Synthesis, characterization and biological evaluation of aurones as potential neuroprotective agents

Abstract

Aim: To synthesize aurone (Ar) derivatives and to demonstrate their effects against diabetes mellitus (DM) and neurodegeneration.

Materials & methods: Five Ar (A–E) derivatives were synthesized, characterized by proton NMR and screened for antioxidant, anti-diabetic and anti-cholinesterase activities. They were further evaluated for neuroprotective effects in streptozotocin (STZ)-induced neurodegenerative model.

Results: Among the aurone derivatives ArE demonstrated significant reversal of cognitive impairment, oxidative stress and neuroinflammation. Biochemical analysis revealed anti-diabetic and neuroprotective effects, possibly through downregulation of inflammatory markers and upregulation of antioxidant enzymes.

Conclusion: Synthesized Ar (A–E) exhibits promising therapeutic potential against STZ-induced neurodegeneration and DM by modulating inflammatory and oxidative pathways, suggesting a novel avenue for disease management.

Graphical Abstract

Plain language summary

Article highlights.

• Synthesis and characterization of aurone derivatives.

• In vitro evaluation of synthesized aurones for anti-oxidant, anti-diabetic and anti-cholinesterase activities.

• The synthesized aurones reduced neurodegeneration in the streptozotosin induced memory impairment model.

• Among ArC, ArD and ArE demonstrated significant effects in reducing neuroinflammation.

1. Background

Neurodegenerative diseases (NDs) pose a significant risk to the elderly and are projected by the World Health Organization (WHO) to surpass cancer within the next two decades [1,2]. Alzheimer's disease (AD) and Parkinson's disease (PD) are the most prevalent neurological disorders, which are characterized by a variety of pathological processes and accompanied by cognitive declines, movement difficulties and other impairments [3,4]. Moreover, these disorders exhibit shared mechanisms that contribute to neuronal dysfunction and deterioration, such as oxidative stress, free radical production, neuroinflammation, misfolding and aggregation of proteins, impaired energy production and mitochondrial dysfunction [5,6]. The primary cause of dementia in the elderly is AD which is marked by the loss of neurons and synapses in the cerebral cortex and subcortical areas [7]. As per the findings of Alzheimer's Disease International (ADI), the global prevalence of dementia exceeds 50 million people and by the year 2050, dementia is predicted to affect as many as 131.5 million people [8].

Diabetes elevates the mortality rate by 1.5–three-times and is both an independent risk factor and a major co-morbidity of neurological and functional disabilities [9,10]. Interestingly, research has shown that roughly 20% of NDs are linked to diabetes [11]. Furthermore, individuals affected by neurodegenerative disorders display notably higher rates of diabetes mellitus (DM), although precise comprehensive statistics are currently unavailable.

Neuroinflammation is linked to the degeneration of neurons, leading to disruptions of synaptic activities and memory functions. Numerous studies suggest that neuroinflammation induced by lifestyle factors worsens the progression of AD pathologies and dementia via associations with environmental factors and aging [12–14]. Excessive accumulation of reactive oxygen species (ROS) induces neuroinflammation via oxidative stress, which may lead to lipid peroxidation. In this state, mitochondrial dysfunction boosts ROS generation and diminishes levels of natural antioxidants such as glutathione S-transferase (GST) and glutathione (GSH) [15,16]. Furthermore, the progression of AD is significantly mediated by oxidative stress and inflammation [17]. In AD, extensive neurodegeneration results from an inflammatory response, as several studies linked the exacerbation of AD to the release of inflammatory mediators, including ILs, TNF-α and NF-κB [18]. Moreover, the ‘one-molecule and one-target’ approach appears less effective in addressing conditions like AD as this approach might alleviate clinical symptoms, but it fails to impede the progression of the disease. Therefore, it is essential to develop multi-target-directed ligands (MTDLs) to develop novel drugs that have the potential to alleviate complex conditions by integrating two or more biological functions [19]. Numerous heterocyclic compounds have been shown to play a substantial role in preventing the progression of AD, and experimental studies indicate that a number of these compounds have a positive effect on cognitive function [20,21].

