Funding
This work was supported by grant from MIUR-ITALY PRIN 2017 (Project N. 2017A95NCJ).