HPLC–DAD–ESI/MS profiles of bioactive compounds, antioxidant and anticholinesterase activities of Ephedra alata subsp. alenda growing in Algeria

Abstract Ephedra (Ephedraceae) is used in medicine for various purposes as having, antioxidant, anticarcinogen, antibacterial, anti-inflammatory hepatoprotective, anti-obesity, antiviral and diuretic activities. In this study the aim was to investigate chemical constituents of Ephedra alata and understand the possible effects of those constituents in antioxidant activity and alzheimer's disease essay. For this purpose, natural compounds from E.alata were characterized by LC–DAD–ESI-MS/MS using negative and positive ionization modes, while the bioactivity was assessed by acetylcholinesterase (AChE) inhibition study and determining of antioxidant activity; DPPH radical scavenging and β-carotene bleaching assays were used to assess the antioxidant potential. The proposed method of spectrometry provided tentative identification of 27 compounds including alkaloids and phenolic compounds as flavonoids. The methanolic extract showed high contents of total phenolic and exhibited an important antioxidant potential and demonstrated a potent inhibitory effect against acetylcholinesterase (IC50: 11,25 ± 0,25 µg/mL). The results showed that the plant possesses a therapeutic effect. GRAPHICAL ABSTRACT


Introduction
In living systems, oxidation is a basic part of the normal metabolic process and rapid production of free radicals may cause human neurologic and other disorders such as cancer, diabetes, inflammatory disease, asthma, cardiovascular, neurodegenerative diseases (Pinton et al. 2012). Medicinal plants may contain a wide variety of free radical scavenging molecules, such as phenolic compounds, nitrogen compounds, vitamins, terpenoids and some other endogenous metabolites (Zheng and Wang 2001;Cai et al. 2003).
Ephedra has been one of the best-known herbal remedies in Traditional Chinese Medicine for the treatment of allergies, bronchial asthma, chills, fever, flu, headache, colds hay, nasal congestion and central nervous system disorders (Bagheri-Gavkosh et al. 2009;Chebouat et al. 2014). In addition to the known components of the genus ephedra which are alkaloids (Al-Snafi 2017); phytochemical analyzes of E. alata have indicated the presence of phenolic compounds such as flavonoids, cardiac glycosides and reducing sugars (Jaradat et al. 2015). Therefore, the present study aims to characterize the phytochemical content and therapeutic value of the Algerian plant E. alata subsp. alenda.
The analytical technique used was the high-performance liquid chromatographydiode array detector with tandem mass spectrometry (HPLC-DAD-ESI-MS/MS), while the biological effect was evaluated by cholinesterase inhibition and antioxidant activities (DPPH ᭹ scavenging, b-carotene bleaching assays).

Analysis of LC-MS/MS
The metabolic profiling of the aqueous-methanolic extract of E.alata subsp. alenda was performed by using HPLC-DAD-ESI-MS/MS. Figure S1 (Supplementary material) shows the base peak chromatograms (BPC) of E. alata alenda in the negative and positive ionization modes. Tables S1 and S2 (Supplementary material) showed a list of 27 compounds tentatively detected and characterized. These compounds were tentatively characterized by means of MS data, together with the interpretation of the observed MS/MS spectra in comparison with those found in the literature.

Negative ionization
This study showed that flavonoids are useful markers in E. alata alenda. Compound 04 corresponding to gallocatechin, this compound yielded [M-H]at m/z 305. Its MS/MS fragmentations showed the major fragment at m/z 125 (Supplementary material, Table  S1), which resulted from a dissociation at O1-C2 and C4-C10 (Supplementary material, Figure S2) (Yuzuak et al. 2018). The loss of Phloretin moiety occurred in peaks 10 and 13, both with m/z 435, which were characterized as Phloridzin isomer 1 and isomer 2, respectively (Zhao et al. 2014;Sut et al. 2019). Compound 14 was described as apigenin 6, 8-di C-glucoside (vicenin-2 isomer) with an [M-H] À at m/z 593 and typical C-glycosyl fragments at m/z 503, 473 and 383 (Ferreres et al. 2003;Brito et al. 2014). Two other polar compounds were also represented by sinapic acid hexoside at m/z 385 and vanillic acid glucoside at m/z 329, which had in MS/MS fragmentation a major fragment at m/z 167 corresponding to vanillic acid molecule (Ammar et al. 2015).

