Phytochemical and antioxidant activity studies on Ononis angustissima L. aerial parts: isolation of two new flavonoids

Abstract Ononis angustissima aerial parts extract and exudate were subjected to phytochemical and biological studies. Two new natural flavonoids, (3S)-7-hydroxy-4′-methoxy-isoflavanone 3′-β-d-glucopyranoside (1) and kaempferol 3-O-β-d-glucopyranoside-7-O-(2′′′-acetyl)-β-d-galactopyranoside (4), and sixteen known compounds were isolated through a bio-oriented approach. Their structural characterisation was achieved using spectroscopic analyses including 2D NMR. The phytochemical profile of the extracts was also performed, and the antioxidant activity of all compounds was tested by three different assays. To get a trend in the results and to compare the antioxidant capacity among the different methods used, the obtained data were transformed to a relative antioxidant capacity index.


Introduction
Plants rich in antioxidant phenolics have been attracting an increasing attention, and nowadays, the discovery of new sources of safe and inexpensive antioxidants from natural origin is mandatory (Fernández et al. 2006), since some synthetic ones showed potential health risks and toxicity (Safer & AI-Nughamish 1999 Ononis L. is a large genus of perennial herbs and shrubs of the Fabaceae family (Ozenda 1958) known to be very rich in phenolic derivatives (Barrero et al. 1993;Abdel-Kader 2001;Mhamdi et al. 2015). Some Ononis species are favourite food for sheep, goats, donkeys and other animals; the young shoots are succulent and sweet and in some Saharian countries are eaten as vegetables (Burnett 1833;Ozenda 1958). Recently, some herbal teas containing Ononis species as components are sold in the market. Several plants belonging to the Ononis genus are used as folk medicine remedies for the treatment of jaundice, urinary tract inflammations and kidney stones; besides, some species have been used as wounds, eczema and rheumatic complaints healings, against skin cancer and lesions and topically as antiseptic and antimicrobial agents (Süntar et al. 2011). The ethnopharmacology importance and the richness in flavonoids of Ononis genus prompted us to carry out further studies (Bouheroum et al. 2009;Mezrag et al. 2013) on Ononis angustissima Lam., a plant widespread in Sahara regions. An antioxidant-oriented approach was carried out on the aerial part extracts and exudate, leading to the isolation and structural characterisation of two new natural flavonoids, (3S)-7-hydroxy-4′-methoxy-isoflavanone 3′-β-d-glucopyranoside (1) and kaempferol 3-O-β-d-glucopyranoside-7-O-(2′′′-acetyl)-β-d-galactopyranoside (4), together with sixteen known phenolic derivatives (2-3 and 5-18) ( Figure 1). In order to deepen and fully characterise the phytochemical profile of O. angustissima, LC-HReSIMS and LC-HReSIMS/MS analyses were also performed. extracts, fractions and pure compounds' antioxidant activity was tested using three different complementary methods, and the relative antioxidant capacity index (RACI) was also calculated. All extracts were also evaluated for their cytotoxic activity.