Heterocyclic compounds, such as aurones, have gained attention in AD research due to their potential neuroprotective properties. Aurones are classified as a subset of flavonoids that occupy a unique position in the natural world. They are classified as tricyclic flavonoids in which a double bond connects the phenyl group to the benzofuranone ring [22]. Research has shown the promise of aurones as a therapeutic agent for AD, cancer, obesity and antiviral [23,24]. Flavonoids possess antioxidant and anti-inflammatory properties that are potentially beneficial in conditions like AD, where oxidative stress and neuroinflammation play critical roles [25–27]. In recent years, aurones have garnered significant attention due to their diverse bioactivities linked to neurological conditions, particularly AD. Numerous studies have highlighted the robust affinity of both naturally occurring aurones and their chemically synthesized counterparts toward amyloid-beta (Aβ) aggregates [28,29], along with potent monoamine oxidase (MAO) inhibitory activities [30,31]. Lunven et al. reported that aurone derivatives exhibit the potential to prevent the formation of tau aggregates and the progression of neurodegenerative disorders including Alzheimer's disease [31]. In another study, the inhibitory activities of aurones against MAO and Aβ were evaluated [32]. Aβ is a prominent feature of AD due to its presence in the brain's extracellular plaque. Elevated Aβ levels correspond with the cognitive decline in AD. Sporadic AD is linked to deficient Aβ clearance, while familial AD is associated with increased Aβ production [33]. Activated MAO plays a crucial role in AD pathogenesis, contributing to amyloid plaque formation, neurofibrillary tangles (NFTs) and cognitive decline. MAO inhibitors have shown promise in improving cognitive deficits and reducing Aβ pathology by regulating amyloid precursor protein cleavage and decreasing Aβ protein fragments [34]. Aurone structure holds a promise for crafting multifaceted drugs targeting AD. A recent study by Masahiro Ono et al. introduced an analog of aurone featuring a dimethylamino group at 4-position and radioiodine at 5-position. These analogs emerged as a compelling probe for Aβ plaques in AD, demonstrating a good binding affinity (Ki = 6.82 nM) [35]. Furthermore, differently structured aurones previously demonstrated anticholinesterase inhibitory and antioxidant effects [36,37].

Keeping in view the important role, we formulated a range of aurone derivatives envisioned to act as versatile agents, exhibiting anti-diabetic, anti-Alzheimer, antioxidant and anti-inflammatory properties. The derivatives featured in our study comprise new compounds and their evaluation as multifunctional anti-AD agents has not previously been documented.

2. Materials & methods

2.1. Chemical & solvent

All the solvents and chemicals used in this work are of analytical grade that includes 2-hydroxy acetophenone (118-93-4, Sigma-Aldrich, MO, USA), Benzaldehyde (100-52-7, Sigma-Aldrich), p-tolu aldehyde (104-87-0, Sigma-Aldrich), 4-methoxy benzaldehyde (123-11-5, Sigma-Aldrich), 4-chloro benzaldehyde (104-88-1, Sigma-Aldrich), 4-(Dimethylamino) benzaldehyde (100-10-7, Sigma-Aldrich), mercuric acetate (1600-27-7, Sigma-Aldrich), ethanol (64-17-5, Sigma-Aldrich), methanol (67-56-1, Sigma-Aldrich), pyridine (110-86-1, Sigma-Aldrich), tween 80 (9005-65-6, Sigma-Aldrich), streptozotocin (18883-66-4, Sigma-Aldrich), DPPH (1898-66-4, Sigma-Aldrich), ABTS (30931-67-0, Sigma-Aldrich), ascorbic acid (50-81-7, Sigma-Aldrich), alpha-amylase (9000-90-2, Sigma-Aldrich), alpha-glucosidase (9001-42-7, Sigma-Aldrich), glibenclamide (10238-21-8, Merck, Darmstadt, Germany). Acetylcholine esterase from Electric eel (9000-81-1, Sigma-Aldrich), buteryl choline esterase from equine serum (9001-08-5, Sigma-Aldrich), acetylthiocholine iodide (1866-15-5, Sigma-Aldrich), butyrylthiocholine Iodide (1866-16-6, Sigma-Aldrich), 5,5-dithio-bis-nitrobenzoic acid (69-78-3, Sigma-Aldrich), galantamine hydrobromide from Lycoris sp. (1953-04-4, Sigma-Aldrich).

2.2. General procedure for the synthesis of aurone (A–E)

The synthesized aurones Ar (A–E) were synthesized using the Claisen-Schmidt reaction for 2-hydroxy chalcone derivatives [38]. Briefly, chalcones were produced by reacting equimolar quantity of 2-OH acetophenone and their respective benzaldehyde derivatives in ethanol using a 40% NaOH solution as a catalyst. After completion, the solution was poured into ice-cold water and acidified with dilute 1N HCl. Filtration of the precipitated product was done by washing it with cold water and dried to achieve the desired product. In the next step, to a solution of mercuric acetate in pyridine, the already prepared chalcone was added with stirring, followed by refluxing the mixture for 1 h. The reaction mixture was cooled down and poured into ice-cold water followed by acidification with dilute 1N HCl. Filtration of the precipitated solid was done by washing it with cold water and dried to obtain the desired products [39].

