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Apple leaves and their major secondary metabolite phlorizin exhibit distinct neuroprotective activities: Evidence from in vivo and in silico studies
⁎Corresponding author. mansour.sobeh@um6p.ma (Mansour Sobeh)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Apple, Malus domestica Borkh., a fruit-producing and medicinal plant, is reported to have a wide array of biological effects including a neuroprotective potential. In this work, the phytochemical composition of the leaf extract from M. domestica was thoroughly investigated using LC-MS/MS. Phlorizin, the major secondary metabolite was also isolated and characterized using MS and NMR data. Total phenolic content (TPC) and the total flavonoid content (TFC) were determined. The in vivo neuroprotective effect of the extract and its major component phlorizin was evaluated in rats using the novel object recognition (NOR) test. The antioxidant activity of the extract and phlorizin was evaluated in vitro by the well-known DPPH assay and in vivo through determining the total antioxidant capacity (TAC), and some oxidative stress parameters such as the reduced and oxidized glutathione (GSH, GSSG) respectively and the lipid peroxidation marker (MDA) in rats previously treated with dexamethasone (10 mg/kg i.p.) daily for 9 days and the tropane alkaloid scopolamine (1 mg/kg. i.p.) for 2 consecutive days (day 7 and 8). Moreover, the extract and phlorizin were tested to block the activity of β-secretase (BACE1) that is involved in the formation of amyloid-β (Aβ) plaques in silico and in vivo, an important player in the pathogenesis of Alzheimer’s disease. Molecular docking was performed to confirm the potential of phlorizin and other individual components of the extract to bind to and block BACE1. The extract and phlorizin revealed substantial antioxidant potential with IC50 of 6.93 and 5.14 µg/mL, respectively in DPPH assay. They were able to significantly restore TAC, increase GSH, and reduce both GSSG and MDA levels in the dexamethasone/scopolamine treated rats. Both extract and phlorizin were able to inhibit BACE1 with IC50 of 1.65 and 1.18 µg/mL, respectively. Our results provide evidence that M. domestica and its major secondary metabolite phlorizin are promising neuroprotective natural agents for treating Alzheimer Disease and other neurodegenerative disorders especially recognition impairment disorders. More studies are needed to elaborate the mechanism of action.
Keywords
Apple leaves
LC-MS/MS
Neuroprotection
Alzheimer
Novel object recognition (NOR)
1 Introduction
The neurodegenerative Alzheimer’s disease (AD) is characterized by an irreversible loss of cholinergic neurons and is associated with disrupted intellectual abilities of memory and cognition. The disorder is one of the most common causes of dementia until now and represents about 60 to 80% of dementia cases (World Alzheimer report., 2016).
Aggregation of amyloid-β (Aβ) in senile plaque is one of the fingerprints of AD. A main element for Aβ formation is the beta-site amyloid precursor protein (APP)- cleaving enzyme 1, known as β-secretase (BACE1). Therefore, BACE1 inhibition is a pivotal target for discovery and developing novel anti-Alzheimer drugs. Some natural polyphenols demonstrated substantial activities as specific BACE1 inhibitors, among them 2,2′,4′-trihydroxychalcone that was identified in Glycyrrhiza glabra (Zhu et al., 2010; Vassar et al., 2009).
The rose family, Rosaceae, comprises about 91 genera, with the genus Malus having 55 species of small deciduous trees or shrubs. The apple tree, Malus domestica Borkh., is cultivated worldwide and is one of the most economically relevant fruit crops with around 7500 cultivars (Lim, 2012). Apples are consumed fresh and as derived dietary products (jam, cider and juice). They are rich in antioxidant polyphenols, which possess a plethora of biological activities in treating inflammation, cardiovascular diseases, obesity, diabetes, and neurodegenerative brain disorders including Alzheimer disease (Toda et al., 2011; van Wyk and Wink, 2015). A polyphenol rich extract from apple fruits exhibited neuroprotective effects against aluminum-induced neurotoxicity which has long been linked to the development of Alzheimer disease (Cheng et al., 2014).
Apple leaves, which are traditionally used in China to treat menoxenia (abnormal menstruation) accumulate a large amount of polyphenols including phlorizin, isoquercitrin, quercetin-3-D-xyloside, quercetin-3-D-rhamnoside and quercetin-3-D-arabinoside that exhibited substantial antioxidant activity against H2O2-induced oxidative stress (Lu et al., 2019). Phlorizin was studied pharmacologically, evaluated as a therapeutic agent to treat diabetes and proved to inhibit the sugar transporters SGLT1 and SGLT2 (Ehrenkranz et al., 2005).