Results and discussion
In this study, all extracts of O. angustissima were subjected to the 2,2-diphenyl-1-picrylhydrazyl (DPPH), β-carotene bleaching (BCB) and ferric reducing antioxidant power (FRAP) assays to screen their antioxidant activity. As previously demonstrated (Milella et al. 2014), a single assay cannot determine the antioxidant activity of a phytocomplex; therefore, three complementary approaches were used to study the antioxidant potential of O. angustissima. Total phenolic content (TPC) was also measured, and the results were included in RACI calculation. In this way, RACI provided a more comprehensive assessment of the whole extract antioxidant potential. Both n-BuOH and crude exudate (ex-ONA) extracts showed an interesting antioxidant activity and to be a good source of phenolics (Table S1); on the basis of these results, they were further analysed. The n-BuOH extract was subjected to Sephadex LH-20 column chromatography collecting five major fractions (A-e) that were consequently evaluated for their TPC and antioxidant activity (AA) ( Table S1). Fraction e resulted the richest in terms of TPC (199.5 mggAe/g) followed by fractions C and D (121.5 and 110.9 mggAe/g, respectively). Fraction e showed the highest DPPH value (143.7 mgTe/g of extract), while the highest BCB value was registered for fraction D demonstrating 57.9% of AA. On the other hand, the highest FRAP value was obtained from fraction C (29.6 mgTe/g). On the basis of RACI results (Figure 2(a)), fractions B-e were purified and a total of nine compounds (1-9) were characterised ( Figure 1) of which two were new natural flavonoids (1 and 4). Fraction e showed also the presence of highly polymerised polyphenols. Following the same approach, the TPC content and the AA of ex-ONA fractions (AA-NN) were also studied, demonstrating a higher TPC content than the n-BuOH one, according to the respective extract results (Table S1). The highest TPC value was observed for fractions II and HH with 639.1 and 527.8 mggAe/g, respectively; these two fractions resulted also the most active in DPPH test (93.3 and 78.2 mgTe/g, respectively), together with the NN fraction (190.5 mgTe/g). On the other hand, ex-ONA fractions CC and DD demonstrated the highest AA when measured by BCB test. This phenomenon can be explained by the affinity of the antioxidant complex for the lipids, and thus, the lipophilic nature of BCB test could be the  In the 1 H NMR, the presence of an anomeric proton at δ 4.84 (1H, d, J = 7.6 Hz) and a methoxy group at δ 3.85 (3H, s) was also observed. The 1D-TOCSY spectrum together with information obtained from 13 C NMR led to establish the presence of a β-glucose unit. Hydrolysis of 1 with 1 N HCl, followed by gC analysis through a chiral column of the trimethylsilylated monosaccharide, led to the assignment of the glucose configuration. All the NMR assignments were obtained using 1D-TOCSY, COSY, HSQC and HMBC NMR correlations. The presence of the methoxy group at C-4′ was confirmed by NMR chemical shifts and the HMBC correlation between OMe and C-4′, while the correlation of H-1 glc and C-3′ located the glucose moiety at C-3′. In order to establish the absolute configuration at C-3, the CD spectrum was recorded (Slade et al. 2005). The negative Cotton effect at 312 nm led to establish the (3S) configuration at C-3 of compound 1. Thus, the structure of 1 was elucidated as (3S)-7-hydroxy-4-methoxyisoflavanone 3′-β-d-glucopyranoside.
The  (Table S2) showed 29 signals, of which 12 were assigned to a saccharide portion, 2 to an acetyl group and 15 to a flavonol moiety. The 1 H and 13 C NMR spectra (Table S2) of 4 clearly indicated that kaempferol was the aglycone (Agrawal 1989;Le gall et al. 2003). Analysis of 1D-TOCSY and DQF-COSY spectra allowed the complete assignments of all proton resonances of the glucose unit starting from the anomeric proton signal at δ 5.09 and of the galactose unit starting from the H-1 signal at δ 5.54, and the H 2-6 signals at δ 3.70 and 3.56, showing that the hydroxyl group at C-2 of galactose was acetylated. These results were consistent with the typical esterification shift of H-2 gal signal at δ 5.29 and C-2 gal at 73.5 ppm (D'Agostino et al. 1992). The HMBC cross-peaks were useful in the determination of the aglycone substitution pattern. Diagnostic long-range correlations were observed between H-1 glc and C-3, H-1 gal and C-7, and H-2 gal and COO. The sugar configuration was established as reported for 1. On the basis of the above results, 4 was characterised as kaempferol 3-O-β-d-glucopyranoside-7-O-(2′′′-acetyl)-β-d-galactopyranoside.
