A new iridoid diglycoside from Sambucus ebulus L

Abstract The phytochemical examination of the polar constituents of Sambucus ebulus L. leaves led to the identification of patrinoside (1) and of a new diglycoside iridoid, patrinoside-aglycone-11-O-[β-D-glucopyranosyl-(1→6)-2′-deoxy-β-D-glucopyranoside] (trivially named as sambuloside) (2). Both of these structures have been assigned by spectroscopic means (NMR and MS). Graphical Abstract


Introduction
The genus Sambucus L. (family Adoxaceae) comprises about 22 accepted species, mainly deciduous shrubs and small trees, distributed in the temperate and subtropical areas of the Northern Hemisphere (Eriksson and Donoghue 1997). Within it, the species Sambucus ebulus L., commonly known as 'dwarf elder', is a perennial herbaceous plant which has its original growth area in Southern and Central Europe and Southwestern Asia. From the botanical standpoint, this species is a perennial rhizomatous herb, up to 150 cm tall, characterised by an unpleasant smell. The leaves are opposite, imparipinnate, with 5-9 oblong lanceolate leaflets. The flowers are white, occasionally pink, grouped in wide terminal corymbs. The fruit is a globose berry, shiny and black when ripe. Different parts of the plant (leaves, rhizomes and fruits) have been used in traditional medicine mainly for the treatment of inflammation-related gastrointestinal disorders, influenza, rheumatoid arthritis and other diseases (Tasinov et al. 2013). In addition, the pharmacological studies on different extracts of S. ebulus have confirmed interesting biological activities related to their phytochemical content, such as the wound healing, antiinflammatory, antioxidant, hypotensive, cytotoxic and anti-angiogenic ones (Ebrahimzadeh et al. 2006;Pieri et al. 2009;Shokrzadeh and Saeedi Saravi 2010;S€ untar et al. 2010).
In the past, some phytochemical investigations on S. ebulus have resulted into the isolation of various secondary metabolites such as flavonoids, steroids, cyanogenic glucosides (Shokrzadeh and Saeedi Saravi 2010;Zahmanov et al. 2015), secoiridoids ) and several 'Valeriana-type' iridoids Gross et al. 1987;Pieri et al. 2009). In a previous study carried out in our laboratory some years ago (Tomassini et al. 2013), we analyzed the EtOAc extract from the leaves of S. ebulus, identifying two acetylated non-glycosidic iridoids also reported in a further study (Atay et al. 2015). At the present, following the reported informations about the popular use of S. ebulus, since the great majority of the ethnomedicine remedies are prepared as aqueous extracts, we decided to examine, in a new study, the content of a more polar extract from the plant leaves.
Patrinoside (1), initially isolated from Patrinia scabiosaefolia Fischer (Taguchi and Endo, 1974), is present as base-structure in many iridoids recovered in species belonging to Adoxaceae, including S. ebulus (Pieri et al. 2009;Tomassini et al. 2013;Atay et al. 2015). Yet, this is the first time that this compound is reported as such in S. ebulus.
Sambuloside (2) was identified by means of 1D-and 2D-NMR experiments as well as ESI-MS and ESI-MS/MS analysis. In particular, in the monodimensional proton spectrum, the typical pattern of patrinoside was observed. This is characterized by the H-3 olefinic signal displayed as a singlet at d H 6.32, the H-1 hemiacetalic signal as doublet with a coupling constant of 4.4 Hz at d H 5.99, the oxymethyne signal corresponding to H-7 at d H 4.32, the two hydroxymethylenes splitted signals referring, respectively, to H-10 at d H 3.68 and 3.88 and to H-11 at d H 4.05 and 4.23, the three methynes signals relative to H-5, H-8, H-9 and the methylene signal assignable to H-6.
The 13 C-NMR spectrum showed the corresponding carbon resonances of these protons. Yet, both in the proton and in the carbon spectra, additional resonances showed the presence of an isovaleric moiety (with a carboxyl group engaged in an ester bond due to the signal falling at 173.4 ppm) and of two monosaccharide units, with two proton signals at d H 4.58 and 4.30 typical of two anomeric protons. By the way, after comparison with data reported in the literature (Pieri et al. 2009), these two saccharide units were seen to correspond to a glucose and a 2 0 -deoxyglucose. In fact, the signals of H-2 0 resonating at about 2.10 and 1.49 ppm and typically coupled with H-1 0 could be clearly observed in the COSY spectrum. Regarding the exact position of this second sugar unit, a clear indication came from the low-field shift of the resonances H 2 -6 0 and C-6 0 of the deoxyglucose (proton signals at d 4.07 and 3.78, and carbon signal at d 70.0) which is typical of two sugar units when linked to each other in sequence.
DEPT, 1 H-1 H COSY, 1 H-1 H TOCSY, 1 H-13 C HSQC and 1 H-13 C HMBC confirmed all these hypotheses and more. In particular, the 1 H-1 H TOCSY allowed to distinguish the signals belonging to each sugar moiety, while the 1 H-13 C long-range correlations, highlighted in the HMBC spectrum, were able to determine without ambiguity the correct sequence of the two sugars, glucose-(1!6)-2 0 deoxyglucose (where the latter was linked to the hydroxyl at position 11) as well as the acylation site of the isovaleric unit on the hemiacetalic hydroxyl on C-1 (Figures S7 and S8 in the Supplementary Material).
Finally, the NOESY experiment allowed to obtain the relative stereochemical assignments for the chiral carbons in the cyclopentanic ring. Positive NOEs were observed among H-5, H-6b and H-9 and, on the other side of the molecule, among H-1, H-8, H-7 and H-6a. Assuming a b configuration for H-5, as in almost all the known iridoids, 7-b-OH and 8-b-CH 2 OH configurations were assigned to 2, also consistent with the stereochemistry of patrinoside (Figures S9 and S10 in the Supplementary Material).
The  Indeed, in the MS/MS experiment, the following peaks could be observed. The first one, presenting a value of 529.42 Da, may be actually reconducted to the loss of the total isovaleric acid unit. At this point, the formed carbocation presents a positive charge on a trivalent carbon and is located near a quaternary carbon having a free proton. This situation favours the formation of a double bond between these two carbons and the loss of the proton. The derived compound has a molecular weight of 506.49 Da which leads to the previously said peak through the formation of a sodium adduct.
The second one presents an m/z value at 485.42 Da which correspond to the potassium adduct of the compound after the breaking of the bond with the glucose residue (P.M ¼ 446.49 Da). Actually, the peak corresponding to the sodium adduct of this structure is also present even if with a lowest intensity (469.42 Da).
The two subsequent peaks at the corresponding m/z value of 383.33 and 367.33 Da are direct consequence of the two precedent losses. In fact, they refer to the same structure where the original compound has lost both the total isovaleric acid unit with the relative following structural rearrangement and the glucose residue. The molecular weight of this structure is 344.36 Da whereas the two peaks represent the potassium and the sodium adducts, respectively.
Minor MS/MS transition peaks were also visible. The two most important ones are surely those presenting m/z values of 323.33 and 221.08 Da. The former peak corresponds to the sodium adduct of the molecule after the loss of only the two saccharide units (P.M. 300.35 Da). The latter peak, corresponds, instead, to the initial molecule after the loss of the isovaleric acid unit as well as of the two saccharide units altogether, plus sodium.