In the current work, we comprehensively characterized the phytoconstituents of apple leaves using LC-MS/MS and isolated its major compound phlorizin. The in vitro activities including the antioxidant potential and the BACE1 inhibition in addition to in vivo neuroprotective activities were evaluated. Moreover, phlorizin and the other major identified compounds in the extract were virtually screened as BACE1 inhibitors via molecular docking and their binding mode and binding energy towards the enzyme were estimated.
2 Materials and methods
2.1 Plant material
Malus domestica Borkh. (cv. Anna) leaves were provided by Horticulture Research Institute, Agricultural Research Center, Ministry of Agriculture and Land Reclamation, Giza, Egypt in December 2014. Voucher samples are kept at Pharmacognosy Department Herbarium, Faculty of Pharmacy, Cairo University, Cairo, Egypt under no. 12.03.2017.01.
2.2 Extraction and isolation
Air dried leaves (1.13 kg) were subjected to cold maceration with 80% v/v ethanol until exhaustion. The ethanol extract was evaporated under reduced pressure yielding a semisolid dark green extract (200 g). A portion of the extract (110 g) was dissolved in distilled water (500 mL) and successively fractionated with n-hexane (8 × 500 mL), chloroform (10 × 500 mL), ethyl acetate (6 × 500 mL), and n-butanol (4 × 500 mL). The four fractions were evaporated to yield 6 g, 8 g, 40 g and 24.5 g, respectively.
About (25 g) of the ethyl acetate fraction was dissolved in a small amount of distilled water using gentle warming and then chromatographed on 100 g polyamide (50–160 μm, Fluka Chemie GmbH, Switzerland) using distilled water only, followed by increasing a methanol portion gradually by 10% till 100% v/v. Eluates were collected to yield 50 fractions (each of 300 mL) that were examined by TLC using ethyl acetate: glacial acetic acid: formic acid: water (10: 1.1: 1.1: 2.6 v/v v/v) as a solvent system. Fractions (16–18), eluted by 50% methanol v/v, were found to contain one major fluorescent spot when examined under short and long UV light. These fractions were pooled and evaporated under reduced pressure to a buff crystalline powder that yielded (4.4 g) of phlorizin upon purification. The chemical structure of phlorizin was confirmed by means of NMR and mass spectroscopy as previously reported (Mabry et al., 1970; Xü et al., 2011).
2.3 LC-ms/ms
A ThermoFinnigan LCQ-Duo ion trap mass spectrometer (Thermo Electron Corporation, Waltham, Ma, USA) with an ESI source (ThermoQuest Corporation, Austin, Tx, USA) was used to run the phytochemical analysis for identifying the constituents of the leaf extract as previously reported (El-Hawary et al., 2020).
2.4 In vitro antioxidant activities
Total phenolic content (TPC), total flavonoid content (TFC) and DPPH assay were carried out according to Ghareeb et al. (Ghareeb et al., 2018).
2.5 In vitro BACE1 inhibition activity
The assay was carried out using ab133072 – Beta Secretase inhibitor screening assay kit, Abcam® Co. Cambridge. UK in the confirmatory diagnostic unit of VACSERA (Egypt) according to the manufacturer’s instructions.
2.6 Acute toxicity
The toxicity of the extract was evaluated in 2-month-old male and female mice with an average weight of 20 g (variation did not exceed ± 20% of the mean weight). Mice were divided into 4 groups (5 animals each), acclimated for 5 d prior to the test experiments and fasted overnight prior to dosing. The mice received a single dose (2 g/kg) of the tested extract by gavage and were observed for 14 d for changes in the ears, skin, mucous membranes, eyes, respiration, circulatory, autonomic, central nervous system, and in somatomotor activities. Behavior pattern were observed particularly for convulsions, tremors, diarrhea, salivation, lethargy, sleep, and coma. The tested extract did not show any toxicity signs up to an extract dose of 2 g/kg body weight, which indicates that it is safe and nontoxic (Buck, 1976).