2.3. Synthesis of (E)-2-benzylidenebenzofuran-3(2H)-one (ArA)

Yield: 82% (120 mg), light yellow powder, melting point: 100–101°C, solubility: chloroform, ethanol, methanol and ethyl acetate. ¹H NMR (300 MHz, CDCl₃): δ 8.86 (s, 1H. Ar-H), 7.96 (d, 1H. Ar-H), 7.85 (d, 1H. Ar-H) 7.82 (d, 1H. Ar-H), 7.35–7.66 (m, 4H. Ar-H), 6.93 (s, 1H C=CH) [40].

2.4. Synthesis of (E)-2-(4-methylbenzylidene)benzofuran-3(2H)-one (ArB)

Yield: 73% (102 mg), yellow powder, melting point: 93–94°C, solubility: chloroform, ethanol, methanol and ethyl acetate. ¹H NMR (300 MHz, CDCl₃): δ 7.86 (d, 1H. Ar-H), 7.84 (d, 1H. Ar-H), 7.81 (d, 1H. Ar-H), 7.30 (d, 2H. Ar-H), 7.26 (d, 1H. Ar-H), 6.93 (s, 1H C=CH), 2.43 (s, 3H, CH₃) [40].

2.5. Synthesis of (E)-2-(4-methoxybenzylidene)benzofuran-3(2H)-one (ArC)

Yield: 75% (105 mg), yellowish-green powder, melting point: 135–136°C, solubility: chloroform, ethanol, methanol, ethyl acetate. ¹H NMR (300 MHz, CDCl₃): δ 7.95 (d, 1H. Ar-H), 7.84 (d, 1H. Ar-H), 7.69 (d, 1H. Ar-H), 7.36 (d, 1H. Ar-H), 7.21 (m, 2H. Ar-H), 7.027 (d, 2H. Ar-H), 6.92 (s, 1H C=CH), 2.89 (s, 3H, OCH₃) [40].

2.6. Synthesis of (E)-2-(4-chlorobenzylidene)benzofuran-3(2H)-one (ArD)

Yield: 80% (117 mg), yellow powder, melting point: 156–157°C, solubility: chloroform, ethanol, methanol and ethyl acetate. ¹H NMR (300 MHz, CDCl₃): δ 7.86 (d, J = 8.4 Hz, 1H), 7.82 (ddd, J = 7.5, 1.5, 0.9 Hz, 1H), 7.76 (ddd, J = 8.4, 7.5, 1.5 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.38–7.31 (m, 1H), 7.23 (dd, J = 7.5, 0.9 Hz, 1H), 6.84 (s, 1H) [41].

2.7. Synthesis of (E)-2-(4-dimethylamino-benzylidene)benzofuran-3(2H)-one (ArE)

Yield: 78% (110 mg), reddish brown powder, melting point: 150–151°C, solubility: chloroform, ethanol, methanol and ethyl acetate. ¹H NMR (CDCl3, 400 MHz): d 3.10 (s, 6H), 6.82 (d, J = 8.7 Hz, 2H), 6.94 (s, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.34 (d, J = 8.3 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.83 (d, J = 7.0 Hz, 1H), 7.88 (d, J = 8.9 Hz, 2H) [42].

2.8. Assessment of anti-oxidant activity

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were used to evaluate the free radical scavenging activity of the synthesized aurone analogs in vitro by following standard protocol assays [43,44]. Different dilutions of the positive control and test samples were prepared in methanol at a concentration ranging from 31.25 to 1000 μg/ml. A 0.002% DPPH methanol solution was prepared, and 1 ml of DPPH solution was added to 1 ml of already prepared concentrations, i.e. sample solutions and positive control solution for individual testing. These solutions were then kept for about 30 min in a dark place. The absorbance of each sample was noted against methanol using a spectrophotometer and DPPH solution as a blank at 517 nm wavelength. The value of IC₅₀ was measured for the test sample. The negative control used was 1 ml of 0.002% DPPH solution. Separate preparation of about 7 mM (383 mg) ABTS and 2.45 mM (66.2 mg) K₂S₂O₈ in methanol (100 ml) was done in the ABTS method followed by proper mixing. To the working dilutions prepared in the above step, 2 ml of ABTS was added. The absorbance was measured at 745 nm after 25 min of incubation and the percent inhibitory activity and IC₅₀ were calculated.

