New Adenosine Derivatives from Aizoon canariense L.: In Vitro Anticholinesterase, Antimicrobial, and Cytotoxic Evaluation of Its Extracts

Aizoaceae is a large succulent family characterized by many psychoactive species. Aizoon canariense L., a wild neglected plant traditionally used in gastrointestinal ailments, has been the subject of a limited number of phytochemical and biological studies. Therefore, herein, we investigated the in vitro cytotoxic, antimicrobial, and anticholinesteraseactivity of the aerial parts of A. canariense L. and analyzed the phytochemical compositions of the lipoidal and alkaloidal fractions. Petroleum ether extract showed the presence of behenic and tricosylic acid, while an in-depth investigation of the alkaloidal fraction revealed the identification of new adenine based alkaloids (1–5), which were isolated and identified for the first time from Aizoon canariense L. Their structures were elucidated based on extensive spectroscopic analyses. The alkaloidal extract showed a powerful cytotoxic effect (IC50 14–28 μg/mL), with the best effect against colon carcinoma, followed by liver and breast carcinomas. The alkaloidal extract also had a potent effect against Candida albicans and Escherichia coli, with minimum inhibitory concentrations (MIC) values of 312.5 and 625 µg/mL. The in vitro anticholinesterase activity was potent, with IC50 < 200 ng/mL for the tested extracts compared with 27.29 ± 0.49 ng/mL for tacrine.


Introduction
African plants have been proven to have encouraging healing powers but are scarce in scientific investigations. Many studies have shown promising antimicrobial, cytotoxic, and enzyme inhibitory effects, correlated with the diversity of their phytoconstituents [1,2] Aizoaceae, the "ice plant", is the largest family of succulent leaves, consisting of 135 genera and 2499 species. Aizoaceae is characterized by its flavonoids, as reported in Aptenia [3,4] and Trianthema [5,6], and alkaloids, as identified in Sceletium species [7], in addition to sterols and triterpenes [8]. Alkaloids of the family Aizoaceae are characterized by their phenolic alkaloid nature [7,9]. Six genera and ten species are recognized from Aizoaceae in Egypt, however are poorly studied [10]. Aizoon is one of the wildly grown genera of the subfamily Aizooideae [11]. Aizoon canariense L. (Gafna or Shafna) was traditionally used to treat gastrointestinal problems and as a hypotensive [12]. Limited Purine bases and nucleosides are produced by the turnover of nucleotides and nucleic acids, as well as from some cellular metabolic pathways [23]. Purine base is not limited to the xanthine alkaloids but it encompasses adenine and guanine glycosides [24]. Combined information from 1D and 2D NMR (COSY, and HSQC) experiments were utilized to predict the structures of compounds 1-5 (Figures 1 and S2-S16). The 1D 1 H-NMR spectra of compound 2 showed the presence of 3 protons corresponding to H-2, H-8, and NH protons at δ H 8.76, 8.58, and 5.67 ppm, respectively, characteristic of alkaloids with an adenine nucleus [25]. A set of 4 aromatic protons was detected at δ H 7.23-7.42 ppm corresponding to H-2 , 3 , 5 , and 6 . H-1 was noticed as a singlet at δ H 3.65 ppm, while H-5 -8 were observed as a broad signal corresponding to 8Hs at δ H 1.29 ppm. H9 was observed at δ H 1.21-1.23 ppm as doublet signal counting for 3Hs. H-2 and 4 were displayed as two singlet signals at δ H 2.88 and 2.11 ppm, respectively, and directly after the broad signal corresponding to acetate methyl groups of ribose at δ H 2.05 ppm and counting for 9Hs. Ribose protons were observed at δ 3.0-3.6 ppm. The later values were in agreement with the reported data by Ciuffreda et al. for adenine nucleoside acetates [25]. The signals arising due to anomeric protons are usually reported to appear in the range of 4.0-5.9 ppm, while the protons of α-glycosides typically resonate 0.3-0.5 ppm downfield from those of the corresponding β-glycosides [26]. In compound 2, the anomeric H1 appeared as a doublet signal at δ H 4.03-4.05 ppm (J = 8.0 Hz), which is in the range of β-glycosides. Additionally, the coupling constant between H-1 and H-2 is 8.0 Hz. Ciuffreda et al. introduced further confirmation of anomeric configuration via the evaluation of differences between δ-values of H-2 and H-1 . The magnitude of these differences is larger than 2.15 ppm in β-derivatives and smaller than 1.85 ppm in the α-ones [25]. In our case, the difference between δ-values of the H-2 and H-1 was 4.72, which is more than 2.15, so it should be a β-anomer. The 1D APT 13 C-NMR of compound 2 demonstrated the sugar moiety methines at δ c 70-83 ppm and were phased negatively, while C-2 , 4 , and 1 methylenes were reported at δ c 50.52, 52.38, and 59.62 ppm, respectively, and were phased positively. Further methylenes (C-5 -9 ) were reported at δ c 25-32 ppm. The methyl at C-10 was observed at δ c 20.82 ppm and further methyls of acetates were detected at δ c 25.39 and 31.69 ppm and were phased negatively. The C-3 carbonyl was noticed at δ c 206 ppm and was phased positively, while acetate carbonyls were observed in HSQC ( Figure S5). Ester carbonyls appeared at δ c 174-177 and showed coupling with both sugar and methyl Hs (Table 1). Additionally, the coupling of sugar Cs and Hs of adjacent carbons was spotted in HSQC. The characteristic coupling of ketone carbonyl C-3 , H-4 , and H-2 was noticed in HSQC (207.51, 2.11 and 207.29, 2.88 ppm, respectively). In the HSQC, 6 couplings in the aromatic region were noticed that corresponded to H-2, 8, 2 , 3 , 5 , and 6 . A characteristic coupling between C-2 and H-2 was observed at δ H 2.88, 50.45 ppm. Additionally, couplings of ribose carbons and protons were noticed. The positive MS spectrum revealed an [M] + peak at m/z 625, suggesting a molecular formula of C 31 H 39 N5O 9 ; therefore, compound 2 was identified as N-(4 -((3 -oxononyl)oxy)phenyl)β-adenosine-2 ,3 ,5 -triacetate, isolated for the first time from aerial parts of A. canariense L.