2.7 In vivo biological experiments
2.7.1 Animals
Female albino Wistar rats, weighing 100–150 g, were received from the animal house of the National Organization for Drug Control and Research, Giza, Egypt. Rats were housed and maintained under standard laboratory conditions with free access to food and water ad libitum for at least 1 week before starting the behavioral test. All experimental protocols were approved by the Faculty of Pharmacy, Cairo University (FOPCU) Ethical Committee for animals use and care, Cairo University, Cairo, Egypt and the Medical Research Ethics Committee (MREC), NRC, Egypt.
2.7.2 Experimental design
Forty-eight rats were randomly allocated into six treatment groups (8 rats/ group) as follows: G1 (vehicle group): animals orally received saline daily for 9 days. G2 (dexamethasone/ scopolamine): animals orally received dexamethasone (10 mg/kg i.p.) daily for 9 days and hyoscine butylbromide (1 mg/kg. i.p.) for 2 consecutive days (day 7 and 8). G3 and G4: animals orally received leaf extract (200 mg/kg BW, 400 mg/kg BW, respectively) plus dexamethasone (10 mg/kg i.p.) for 9 days and hyoscine butylbromide (1 mg/kg. i.p.) for 2 consecutive days (day 7 and 8). G5 and G6: animals orally received phlorizin (20 mg/kg BW, 40 mg/kg BW, respectively) plus dexamethasone (10 mg/kg i.p.) for 9 days and hyoscine butylbromide (1 mg/kg. i.p.) for 2 consecutive days (day 7 and 8). We preferred to use female rats for the behavioral studies because they showed better performance than males in some specific cognitive tasks, particularly NOR (Sutcliffe et al., 2007). The rat’s ability to perform the NOR or the reversal learning tasks was not affected by the stage of the oestrous cycle (Sutcliffe et al., 2007; McLean et al., 2009).
2.7.3 Locomotor activity
The utilized test apparatus (NOR chamber) has the same layout as described before (Ennaceur and Delacour, 1988; Arnt et al., 2010), however, all details about the procedure were reported by (McLean et al., 2009; Grayson et al., 2007) and modified by (Antunes and Biala, 2012). The full procedure was carried out over three phases, i. Habituation phase: the apparatus was fully explored by each rat for 2 min on three consecutive days to exclude false or hesitated movements on the testing day, ii. Exploration trial 1 (T1) phase: 30 min after scopolamine injection, each rat was placed in two opposite corners about 6 cm off the walls of NOR chamber, the results were recorded for each rat and expressed in “T1” as obA and obB records. There was an interval period between this phase and the next one during which rats were returned to their home cages for 1 h, iii. Exploration trial 2 (T2) phase: in which one of the two identical objects mentioned above was replaced by another different one (novel object). The resident object from the previous trial was called (familiar object). The rats were permitted to explore the unlike familiar (F) and novel (N) objects, the recorded results in this phase are expressed in “T2” as N and F records. In the exploration trials we recorded sniffing, licking, touching or directing the nose for not more than 2 cm towards an object. Any rat failed exploring an object in either trial was excluded from the experiments and the box was cleaned with 10% ethanol–water solution between each other animal. Two stopwatches were used to record the exploration time(s) of each object in each trial manually and the following factors were calculated: T1 = the total exploration time of both identical objects (obA, obB) and T2 = the total exploration time of both different objects (N, F). Comparing the time spent in exploring F to that spent in exploring N led to obtaining the discrimination index (DI)= (N–F)/ (N + F). Additionally, the total number of sectors or line crossings by the animal in both acquisition and retention trials was used for recording the locomotor activity. Object exploration was determined as rats sniffing, licking, or touching the objects using their forepaws, but not standing or sitting on the objects, turning around, or leaning against them. The exploration time(s) of the objects in each trial was manually recorded.
2.7.4 Blood collection and tissue preparation
Blood samples were collected in test tubes from the rats’ retro-orbital venous plexus under ether anesthesia. After clotting, samples were centrifuged at 3000 rpm for 10 min. Separated serum was stored at − 20 °C to be used for determining TAC. After collection of blood samples, rats were sacrificed by cervical dislocation and their brains were removed immediately on ice and homogenized in ice-cold phosphate buffer saline (20% w.v). This homogenate was used for the determination of MDA, GSH, GSSG and Beta Amyloid 1–42.