2.9. Enzyme inhibitory activities

The evaluation of enzyme inhibitory activity was performed on two separate enzymes, namely α-amylase and α-glucosidase. For α-amylase, the established protocols were followed with some modifications [45]. 40 μl amylase solution was added to 40 μl of the test sample from different concentrations ranging from 31.25 μg/ml to 1000 μg/ml, followed by incubation at 37°C for 30 min. Then, 40 μl of starch solution (0.1%) was added to this mixture followed by the addition of 1M HCl. Finally, iodide solution was added and at 580 nm absorbance was recorded. Acarbose was used as a positive control and IC₅₀ and percent inhibitory activity were calculated.

Alpha-glucosidase inhibitory activity was carried out according to the Pistia-Brueggeman and Hollingsworth protocol [46]. The synthesized compounds of various dilutions ranging from 31.25 μg/ml to 1000 μg/ml were incubated at 37°C with an already prepared solution of α-glucosidase, followed by the addition of 1M of 4-Nitrophenyl β-D-glucopyranoside (pNPG) as a substrate to initiate the reaction and then finally incubated for 30 min. To stop the reaction, 0.1N Na₂CO₃ was added. The absorbance was recorded at a wavelength of 405 nm. Acarbose was used as a positive control and IC₅₀ and percent inhibitory activity were calculated.

2.10. Assessment of choline esterase enzyme inhibition activity

For cholinesterase inhibitory potentials, the synthesized compounds were tested as per Ellman's assay [47]. Hydrolysis of acetylthiocholine iodide (ATCI) or butyrylthiocholine iodide (BTChI) by specific acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes is the basis of this test after the addition of the 5-thio-2-nitrobenzoate anion forming a yellow complex. Briefly, the aurone analogs were dissolved in methanol at concentrations of 62.5 to 1000 μg/ml. AChE (518 U/mg) and BChE (7–16 U/mg) were freshly prepared. Meanwhile, distilled aqueous solutions of BTChI (0.5 mM), 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) (0.2273 mM) and ATCI were prepared and stored in airtight Eppendorf tubes for 15 min in a refrigerator at 8°C as the final solution. In methanol, the positive control drug (galantamine) was dissolved at the same concentration.

2.11. Molecular docking

From the protein data bank, the 3D structures of human pancreatic α-amylase (Protein Data Bank [PDB] ID: 2QV4) and α-glucosidase from sugar beet (PDB ID: 3W37) were downloaded. The structure was cleaned of heteroatoms, water molecules and other unwanted compounds. 2D structure of synthetic compounds (A, B, C, D, E) was prepared in Discovery Studio v4.5 (DS). The promising best pose of α-amylase (PDB ID: 2QV4) was obtained which were ILE5, TRP58, TRP59, TYR62, GLN63, HIS101, GLY104, VAL107, TYR151, LEU162, ARG195, ASP197, ALA198, HIS201, GLU233, ILE235, PHE256, ASN298, ASP300, ASN301, HIS305, GLY306 and ALA307. Similarly the actives sites were LYS156, SER157, TYR158, VAL232, ASP233, SER240, ASP242, PRO243, LEU246, HIS280, SER304, ASP307, VAL308, GLY309, THR310, SER311, PRO312, LEU313, PHE314, ARG315, TYR316, VAL319, PRO320, PHE321, ASP325, ALA329, GLU411 and ASN415 for α-glucosidase.

3. In vivo biological activities

3.1. Experimental animals

From the National Institute of Health (NIH), adult male Sprague Dawley rats were obtained, weighing 210–230 g, 8–9 weeks old and were homed in a standard laboratory environment (temperature range 21–23°C; humidity 40–60%; 12:12 h light/dark cycling; allowed access to water and food). NIH Guidelines for the Care and Use of Laboratory Animals were followed for procedures involving animals and their care. According to the guidelines given in ‘Animals Byelaws 2008 of the University of Malakand (Scientific Procedures Issue-I)’, the Ethics Committee of the Department of Pharmacy approved the study based on the Animals Byelaws 2008 University of Malakand and awarded Notification No: Pharm/EC-Aur/07-22/110.

3.2. Acute toxicity studies

To assess the acute toxicity of the synthetic compounds, we utilized 10 nulliparous nonpregnant female rats, after their division into treatment and control groups. Following an overnight deprivation of water and food, one rat received a limited oral dose of 500 μmg/kg in adherence to OECD guidelines 425 [48,49]. After a 24-h observation period to ensure survival, the same procedure was repeated for the remaining rats. Initial monitoring spanned 48 h for any distress or mortality indicators, followed by daily observations over 14 days for signs of toxicity such as squinted eyes, salivation, writhing, tremors, loss of fur, altered behavior, convulsions, stress and mortality. On the 15th day, a cardiac puncture was performed to collect blood samples for various biochemical analyses encompassing liver and renal function tests, as well as a hematological profile.