Determination of the Lipoidal Matter
Fatty acids have a vital role in maintaining the structural integrity of cellular membranes, as well as being a great energy source and being present in signaling molecules Studies showed that Alzheimer's disease patients have reduced levels of polyunsaturated and monounsaturated fatty acids [29,30], therefore supplementation of such fatty acids may ameliorate cognitive functions. GLC analysis of A. canariense L. petroleum ether extract allowed the identification of 91.7% of its fatty acid content, characterized by being long (C14-C22) and very long-chain fatty acids (more than 22 carbons) ( Table 2). Saturated fatty acids showed their predominance, with tricosanoic acid (C23) being the major identified saturated fatty acid (43%), followed by behenic acid (C22), which has a role in skincare as an emollient and is able to restore the skin's natural oils and improve overall levels of hydration [31]. Nervonic acid (C-24:1 ∆15, cis-15-tetracosenoic acid) is the major unsaturated fatty acid. It is a monounsaturated analog of lignoceric acid, which is known to enhance brain function and prevent demyelination. Additionally, it was proven to ameliorate memory function and to improve the activity of γ-glutamate cysteine ligase in the cerebral cortex [32,33]. Eicosenoic acid (C-20), also called gondoic acid, is a monounsaturated omega 9 fatty acid, which was reported to cause a mild reduction in NO levels and to reduce LPS-induced increase in iNOS, therefore having a mild anti-inflammatory effect [34]. The different A. canariense L. extracts were screened for their cytotoxic, antimicrobial, and acetylcholinesterase inhibitory activities (Table 3). Cytotoxic activity was evaluated for alkaloidal as well as methanolic extracts of A. canariense L., against three cancer cell lines, namely breast carcinoma (MCF-7), hepatocellular carcinoma (HepG-2), and colon carcinoma (HCT-116), using sample concentrations ranging from 0 to 500 µg/mL. The results of the cytotoxic activity of A. canariense L. extracts revealed that the alkaloidal extract had the most powerful effect (IC 50 14-28 µg/mL), with the best effect against HCT-116, followed by HepG-2 then MCF-7. The methanolic extract showed comparable results with higher IC 50 ( Table 3). The powerful cytotoxic potential of A. canariense L. was previously demonstrated against human CCRF-CEM leukemia cells [35] and HepG2 [15].