2.8 Oxidative stress parameters
Total antioxidant capacity (TAC), oxidized glutathione (GSSG) were assayed colorimetrically by a kit from Bio Vesion Incorporated, San Francisco, USA). In the brain homogenate, the Rat Beta Amyloid 1–42 was determined by ELISA kit (Sandwich ELISA, Lifespan BioSciences, Seattle, USA). The lipid peroxidation marker (MDA) and reduced glutathione (GSH) were determined colorimetrically using kits of Biodiagnostic (Cairo, Egypt). All experiments were performed according to the manufacturer’s instructions.
2.9 Molecular docking
The identified X-ray crystallographic structure of BACE1 (PDB code: 4IVS) with the co-crystallized indole acylguanidine inhibitor ligand was downloaded from PDB (Protein Data Bank, www.rcsb.org). Molecular Operating Environment (MOE, 2010.10; Chemical Computing Group Inc., Montreal, Canada) software was used to run the docking experiments. The downloaded protein was prepared for docking by adding the hydrogen atoms and computing the partial charges. The chemical structures of the major identified compounds in M. domestica extract were drawn using the builder tool of MOE or downloaded directly from PubChem (https://pubchem.ncbi.nlm.nih.gov). Ionization state of every compound at pH 7.0 was checked and the partial charges were assigned accordingly. The force field MMFF94x was used to perform the energy minimization of the compounds and the default settings of MOE docking protocol including the placement method (Triangle Matcher), scoring function (London dG), and refinement were used to run the docking trials.
2.10 Statistical analysis
All results of object recognition test and antioxidative stress parameters were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism software (Version 7.00). Comparison between groups and within each group in terms of the exploration time of the identical objects in T1 and that of F and N objects in T2 was carried out using student’s t-test. Each group was initially tested for significance by two-way analysis of variance (ANOVA). In addition, One-way ANOVA was used for discrimination index and cross lining in object recognition test. All evaluations were followed by Tukey test for determination of statistical differences among the experimental groups. The difference was considered significant at p < 0.05.
3 Results and discussion
The apple leaf extract was analyzed phytochemically using LC/MS-MS. Altogether, 30 secondary metabolites were annotated according to their molecular weights and mass fragmentation patterns (Fig. 1 and Table 1). The extract is apparently rich in the dihydrochalcone phlorizin (accounting for 61% of the extract) along with several other polyphenols including phloretin, quercetin, kaempferol, and isorhamnetin glucosides. Several previously reported compounds including rutin, avicularin, quercitrin, isoquercitrin, chlorogenic acid were also detected in the extract (Lu et al., 2019; Liaudanskas et al., 2014; Rana et al., 2016).
LC-MS chromatogram of M. domestica leaf extract. Peak numbers indicate substances listed in Table 1.
No.
tR
Tentative identification
[M−H]-
MS2fragments
1
1.12
Quinic acid
191
85, 127, 173
2
1.53
Malic acid
133
115, 133
3
6.23
Phloretic acid 3-O-glucoside
327
147, 165
4
7.2
p-Hydroxybenzoic acid 3-O-glucoside
299
137, 179, 239
5
9.89
Chlorogenic acida
353
179, 191
6
10.7
Caffeoylglucose
341
179, 191
7
15.48
Sinapic acid 3-O-glucoside
385
385, 223, 161
8
15.87
Apigenin 7-O-rhamnoside
415
161, 269
9
29.78
(epi)Catechin glucoside
451
289
10
30.77
Quercetin rutinoside (Rutin)a
609
300, 301
11
31.62
Isoquercetina
463
300, 301
12
31.95
Avicularina
433
300, 301
13
33.22
Kaempferol rutinoside
593
284, 285
14
34.37
Isorhamnetin 3-O-glucoside
477
314, 315, 357
15
35.40
Quercitrina
447
179, 301
16
36.93
3-hydroxy phloretin
289
125, 167, 271
17
38.26
Kaempferol 3-O-glucoside
447
285, 286, 327
18
38.84
Phloretin-2-O-xyloglucoside
567
273, 405
19