3.3. Streptozotocin-induced diabetic neurodegenerative model

Sprague-Dawley rats were taken and placed under standardized conditions (12 h light/dark cycle, 25°C, 35–60% humidity) and fed on a high-fat animal diet and water ad libitum for 21 days. On the 22nd day of the protocol, rats were intraperitoneally injected with streptozotocin (STZ) at a dose of 50 mg/kg. 48 h after STZ treatment, rats were fasted overnight and blood glucose levels were assessed from the tail vein using a glucometer to verify diabetes. Once the glucose level exceeded 400 mg/dL, type II diabetes was identified. Following this, animals were continued on their diet and left for 4 weeks for the development of memory impairment (neurodegeneration), and glucose levels were measured during each week (1st week [day 7], 2nd week [day 14], 3rd [day 21] and 4th [day 28] after induction). Four weeks after STZ, the diabetic control group received a citrate buffer for another two weeks, while the treated group received the test drugs at a dosage of 40 mg/kg. Normal control groups were treated with a combination of citrate buffer and normal saline (containing tween 20).

3.4. Animal grouping & treatment

Group I was considered the normal control group that received tween 80 in normal saline, Group II was considered as a disease group (diabetic control), group III–VII were treated groups and received ArA, ArB, ArC, ArD and ArE at doses of 40 mg/kg respectively and group VIII receive the standard drug (glibenclamide) at a dose of 5 mg/kg per body weight.

4. Behavioral studies

4.1. Novel object recognition test

In adult male rats, this procedure is carried out to find the effect of compounds on novel object recognition test (NOR). Briefly, an open plexiglass box (33 × 33 × 20 cm) and different materials (different in texture and shape) were used for the experiment. Exploratory behaviors were defined as observations where the rat's nose was oriented toward the object within a distance of 2 cm, and the rat was positioned directly in front of the object. A video camera was used to record all locomotor activities (during the dwell time) and time spent with each object. One day before the training step, the habituation training of the NOR task started. Then, in the absence of objects each rat was introduced into the box for 10 min for habituation, and locomotor activity was recorded. The rats were then subjected to a training session 24 h later in which 2 same objects were located in the box to explore the objects and the total time spent was recorded over 10 min. The rats were returned to the same task in the retention session, the following day after the training endeavor, but one of the identical objects was replaced by a new object. Discrimination index DI = (N - F)/(N + F), was used for the calculation of recognition memory where N is time spent exploring the new object and F is time spent exploring the familiar object in retention sessions [50].

4.2. Y-maze test

The Y-maze test has been categorized as a task to assess spatial learning and memory, as well as a working memory test [51,52]. The memory function of the Y-maze task is that rats must not forget which arm was last visited and therefore rat has to switch arms [53]. Studies have shown that hippocampus-related cognitive impairment is associated with rats performing this task [54]. The maze has a central arena and three branches that are connected. In a randomized order each animal was tested once. After 15 min of acclimatization to the test enclosure, the animals were placed on an arm alone and allowed to cross the arm for 8 min. Arm entry is counted when the hind paw is completely under the arm. Change is defined as access to the three arms of the overlapping triad (i.e., A, B, C, B, C, A, etc.).

4.3. Biochemical studies

The rats were weighed and anesthetized at the end of the experiment, through an intraperitoneal injection of a xylazine/ketamine mixture. About 2 ml of blood samples were obtained via cardiac puncture to study blood parameters. Plasma was separated by centrifugation at 4000 rpm for 10 min and afterward stored at -70°C in a freezer for subsequent analysis. Brain tissues were isolated in 0.1M phosphate buffer saline and were homogenized for further polymerase chain reaction (PCR) studies.

4.4. Assessment of anti-oxidant & liver function test

Various liver function tests (LFTs) were conducted on different groups of animals, examining parameters like total bilirubin, urea, cholesterol, alanine transaminase (ALT), creatinine, total glyceride, alkaline phosphatase (ALP), uric acid and aspartate aminotransferase (AST) using blood samples. Additionally, antioxidant parameters such as GSH, GST, Catalase and lipid peroxidation (LPO) were evaluated using brain tissue from these diverse animal groups.

4.5. Real-time polymerase chain reaction

As discussed previously, from the rat cortical tissue, total RNA was extracted in TRIzol. M-MuLV reverse transcriptase (20 μl) was used to dilute 1 microgram of RNA and then mix to synthesize cDNA with the help of a cDNA synthesis kit (vivantis cDSK01-050 Sdn. Bhd, Malaysia). Real-time PCR was performed to estimate the gene expression quantitatively, using the 2X HOT SYBR Green qPCR master mix (Solar Bio cat #SR1110) and real-time Mic PCR (BioMolecular System) according to the specifications of the manufacturer.