Antimicrobial Activity
The antimicrobial activity levels of both methanolic and alkaloidal extracts were evaluated using the disc diffusion method against Gram-positive, Gram-negative, and fungi compared with reference antimicrobial and antifungal agents. MIC values were estimated for the most sensitive micro-organisms (Tables 4 and 5). Through this study, the alkaloidal extract showed high activity against Candida albicans and Salmonella typhimurium and moderate activity against Bacillus subtilis, while the methanolic extract showed promising antifungal activity against Aspergillus flavus, which was comparable to ketoconazole, as well as moderate activity against Staphylococcus aureus and Escherichia coli. Comparing the MIC values of the alkaloidal and the methanolic extracts of A. canariense L., the alkaloidal extract had better activity against Candida albicans and Escherichia coli, with MIC values of 312.5 and 625 µg/mL, respectively, while the methanolic extract showed better activity against Staphylococcus aureus and Bacillus subtilis, with MIC values of 625 µg/mL for both. The potent antifungal effect was supported by a previous report demonstrating a powerful effect against A. fumigatus [16]. Table 4. Mean inhibition zones in mm of alkaloidal and methanolic Aizoon canariense L. extracts.

Anticholinesterase Activity
The increase in acetylcholinesterase (AChE) activity is the most characteristic change that occurs in Alzheimer's disease. AChE is the enzyme responsible for acetylcholine hydrolysis, from both cholinergic and non-cholinergic neurons of the brain. The increase in acetylcholine level can be achieved by inhibition of AChE, which helps in the treatment of Alzheimer's disease. The inhibitory activity of acetylcholinesterase was assessed using BioAssay Systems' QuantiChromTM Screening kit based on an improved Ellman method. Through this work, the anti-Alzheimer's activities (AChE enzyme inhibition activity) of the crude methanolic, dichloromethane, alkaloidal, as well as aqueous alkaloidal extracts of A. canariense L. were evaluated in vitro and compared with that of the standard tacrine (AChE inhibitor). The results are shown in Table 3 and Figure 2, representing the % inhibition levels at 10-1000 µg/mL and IC 50 for the different extracts. The results demonstrated that the methanolic extract showed significant activity with IC 50 = 112.24 ± 7.73 ng/mL, along with the aqueous alkaloid extract with IC 50 =139.27 ± 21.40 ng/mL. Moreover, the dichloromethane extract showed the highest (very potent) and most promising antiacetylcholinesterase activity with IC 50 = 62.48 ± 1.31 ng/mL compared to the standard drug (AChE inhibitor) tacrine, with IC 50 = 27.29 ± 0.49 ng/mL (Table 3). Taken together, these results demonstrated a considerable anti-Alzheimer's activity of the extract. The ability of Aizoaceae plants to manage Alzheimer's was previously demonstrated with Trianthema portulacastrum, mainly in the phenolic-rich fraction, where docking studies confirmed the significant binding affinity of chlorogenic acid towards AChE [36], while an in vivo model using Sceletium tortuosum showed cognitive set flexibility and executive function and positive changes in mood and sleep compared with the placebo group [37].

General Experimental Procedures
NMR analyses ( 1 H, 13 C, COSY, and HSQC) were performed on a Bruker instrument (Billerica, MA, USA; 400 and 100 MHz for 1 H-and 13 C-NMR, respectively) using DMSO-d 6 as a solvent and with chemical shift values given in δ (ppm) and referenced to the TMS signal as an internal reference. All samples were prepared in suitable deuterated solvents. An ultra-mass spectrometer was used (Thermo FisherScientific, Bremen, Germany), equipped with a Nanomate electrospray ionization (ESI) interface (Advion). An electrospray voltage of 1.7 kV (+/−) and a transfer capillary temperature of 200 • C were applied. Chromatographic analysis was carried out on TLC plates (Merck, Germany) using CH 2 Cl 2 -MeOH at different ratios, while column chromatographic separation was performed using a silica gel column with CH 2 Cl 2 and gradient increase of MeOH. The analysis of fatty acid methyl esters was performed on an Agilent 19091J-413 gas chromatography instrument equipped with a flame ionization detector (FID), for which an HP-5 5% phenyl methyl siloxane capillary column (30 m × 320 µm × 0.25 µm) was used. The injector temperature was 250 • C, with an average velocity of 27 cm/s. H2 was the carrier gas, with a flow rate of 30 mL/min. The detector operated at a temperature of 280 • C.

Determination of Lipoidal Matter
Here, 1 g of petroleum ether extract was refluxed with KOH for three hours, then the mixture was partitioned with diethyl ether. The aqueous layer was acidified with HCl, extracted with diethyl ether, and then the ethereal extract was esterified by refluxing with H 2 SO 4 -MeOH at a ratio of 3:50 for three hours. The ethereal layer was then collected and the residue was kept for GLC analysis. The identification of the fatty acid methyl esters was carried out by comparing retention times with the applied authentic sample. The quantitative estimation of each peak was achieved by using a computer integrator, adopting the internal normalization procedures [39].