39.71
Quercetin rhamnoside
447
179, 301
20
42.06
Phlorizina
435
167, 273,301
21
43.20
Phlorizina
435
167, 273,301
22
48.79
Eriodictyol
287
151, 269
23
51.99
Quercetina
301
151, 179, 301
24
57.48
Phloretin caffeoyl-6-O-glucoside
597
273, 323, 449
25
61.85
Phloretin coumaroyl-6-O-glucoside
581
273, 419
26
62.11
Phloretin coumaroyl-5-O-glucoside
581
273, 419
27
63.23
Phloretin feruloyl-5-O-glucoside
611
273, 337
28
67.04
Pomaceic acid
501
325, 409, 483
29
70.21
Euscaphic acid
487
471, 469, 325
30
73.26
Pomolic acid
471
453, 441, 427
3.1 Biological activities
3.1.1 In vitro activities
Natural antioxidants are key player against oxidative stress, which is considered a lead cause to many neurodegenerative conditions (Grimmig et al., 2017; Arab et al., 2016; Bashandy et al., 2020). The extract and its major compound phlorizin revealed substantial antioxidant activity in DPPH assay and BACE1 inhibitory activity (Table 2). Phlorizin inhibited the β-secretase activity with an IC50 of 1.18 µg/ ml, an effect that is comparable to the strongest natural BACE 1 inhibitor, trihydroxychalcone (Zhang, 2012). Total phenol and total flavonoid contents of apple leaf extract were found to be 245 mg gallic acid equivalent/g in Folin-Ciocalteu analysis and 108.19 mg/quercetin equivalent in FeCl3 assay, respectively. Similar results had been reported from the extract of apple leaves before (Lu et al., 2019; Liaudanskas et al., 2014).
Sample
DPPH
BACE 1 inhibition
IC50 µg/ ml
Extract
6.93 ± 18
1.65
Phlorizin
5.14 ± 21
1.18
Ascorbic acid
4.06 ± 03
–
Reference compound
–
0.0145
3.1.2 In vivo antioxidant activity
Injection of dexamethasone/ scopolamine reduced TAC and GSH by 81% and 64%, respectively and increased MDA and GSSG by 186% and 300% respectively as compared to the untreated group. As expected, the extract and phlorizin significantly counteracted the deleterious effects of dexamethasone/ scopolamine in rats (p-value < 0.001). The extract (200 mg/kg) elevated TAC by 19% and decreased MDA and GSSG by 92% and 215% and finally increased GSH by 27%. However, TAC was elevated by 33% and MDA and GSSG were reduced by 132% and 255% and finally, GSH was increased by 38% upon using the high dose (400 mg/kg) of the extract. In case of phlorizin, TAC was increased by 29% and MDA and GSSG were reduced by 107% and 251% with the low dose (20 mg/kg) while GSH was increased by 36%. With the high dose (40 mg/kg), TAC was elevated by 48%, MDA and GSSG were decreased by 138% and 291% and GSH was increased by 43%, (Table 3). Data are expressed as the mean ± SEM (n = 8) and were analyzed by ANOVA followed by post-hoc Tukey test. a Statistically significant from control negative at p < 0.001. b Statistically significant from dexamethasone/ scopolamine at p < 0.001.
Group
TAC
MDA
GSH
GSSG
nmol/µL
nmol/mg protein
µmol/mg protein
µg/mg protein
Control (vehicle)
2.01 ± 0.06
12.46 ± 0.73
134.32 ± 2.81
0.45 ± 0.02
Dexamethasone (10 mg/kg)/ scopolamine (1 mg/kg)
0.38 ± 0.07a
35.60 ± 0.61a
48.66 ± 1.93a
1.8 ± 0.10a
Extract
200 mg/kg
0.77 ± 0.03a
24.11 ± 0.39ab
84.99 ± 1.90ab
0.83 ± 0.01ab
400 mg/kg
1.05 ± 0.04ab
19.2 ± 0.44ab
99.49 ± 1.74ab
0.65 ± 0.02b
Phlorizin
20 mg/kg
0.97 ± 0.07ab
22.23 ± 0.28ab
97.66 ± 2.01ab
0.67 ± 0.01b
40 mg/kg
1.35 ± 0.11ab
18.40 ± 0.35ab
106.65 ± 2.36ab
0.49 ± 0.01b
3.1.3 Beta amyloid (Aβ) 1–42
Several studies have highlighted the strong link between the oxidative stress and the etiology of Alzheimer’s disease. ROS, in particular the hydroxyl radical which is the most reactive one, may contribute to oxidative damage through its impact on both the (Aβ) peptide itself and the surrounding molecules (proteins, lipids) (Cheignon et al., 2018). Injection of dexamethasone/ scopolamine elevated (Aβ) by 207% as compared to the untreated group. The extract and phlorizin significantly counteracted the deleterious effects of dexamethasone/ scopolamine in rats in a dose dependent manner (Fig. 2).
