Chemical composition, in vitro antioxidant, anticholinesterase and antibacterial activities of Linaria scariosa Desf

Abstract The study was performed on the dichloromethane (DCM), ethyl acetate (EAc) and n-butanol (Bu) fractions (F) obtained from the 80% ethanol extract of Linaria scariosa Desf. aerial parts, collected in the North Eastern region of Algeria. Remarkable total phenolic and flavonoid contents were obtained, mainly for EAcF. These results were in accordance with the antioxidant activity of EAcF against DPPH, ABTS, CUPRAC and reducing power tests. DCMF and BuF exhibited significant cholinesterase activity inhibition of BChE and AChE. Moreover, EAcF displayed only moderate antibacterial activities, especially against S. aureus. The biological results were correlated to the chemical components, deduced by both GC-MS analysis of the fractions and the isolation of hemipholin, pectolinarigenin, antirride, antirrinoside, pectolinarin and linariosise, some of which known to exhibit potent effects on the tested biological activities. The study provides the first biological and chemical investigation on Linaria scariosa Desf (unresolved name). Graphical Abstract


Introduction
The genus Linaria Mill. is common in the folkloric medicine of different nations for the use as tonic, anti-scorbutic, laxative, anti-diabetic and diuretic (Cheriet et al. 2015a). It is known for the presence of iridoids (Bianco et al. 2004;Guiso et al. 2007), and flavonoids (Cheriet et al. 2014(Cheriet et al. , 2015a as the major constituents with various biological activities such as anti-tumor (Tundis et al. 2005), anti-AChE (Loizzo et al. 2007), antiinflammatory , antioxidant (Hanfer et al. 2016; and antidiabetic ). In the present study, we examined for the first time the in vitro antioxidant, anticholinesterase and antibacterial activities of different fractions obtained from the aerial parts of Linaria scariosa Desf. (unresolved name) (synonyms: Kickxia dentata (Vahl) D.A. Sutton and Antirrhinum dentatum Vahl.) supported by a chemical investigation including the isolation and identification of six compounds, the phenolic and flavonoid contents as well as the GC-MS profile of each fraction with the detection of various metabolites.

Results and discussion
The total phenolic content (TPC) and flavonoid content (TFC) in DCM, EAc and Bu fractions were determined by Folin-Ciocalteu reagent and expressed as gallic acid (GAE) and quercetin (QE) equivalents, respectively. Ethyl acetate fraction (EAcF) showed the highest TPC (143.6 ± 0.002 lg GAE/mg extract) and TFC (30.19 ± 0.006 lg QE/mg extract), followed by butanol fraction (BuF) (99.14 ± 0.03 lg GAE/mg extract and 26.39 ± 0.01 lg QE/mg extract, respectively) and dichloromethane fraction (DCMF) (81.64 ± 0.01 lg GAE/mg extract and 14.98 ± 0.01 lg QE/mg extract, respectively). Based on the known ability of phenolic and other primary and secondary metabolites to act as antioxidants, these data correlate quite properly with the antioxidant activity of the studied fractions (Table 1). EAcF displayed the highest activity against CUPRAC and ABTS, while moderate activity was observed in its reducing power evaluation and DPPH tests (Table 1). Detailed inhibition assays of DPPH, ABTS, CUPRAC and reducing power (Table S1), and of metal chelating (Table S2) have been also provided for the L. scariosa fractions at different weight values.
The fractions were screened for their inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes, related to galantamine, a drug used for the treatment of Alzheimer's disease and other cognitive impairments ( Table 2). The most interesting anti-AChE data were obtained for BuF. It is notable that similar studies on L. reflexa extracts showed instead the most remarkable activity for EAcF, containing linarin, isolinariin A and B, flavones with rutinose moiety as common part which is known to interact with AChE enzyme (Loizzo et al. 2007). This could explain why the BuF, which contains pectolinarin, exhibited a higher activity compared with EAc and DCM fractions. While EAc and Bu fractions resulted inactive against BChE at all tested concentrations, the moderate effect of DCMF can be related to its chemical composition determined by GC-MS analysis (Table S4) In the evaluation on a series of pathogenic bacteria strain, BuF displayed no activity, only a moderate inhibition was observed both for EAcF against Morganella morganii, S. aureus, Bacillus subtilis and Proteus vulgaris, and for against B. subtilis (Table S3).  These results are in agreement with the behaviour observed for most Linaria species (Cheriet et al. 2015a).
Six pure metabolites were isolated and identified by NMR and MS analyses, in comparison with reported data. They were hemipholin (1) (Chen et al. 2015) and pectolinarigenin (2) (Cheriet et al. 2014) from EAcF, antirrhide (3), antirrhinoside (4) , pectolinarin (5) ) and linarioside (6) (Bianco et al. 2004) from BuF. Notably, this is the first report deals with the isolation of hemipholin from the Plantaginaceae family and the first C-glycosylated flavonoid in the genera Linaria Mill., Kickxia and Antirrhinum. Also a high similarity was observed in comparison with the chemical composition of some Kickxia like Kickxia spuria (Venditti et al. 2018) and Antirrhinum like Antirrhinum majus (Drøhse Høgedal and Mølgaard 2000).
The phytochemical screening by GC-MS analysis allowed to identify a wide series of molecules present in the three factions, reported in Tables S4-S6.

Conclusion
In this study, we report for the first time the antioxidant, anticholinesterase and antibacterial activities of Linaria scariosa extracts, relating them to the chemical profile established by total phenolic and flavonoid contents, by GC-MS analysis of EAc, DCM and Bu fractions, and isolation of pure metabolites. These preliminary data are promising for further studies.