4.6. Enzyme-linked immune-sorbent assay

Following the instructions of the manufacturer, rat enzyme-linked immune-sorbent assay (ELISA) kits for TNF-α and rat IL-1β were purchased from Elabscience Biotechnology and Yuchun Biotechnology, Shanghai, China, respectively. Brain tissue was homogenized, and concentrations of respective proteins were determined in 96-well plates. Following the reaction between the enzyme as well as substrate, the concentrations of TNF-α and rat IL-1 were assessed using an ELISA microplate reader. Picograms of cytokines/milliliter (pg/ml) were used to represent values and each procedure was run in triplicate.

4.7. Statistical analysis

Data were expressed as mean ± SEM and subjected to analysis of variance (ANOVA), followed by a post hoc Tukey test to identify significant differences for each measurement. Results were considered significant when p < 0.05.

5. Results

5.1. Synthesis of aurones

Five new aurone derivatives Ar (A–E) were synthesized (Figure 1). Initially, chalcones were obtained by reacting equimolar amounts of 2-hydroxyacetophenone and the corresponding benzaldehyde derivatives in ethanol, utilizing a 40% NaOH solution as a catalyst. Subsequently, the resultant mixture was refluxed with mercuric acetate solution in pyridine while being stirred continuously for 60 min. The resulting reaction mixture was then cooled and poured into ice-cold water (50 ml), followed by acidification with a 1N HCl solution. The precipitated solid was filtered, and washed with cold water and the pure products were obtained after drying.

Figure 1.

Synthesis of aurone derivatives.

The chemical conversion of basic chalcones into aurones is appealing because it can address the limitations associated with enzyme catalysis [55]. However, the catalytic conversion of chalcones into aurones has not been widely reported. We have reported here the synthesis of various aurone derivatives, which were further tested for their anti-diabetic and anti-oxidant potential.

5.2. Screening for anti-oxidant potential

The synthesized Ar (A–E) were screened for antioxidant potential using DPPH and ABTS free radicals in vitro assay and it was recorded that among the synthesized compounds ArC showed a prominent response with IC₅₀ value of 8 ± 2 μg/ml followed by ArD with IC₅₀ 12 ± 2 μg/ml and then ArB IC₅₀ 13 ± 2 μg/ml when compared with positive control against DPPH free radical (Table 1). The other compounds ArA and ArE showed moderate antioxidant response, while ascorbic acid was a positive control having an IC₅₀ value of 5 ± 1 μg/ml. Similarly, in the ABTS assay compound ArC again showed maximum antioxidant response among the synthesized aurone derivatives having IC₅₀ 12 ± 2 μg/ml followed by ArD IC₅₀ 16 ± 3 μg/ml and then ArE IC₅₀ 18 ± 2 μg/ml, while compound ArA and ArB showed poor antioxidant response. The positive control ascorbic acid IC₅₀ 4 ± 2 μg/ml also showed a prominent response against ABTS free radical.

Table 1.

Antioxidant potential of all synthesized Ar (A–E) using the 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) assay. 

Sample	DPPH IC50 (μg/ml)	ABTS 50 (μg/ml)
ArA	47 ± 2	36 ± 2
ArB	13 ± 2	30 ± 2
ArC	8 ± 2	12 ± 2
ArD	12 ± 2	16 ± 3
ArE	20 ± 2	18 ± 2
Ascorbic acid	5 ± 1	4 ± 2

Data is presented in the form of mean ± SEM after analysis by one-way ANOVA.

ArC exhibited a significant response, followed by ArD in DPPH and ABTS antioxidant assay.

ANOVA: Analysis of variance; DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid).

5.3. In vitro anti-diabetic assay of the synthesized aurones

In vitro, anti-diabetic assay of the synthesized Ar (A–E) was performed against α-amylase and α-glucosidase enzymes (Table 2). During the α-amylase assay, it was noted that among the synthesized compounds ArC showed more potential having an IC₅₀ value of 10 ± 2, followed by ArB 13 ± 2 and ArD 19 ± 2 μg/ml while ArA and ArE showed poor response with IC₅₀ of 107 ± 1 and 60 ± 2 μg/ml respectively. The positive control acarbose showed a significant response with an IC₅₀ value of 7 ± 2 μg/ml. Similarly, in the α-glucosidase assay, compounds ArC, ArB, ArD and ArE showed prominent responses with IC₅₀ values of 25 ± 2, 30 ± 2, 32 ± 2 and 38 ± 2 μg/ml respectively. Compounds ArA showed poor response while the positive control acarbose showed a significant response with IC₅₀ of 5 ± 2 μg/ml.