Cell Culture
All cell lines used in this study were obtained from Nawah Scientific, Inc. (Mokatam, Cairo, Egypt). Cells were maintained in DMEM media supplemented with 100 mg/mL of streptomycin, 100 units/mL of penicillin, and 10% heat-inactivated fetal bovine serum in a humidified 5% (v/v) CO 2 atmosphere at 37 • C.

Screening of Cytotoxic Activity
The cytotoxic activity of both methanolic and alkaloidal extracts was estimated using 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay against human breast cancer (MCF-7), liver cancer (HEPG2), and colon cancer (HCT-116) cell lines [40,41]. Principally, the MTT assay measures cell viability through the determination of the mitochondrial function of cells by measuring the activity of various mitochondrial enzymes (Stone V). Cell viability was determined using a cell proliferation kit [40] according to the manufacture's protocol, while the optical density was measured at 590 nm with the microplate reader (SunRise, TECAN, Inc, Mannedorf, Switzerland) to determine the number of viable cells and the percentage of viability was calculated as (1 − (ODt/ODc)] × 100%), where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The relationships between surviving cells and drug concentrations were plotted to get the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC 50 ), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose-response curve for each concentration using GraphPad Prism v.8.4.2.
(San Diego, CA, USA), as indicated. Here, p values < 0.05 were considered statistically significant.

Anticholinesterase Activity
The most important enzyme controlling acetylcholine (ACh) levels in healthy brains is acetylcholinesterase (AChE), while butyrylcholinesterase (BChE) is involved to a lesser extent [44]. The anticholinesterase activity levels of the different A. canariense L. extracts were estimated using a QuantiChromTM kit, IACE-100, (BioAssay Systems, Hayward, CA, USA). The acetylcholinesterase inhibitor screening kit is dependent on an enzyme-catalyzed kinetic reaction [45]. The enzyme source, according to the manufacturer's instructions, is E. electricus. The enzyme hydrolyzes the substrate acetylthiocholine, resulting in the production of thiocholine, which reacts with 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) to form 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate, which can be detected at 412 nm [46,47]. Briefly, and according to the manufacturer's instructions, 45 µL samples of AChE (400 U/L) were incubated with 5 µL samples of tested extracts at a series of concentrations ranging from 1 to 500 µg/mL or 5 µL of 40 v% DMSO in a 96-well microplate. While in a separate well, 45 µL of assay buffer was used instead of AChE to achieve 100% inhibition in the negative control. The reaction mixture was incubated for 15 min at 37 • C. For each well, 150 µL of assay buffer was added, containing 1 µL substrate and 0.5 µL DTNB. The thiocholine produced by the action of acetylcholinesterase forms a yellow color with DTNB. The intensity of the produced color measured at 412 nm is proportionate to the enzyme activity in the sample. The optical density of the tested extracts was measured at 412 nm at 0 and 10 min in a plate reader compared with tacrine (Santa Cruz Biotechnology Cat# sc-200172) as standard (AChE inhibitor). The anticholinesterase activity was calculated as follows: % inhibition = 1 − (∆OD test /∆OD control ) ×100 [48].

Statistical Analysis
Statistical analysis of the data was performed using one-way ANOVA, followed by Tukey's multiple range test for post-hoc comparisons (GraphPad Prism, version 8.4.2). All the data are presented as the means of 3 determinations ± SE. The p value significance levels are represented as asterisks (*) for p < 0.05, (**) p < 0.01, and (***) p < 0.001.

Conclusions
This study provides the first report for the isolation and characterization of five adenine-based alkaloids from the polar alkaloidal fraction after acid-base extraction of the aerial parts of A. canariense L. The alkaloidal fraction of A. canariense L. showed a promising cytotoxic effect against HCT-116, MCF-7, and, HepG-2, in addition to significant antimicrobial effects. Furthermore, the alkaloid fraction, as well as dichloromethane (flavonoid containing fraction), showed a significant effect against Alzheimer's disease, which requires further in vivo studies. The predominance of behenic and tricosanoic acids in the non-polar fraction, as well as the adenine-based alkaloids, may correlate to the potential effects in cerebral disorders. Our work revealed A. canariense L. a potential candidate for the treatment of many ailments.