Assessment of Beta Amyloid 1–42 (pg/mg protein) in brain homogenates. Data are expressed as the mean ± SEM (n = 8) and were analyzed by ANOVA followed by post-hoc Tukey test. aStatistically significant from control (p < 0.001). bStatistically significant from dexamethasone/ scopolamine (p < 0.001).
3.1.4 In vivo locomotor activity
The studied extract (200 mg and 400 mg/kg, p.o.) and its major compound phlorizin (20 mg and 40 mg/kg, p.o.) were able to reverse the rats’ dexamethasone/scopolamine-induced cognitive impairment in the NOR test. These results confirm the pro-cognitive effect of the present extract and its major compound phlorizin associated with modulating cholinergic mechanisms. Previous studies suggested that pro-cognitive effect could be due to reversing the cognitive impairment caused by the muscarinic receptor antagonist scopolamine, whose effect mimics that of the reported 5-HT6 receptor antagonists (Woolley et al., 2003). The vehicle saline-treated rats easily differentiated between the familiar and novel objects during exploration trial 2, in which they obviously directed exploration towards the novel object (Fig. 3b, p < 0.05). Sub-chronic dexamethasone/ scopolamine treatment impaired the response in the NOR test. This was shown as discrimination failure between familiar and novel objects in the exploration trial T2 (Fig. 3b, d). These actions were restored by pre-treatment with the extract (200 and 400 mg/kg BW, p.o.) which resulted in a dose dependent reversal of the dexamethasone/scopolamine-impaired response. While the low dose (200 mg/kg BW) was less effective, the 400 mg/kg dose showed full restoration of the normal levels seen in the vehicle control group, p < 0.001 (Fig. 3b, d). No significant changes were observed in initial exploration nor locomotor activity (Fig. 3a, c). The pre-treatment with both doses of phlorizin (20 and 40 mg/kg BW, p.o.) induced the reversal effect of dexamethasone/scopolamine-impaired responding. Unlike the parent extract, both phlorizin doses did not show significant changes in locomotor activity (Fig. 3a, c) confirming that both doses restored preferential investigation of the novel object in the choice trial following muscarinic receptor antagonism impairment.
(a) Mean exploration time of identical objects in the 3-min acquisition phase of the novel object recognition (NOR) task after treatment with the extract (200 and 400 mg/kg, p.o.) and phlorizin (20 and 40 mg/kg, p.o) in sub-chronic dexamethasone/scopolamine. (b) The ability of the extract (200 and 400 mg/kg, p.o.) and phlorizin (20 and 40 mg/kg, p.o) to reverse the effect of sub-chronic dexamethasone/scopolamine on the exploration time(s) of a familiar object and a novel object in the 3-min retention trial in female rats. (d) The effect of the extract (200 and 400 mg/kg, p.o.) and phlorizin (20 and 40 mg/kg, p.o) in combination with sub-chronic dexamethasone/scopolamine on the discrimination index (DI). (c) The effect of the extract (200 and 400 mg/kg, p.o.) and phlorizin (20 and 40 mg/kg, p.o) in combination with sub-chronic dexamethasone/scopolamine on the total number of line crossings in the NOR task (acquisition + retention trial). Data are expressed as the mean ± SEM (n = 8) and were analyzed by ANOVA followed by post-hoc Tukey test. aStatistically significant from control negative (p < 0.001). bStatistically significant from dexamethasone/scopolamine (p < 0.001).
Our experiments revealed that the groups treated with phlorizin exhibited a higher discrimination index than those of dexamethasone/scopolamine group and less line crossings than dexamethasone/scopolamine group. This indicates that rat groups treated with phlorizin exhibited better behavioral activities, which explains better neural functions and finally confirms the neuroprotective effect of phlorizin. These promising pro-cognitive results are proportionally linked to the antioxidative state achieved by the extract and its major compound phlorizin. Previous studies assumed that the antioxidant ingredients of herbal treatments rich in polyphenols such as proanthocyanidins among others have a significant role in modulating the brain oxidative stress induced by large dose of dexamethasone and/or scopolamine (Assaf et al., 2012; Xiao et al., 2015 Aug; Goverdhan et al., 2012).