Table 2.

In vitro enzyme inhibitory activity Ar (A–E) for α-amylase and α-glucosidase.

Sample	α-Amylase IC50 (μg/ml)	α-Glucosidase IC50 (μg/ml)
ArA	107 ± 1	116 ± 1
ArB	13 ± 2	30 ± 2
ArC	10 ± 2	25 ± 2
ArD	19 ± 2	32 ± 2
ArE	60 ± 2	38 ± 2
Acarbose	7 ± 2	5 ± 2

Data is presented in the form of mean ± SEM after analysis by one-way ANOVA.

ArC showed more potential, followed by ArB and ArD against α-amylase assay.

In the α-glucosidase assay, compounds ArC, ArB, ArD and ArE showed comparatively same responses.

ANOVA: Analysis of variance.

5.4. Evaluation of synthesized aurones for anti-cholinesterase activity

The synthesized aurones were screened for AChE and BChE inhibition activities. The results (Table 3) showed that compounds ArE and ArD showed potent inhibition activities with IC₅₀ values of 33 ± 0 μg/ml and 51 ± 1 μg/ml against AChE and 70 ± 1 μg/ml and 103 ± 1 μg/ml against BChE while ArC showed a potent effect against only AChE with IC₅₀ of 55 ± 1 μg/ml. Galantamine was used as a positive control for the calculation of their IC₅₀ values.

Table 3.

In vitro cholinesterase inhibition effect of all synthesized Ar(A–E).

Sample	Acetylcholinesterase (AChE) inhibition IC50 (μg/ml)	Butyrylcholinesterase (BChE) inhibition IC50 (μg/ml)
ArA	585 ± 2	625 ± 1
ArB	770 ± 3	480 ± 1
ArC	55 ± 1	555 ± 0
ArD	51 ± 1	103 ± 1
ArE	33 ± 0	70 ± 1
Galantamine	27 ± 0	21 ± 1

Data is presented in the form of mean ± SEM after analysis by one-way ANOVA.

ArE and ArD showed potent inhibition activities against AChE and BChE while ArC showed a potent effect against only AchE.

ANOVA: Analysis of variance.

5.5. Molecular docking analysis

Our results demonstrated the binding affinities of synthetic compounds with α-amylase and α-glucosidase (Supplementary Table S1 & Figure 2). Exploration of structures was performed utilizing Discovery Studio, AutoVina and Pyrex. As illustrated in Figure 2, molecular docking analysis revealed that synthetic compounds established hydrogen bonds in addition to hydrophobic interactions. The optimal configurations, which were determined via traditional hydrogen bonding, displayed a range of binding affinities (Supplementary Table S1). In general, the results demonstrate that synthetic compounds exhibit a strong affinity for binding with amylase (Figure 2).

Figure 2.

Molecular docking analysis showing interactions between synthetic compounds and α-glucosidase and α-amylase. Computational analysis of molecular docking studies showing 2D and 3D interactions.

5.6. In vivo toxicity profile of synthesized aurones

Further, the in vivo toxicity profile of the synthesized compounds was determined by evaluating the changes in body weight (Supplementary Table S2), lipid profile, LFTs and renal function test (RFTs) (Supplementary Table S3) to demonstrate the in vivo safety profile of these aurones. Supplementary Table S2 demonstrates that a significant decline in body weight was noticed in the diabetic control group which was significantly reversed by ArB, ArC and ArD on day 14 while ArA and ArE on day 21. Moreover, all Ar (A–E) have comparably significant lowering effects on serum triglycerides and cholesterol. The LFT profile showed that all aurones have a significant effect on total bilirubin while having a less significant effect on ALT and ALP. Similarly, the RFT profile showed that the diabetic control rats exhibited a significant elevation in the levels of creatinine, urea and uric acid compared with normal. All the Ar (A–E) have reduced uric acid values significantly, while only ArC, ArD and ArE reduced the creatine and urea levels significantly.

5.7. In vivo anti-diabetic assay of the synthesized aurones

The in vivo study reveals that the compound ArC showed a potent anti-diabetic effect by reducing blood sugar level from 355 ± 2 to 100 ± 3 after glibenclamide which reduced sugar level from 362 ± 3 to 100 ± 2 (Table 4). ArE showed good activity by reducing the sugar level from 337 ± 2 to 106 ± 2 while ArB and ArD showed equally good effects by reducing the sugar level from 350 ± 3 to 122 ± 2 and 330 ± 2 to 103 ± 2 respectively.

Table 4.

Evaluation of blood glucose level (mg/dL) of normal diabetic and treated groups.