Positive ionization
The Ephedra genus is known for its alkaloids, this is why a positive ionization study was necessary. Table S2  Positive mode revealed also the presence of other polar compounds such as cinnamic acid derivative, citropten, cyanidin rutinoside and Quercetin 3-O-rutinoside (Ammar et al. 2015).

Total phenolic and flavonoid contents
The results obtained of TPC, TFC of E. alata alenda extract are presented in Table S3 (Supplementary material). The total phenol content was significantly higher than total flavonoid. The amount of TPC and TFC reached to 358,06 ± 10,74 lg GAE/mg dry extract) and 22,93 ± 0,23 lg GAE/mg extract) respectively.

Antioxidant activity
The importance of conducing phytochemical studies, is to not only used for the chemical characterization but also important for correlating the chemical contents with specific functional properties. For these reasons, antioxidant activities were evaluated by using two methods; Diphenylpicrylhydrazyl radical (DPPH) scavenging and b-Carotene bleaching assay. According to the results presented in Table S4 (Supplementary material); hydro-methanolic extract showed a powerful antioxidant activity in DPPH scavenging assay with IC 50 value 12,25 ± 1,09 lg/mL, which is lower compared with standard used BHT. Interestingly, the results presented in Tables S3 and S4 (Supplementary material) indicates that methanolic extract of Ephedra alata var. alenda contains large quantities of polyphenols. This suggests that these secondary metabolites may be responsible for the observed radical scavenging activities.

Acetylcholinesterase inhibitory activity
The inhibition of acetylcholinesterase (AChE) is considered the primary approach to treat Alzheimer's disease (AD). Methanolic extract of Ephedra alata var. alenda showed a high potential to inhibit AChE as seen in Table S5 (Supplementary material), the inhibition IC 50 value was 11,25 ± 0.25 mg/mL which is close to that of Galanthamine standard.

Molecular modeling
Docking studies were performed in order to get insights into the possible binding mode of the identified alkaloids to hAChE (PDB: 4EY6). The obtained results are shown in Table S6 and Figures S5 and S6 (Supplementary material). As can be seen, all the studied compounds exhibit good affinity for the active site of hAChE with low binding energies ranging from À4.9 to À11.6 kcal/mol. Norephedrine and ephedrone form a favorable interaction at the anionic site with the residue TRP86. Ephedrine and methylephedrine could bind favorably with the residue SER125. While 2-(5-ethyl-2-pyridine)ethyl form a hydrogen bond with the residue of the oxyanion hole SER203. This indicates that the studied alkaloids are potential inhibitors of hAChE, which may explain the promising activity of the methanolic extract of E. alata var. alenda.

Conclusion
More than 27 compounds were characterized using the HPLC-ESI-MS/MS method from of the methanolic extract of E. alata Most of them presented flavonoids as O-glucoside quercetin isomers, Herbacetin 7-O neohesperidoside and Phloridzin isomers. This study showed also that alkaloids are useful markers of this species notably ephedrine and pseudoephedrine. This extract showed a good AChE inhibitory activity with an IC 50 of 11,25 ± 0,25 mg/mL in a quantitative assay. A strong antioxidant activity was demonstrated by the two used methods namely, DPPH (IC 50 12,25 ± 1,09 lg/mL) and b-Carotene bleaching assay (IC 50 20,28 ± 2,63 mg/mL). The docking study indicated that the identified alkaloids are potential inhibitors of AChE. The obtained results show that E. alata. alenda is very rich in polyphenols and flavonoids (TPC, 358,06 ± 10,74 lg of GAE/mg dry extract, TFC, 22,93 ± 0,23 mg lg of GAE/mg dry extract), which explains the powerful antioxidant effect of the studied extract. It can be concluded that E. alata var. alenda is a rich source of beneficial phytochemicals and antioxidants.