Compound 2 had a molecular ion peak at m/z 463.1251 (HReSIMS), corresponding to a molecular formula of C 22 H 22 O 11 . The HReSIMS/MS spectrum showed one peak at m/z 301.04 [(M-162)+H] +-, due to the loss of one hexose moiety, and a fragment at m/z 245.03 [ (M-162-56)+H] + , diagnostic of the presence of an isoflavone skeleton (Kuhn et al. 2003). From 1D and 2D NMR data (Table S2), its structure was deduced to be 5′,7-dihydroxy-2′-methoxy-isoflavone 4′-O-β-d-glucopyranoside, recently described by ghribi et al. from O. angustissima roots (ghribi et al. 2015). However, chemical shifts of glucose moiety were completely wrongly attributed by these authors; compound 2 correct 1 H and 13 C NMR values are now assigned in Table S2, and all its experimental data are listed in Supplementary material.
The antioxidant activity of all pure compounds using DPPH, BCB and FRAP tests was assayed (Table S1). Among isolated compounds from the n-BuOH fractions (1-9), compound 2 showed the highest DPPH and FRAP values, while the other isoflavones 1 and 3 were less active (Table S1). This difference could be ascribed to the loss of free -OH on aromatic rings in 1 and 3. Our data also confirmed that the presence of 2,3 double bond increases the activity of compound 2 when it is compared with that of 1. These results are in agreement with those recently shown on similar molecules (ghribi et al. 2015). Moreover, compound 5 showing no free -OH groups and 2,3 double bond displayed the lowest activity among isoflavone derivatives (Table S1). It is possible to assess that 17 and 13 are the most active flavones (Figure 2(b)), both exerting the catechol group on ring B, while compounds 8 and 9 showed the lowest values, both without any free phenolic hydroxyl group. RACI ( Figure  2(b)), combining the antioxidant results obtained from different assays, showed that flavones and isoflavones are the most active compounds and confirmed the importance of the adjacency of two hydroxyl groups in the ortho-diphenolic arrangement (Rice-evans et al. 1996;Heim et al. 2002;Lee et al. 2008). The interesting antioxidant potential obtained for O. angustissima extracts and fractions could be only partially attributed to the activity of pure isolated compounds: the presence of minor constituents could better clarify this activity. Therefore, the n-BuOH extract was subjected to LC-HReSIMS and LC-HReSIMS/MS analyses, in order to provide wider information concerning the polyphenols contents of O. angustissima aerial parts ( Figure S10). Thirty-two main species were revealed, including compounds 1-18. On the basis of accurate molecular weight determination, and taking into account the main fragments observed in the MS/MS spectra, the structure of further fourteen derivatives was proposed (Table S3). Particularly, nine other flavones (compounds a-e, g, h, j and l) and four isoflavones (f, k, m and n) were detected: the latter were distinguished by the loss of 56 amu fragment typically observed only in the MS/MS spectra of isoflavones (Cooper et al. 2002). The hydroxylation pattern on A and B rings could be recognised taking into account the different ions 1,3 A + related to C-ring fragmentation (Wu et al. 2004). The following ions suggested the hydroxylation pattern of ring A for compounds a-h and j-n: m/z 137 (C 7 H 5 O 3 ) + indicated one hydroxy, 153 (C 7 H 5 O 4 ) + two hydroxy, 167 (C 8 H 7 O 4 ) + one hydroxy and one methoxy, 181 (C 9 H 9 O 4 ) + two methoxy, 183 (C 8 H 7 O 5 ) + two hydroxy and one methoxy, 197 (C 9 H 9 O 5 ) + two methoxy and one hydroxy, 213 (C 9 H 9 O 6 ) + two methoxy and two hydroxy, 227 (C 10 H 11 O 6 ) + three methoxy and one hydroxy groups, respectively. Moreover, one putative aurone (compound i) was also proposed. These results are in agreement with the detected antioxidant activity.
Finally, the cytotoxic activity of crude O. angustissima extracts was investigated against healthy PBMC and three human cancer cell lines HeLa, Jurkat and MCF7. All the extracts were inactive at the tested concentrations. The absence of cytotoxicity on healthy PBMC could suggest the use of O. angustissima in herbal drugs preparation.

Conclusion
Spectrometric and spectroscopic analyses including 2D NMR allowed to isolate and identify two new natural secondary metabolites and sixteen known compounds from O. angustissima aerial parts belonging to the flavonoid, isoflavonoid, phenylpropanoid, spinonin and chalcone classes. Flavonoids and isoflavonoids reported the highest RACI and some structure-activity relationships based on the presence of free OH groups, different substituents on aromatic rings and the presence of 2,3 double bond in C-ring, were established.

Supplementary material
experimental section, NMR spectra of compounds 1 and 4, NMR data of compounds 1, 2 and 4, total ion current LC-HReSIMS chromatograms of n-BuOH fraction of the aerial parts, data of TPC and antioxidant activity of Ononis angustissima extracts, fractions and isolated compounds, and identification of the major compounds observed in aerial parts n-BuOH fraction by HR-LCMS analysis are available as Supplementary material.

Disclosure statement
No potential conflict of interest was reported by the authors.