3.2 Molecular docking
β-Secretase represents the key element in generating the N-terminal of β-amyloid, which further forms the amyloid plaques considered to be a hallmark in AD (Zou et al., 2013). Docking the major identified compounds from the extract was carried out to obtain some insights in their binding mode and binding energy towards β-secretase. The study aims as well to virtually screen components of the extract as β-secretase inhibitors to help spot some lead compounds for designing novel inhibitors of natural origin.
Table 4 shows the docking scores that reflect the binding energy of the docked compounds towards the target enzyme. Except for the triterpenoids euscaphic and pomolic acids, the rest of compounds which were docked to β-secretase fitted well in the binding site and iterate the majority of the amino acid interactions reported with the co-crystallized ligand and other inhibitors such as the H-bonding interactions with Asp32, Asp228, Gly34, Arg235, and the hydrophobic interactions with Tyr71 and Thr72 (Zou et al., 2013; Yen et al., 2019).
Compound
Docking score (kcal/mol)
Eriodyctiol
−10.53
Isorhamnetin
−9.70
Kaempferol
−10.82
Quercetin
−10.72
Phloretin
−9.45
3-Hydroxyphloretin
−12.31
Phloretin-2-O-glucoside (Phlorizin)
−16.21
Phloretin-2-O-xyloglucoside
−14.99
Phloretin caffeoyl-6-O-glucoside
−13.50
Phloretin coumaroyl-6-O-glucoside
−14.12
Phloretin coumaroyl-5-O-glucoside
−15.04
Phloretin feruloyl-5-O-glucoside
−12.91
Quercetin-3-O-glucoside
−13.94
Quercetin-3-O-rhamnoside (Quercetrin)
−13.70
Quercetin-3-O-rutinoside (Rutin)
−16.51
Quercetin-3-O-arabinofuranoside (Avicularin)
−13.23
Kaempferol-3-O-glucoside
−13.45
Isorhamnetin-3-O-glucoside
−14.29
Apigenin-7-O-rhamnoside
−10.36
1-Caffeoylquinic acid
−12.12
2, 2′, 4′-trihydroxychalcone
−8.85
Co-crystallized ligand
−17.47
In general, the glucosides showed higher binding energy than the corresponding aglycones owing to their bulkier structures and more hydroxyl groups that afforded more H-bonding interactions with the amino acids in the binding site of the enzyme. This is obvious when comparing the docking scores of quercetin (-10.72 kcal/mol) and phloretin (- 9.45 kcal/mol) to that of some quercetin glucosides such as quercetrin, rutin, avicularin, and phloretin glucosides such as phlorizin (Table 4).
Rutin and phlorizin showed the best docking scores (- 16.51, − 16.21 kcal/mol, respectively), reflecting the least binding energy to β-secretase and exhibited a binding mode that afforded most of the reported interactions with the amino acid residues in the binding site such as Asp32, Asp228, Thr 231, and Arg235 (Fig. 4).
3D-binding mode of phlorizin (left) and rutin (right) to the binding site of β-secretase.
Because most of the phenolic hydroxyl groups are ionizable at the physiological pH, we considered docking the phenolate ions of the compounds used in the docking study. They showed similar binding and interactions in the binding site of β-secretase, in addition to some salt bridge interactions with some basic amino acid residues such as arginine and lysine. The natural inhibitor 2, 2′, 4′-trihydroxychalcone demonstrated a docking score of − 8.85 kcal/mol. It showed two interactions (H-bonding with ASP32 and Tyr198). This weak performance in docking could be explained by fewer OH groups and more rigid structure due to the double bond.
4 Conclusions
LC-MS/MS analysis of apple leaf extract revealed that it is a substantial source of potent polyphenolics specially phlorizin. The extract and phlorizin exhibited strong antioxidant and BACE1 inhibitory activities. Thus phlorizin is not only a candidate for a potential prophylactic neuroprotective agent but also for treatment that can suppress and delay the onset of neurodegenerative diseases such as AD. Clinical trials of phlorizin are highly recommended in order to better understand its pharmacokinetic and pharmacodynamic as a necessary requirement for its pharmaceutical use.
Funding
This research received no external funding.
Acknowledgments
The authors would like to thank Dr. Abdel Halim Abdel Mogaly, the Agricultural Museum taxonomist, Giza, Egypt for authenticating the plant material. The APC was paid by UM6P.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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