General analytical procedures
1 H and 13 C NMR spectra were recorded on a Bruker Avance 400 NMR spectrometer (Bruker, Karlsruhe, Germany) at a potency of 400 and 100 MHz, respectively. CD 3 OD was employed as deuterated solvent for the NMR analysis and the signal corresponding to CD 2 HOD (d H 3.21, d C 49.0) was used as reference.
ESI-MS and ESI-MS/MS experiments were recorded on a ThermofisherLCQDeca XP Plus ion-trap mass spectrometer (Thermofisher, Waltham, MA, USA). The CID MS experiments were performed on a linear ion trap mass spectrometer equipped with an ESI source and a syringe pump. Operating conditions of the ESI source were the following: ion spray voltage ¼ þ4.0 kV; sheath gas ¼ 5 (arbitrary scale); sweep gas ¼ 5 (arbitrary scale); capillary temperature ¼ 275 C; collision gas was Helium (nominal pressure, 1.4 Â 10 À5 Torr; activation time ¼ 30 s; activation Q ¼ 0.20). The flow rate was 5 lL/min. The final spectrum was the average of about 40 scans, each consisting of two microscans. These settings represent the usual methodology used in our laboratory for this kind of analysis as reported by Francioso et al. (2014).
Thin layer chromatography was performed on silica gel SiF 254 plates which were visualised using 2 N H 2 SO 4 as spray reagent.
All the solvents of RPE grade, if not diversely specified, were purchased from Sigma Aldrich (Milan, Italy) or Carlo Erba Reagenti (Milan, Italy). Silica gel 60 (70-230 mesh ASTM) was purchased from Fluka, instead.

Plant material, extraction and isolation
Sambucus ebulus L. was collected in Rome, on the banks of the river Aniene. Its botanical identity was confirmed by one of us (L.T.) at the University 'Sapienza', Rome where a voucher specimen is deposited (No. 2989-RO General Herbarium).
Fresh leaves and young stems of the plant (400 g) were subjected to extraction with absolute EtOH for a total volume of 3.0 L at room temperature. The extract was filtered and concentrated to dryness. The obtained residue (20.0 g) was suspended in H 2 O (400 mL) and extracted with EtOAc (3 x 250 mL). The aqueous phase was subsequently extracted with n-BuOH and the extract dried obtaining a residue of 4.5 g.
The entire dried extract was subjected to a first chromatographic separation on a silica gel column with the weight of 160.0 g (ratio 1:35 w/w) using a mixture of CHCl 3 / MeOH (8:2 v/v) as eluting system. The column afforded the iridoid glucoside patrinoside (1) (42 mg) from the assembly of fractions 120-124, together with a high polarity not purified mixture of iridoids (112 mg) from the assembly of fractions 130-145.

Conclusions
The interest of this research is focused on the isolation of two iridoids from S. ebulus, with compound 2 that represents even a previously undescribed phytochemical.
The presence of patrinoside-related compounds in this species is not new since similar or derivative compounds have been reported both in its EtOAc extract (Tomassini et al. 2013;Atay et al. 2015) and in higher polarity fractions (Pieri et al. 2009). Yet, Valeriana-type iridoids (with patrinoside, in a particular way) are chemotaxonomic markers of the Sambucus genus and their further isolation in this work confirms this point as well as the current botanical classification of this species as a member of Adoxaceae family (Hillebrand and Fairbrothers 1970;Eriksson and Donoghue 1997).
Regarding the possible role played by patrinoside and its derivatives in the total medicinal properties of the plant, it is important to highlight that these compounds have shown to exert several interesting pharmacologic activities, in particular as choleretic, anti-inflammatory and spasmolytic agents (Takeda et al. 1981;Atay et al. 2017;Takeda and Aburada 1980;Cometa et al. 2009). These last evidences could constitute the starting point towards a deeper study of the activities reported for Sambucus ebulus in traditional medicine.