Sample	DAY- 1	DAY- 7	DAY- 14	DAY- 21	DAY- 28
Normal	95 ± 2	97 ± 1	100 ± 1	106 ± 2	108 ± 1
Diabetic control	367 ± 2#	375 ± 2#	381 ± 2#	389 ± 2#	360 ± 2#
ArA	327 ± 2	118 ± 2*	120 ± 2*	121 ± 1*	117 ± 1*
ArB	350 ± 3	110 ± 2*	115 ± 2*	118 ± 2*	122 ± 2*
ArC	355 ± 2	109 ± 2*	104 ± 3*	105 ± 2*	100 ± 3*
ArD	330 ± 2	119 ± 2*	114 ± 2*	109 ± 2*	103 ± 2*
ArE	337 ± 2	121 ± 2*	118 ± 2*	109 ± 1*	106 ± 2*
Glibenclamide	362 ± 2	114 ± 1*	111 ± 2*	104 ± 3*	100 ± 2*

Data is presented as mean ± SEM and analyzed by one-way ANOVA followed by post hoc Tukey test.

Symbol #represents p < 0.05 relative to the normal while symbol * represents p < 0.05 relative to the diabetic control.

ANOVA: Analysis of variance; SEM: Standard error of mean.

5.8. Evaluation of aurones against cognitive impairment

To assess cognitive performance, a Y-maze test was conducted utilizing percent alteration behavior. All of the selected compounds induced a positive enhancement in the spatial working memory. The spontaneous alteration performance presented in Figure 3A indicates that all Ar (A–E) showed significant effects (p < 0.05) when compared with diabetic control. Similarly, results for the NOR test were presented in Figure 3B, the discrimination index (DI) indicates a highly significant effect for ArA, ArB, ArD and ArE aurones analogs (p < 0.05) in comparison to diabetic control. The result of this study is consistent with previous research studies that exposure to STZ significantly impairs memory, while aurone derivatives mitigated this cognitive decline.

Figure 3.

Aurones attenuated the behavioral deficits. (A) Percent spontaneous alteration performance of aurone. (B) DI (%) value of NOR test for aurones analogues.

Data were analyzed by graph pad prism and presented in the form of mean ± SEM after analysis by one-way ANOVA and afterward by post hoc Tukey test. p < 0.05 was considered significant.

ANOVA: Analysis of variance; DI: Discrimination index; NOR: Novel object recognition test; SEM: Standard error of mean.

5.9. Aurones downregulated inflammatory mediators & upregulated endogenous antioxidant enzymes

The concentration of GSH in the normal group and diabetic control respectively were 76 ± 1 and 35 ± 1, and the concentration of GST was noted as 67 ± 1 and 39 ± 2 in the normal and diabetic control respectively (Table 5). The treatment groups Ar (A–E) elevated the GSH levels to 42 ± 1, 45 ± 2, 57 ± 2, 49 ± 3, and 67 ± 1 and GST levels to 45 ± 1, 48 ± 2, 66 ± 1, 39 ± 2 and 66 ± 1, respectively. Malondialdehyde (MDA), which is produced as a result of LPO destroys cell function and cell membrane integrity. MDA concentration is a crucial metric for quantifying oxidative stress [56]. In the diabetic control group, an increased level of LPO was noticed as 87 ± 1 relative to the normal group as 23 ± 2 (p < 0.05), while the noted levels in treatment groups Ar (A–E) were 63 ± 1, 65 ± 1, 56 ± 2, 66 ± 2 and 46 ± 2, respectively, which shows significant suppression of LPO (p < 0.05, Table 5).

Table 5.

Evaluation of anti-oxidant parameters of the synthesized aurone derivatives.

Sample	Glutathione (GSH) (μ moles/mg of protein)	Glutathione S-transferase (GST) (μ moles CDNB Conjugate + (min/mg of protein)	Catalase (μ moles H202/min/mg of protein)	Lipid peroxidation (LPO) (TBRAS nM/min/mg of protein)
Normal	76 ± 1	67 ± 1	59 ± 1	23 ± 2
Diabetic control	35 ± 1#	39 ± 2#	45 ± 2	87 ± 1#
ArA	42 ± 1	45 ± 1	52 ± 1	63 ± 1*
ArB	45 ± 2	48 ± 2	54 ± 1	65 ± 1*
ArC	57 ± 2*	66 ± 1*	59 ± 2	56 ± 2
ArD	49 ± 3	39 ± 2	46 ± 2	66 ± 2*
ArE	67 ± 1*	66 ± 1*	59 ± 2	46 ± 2*

Data is presented in the form of mean ± SEM after analysis by one-way ANOVA and afterward by post hoc Tukey test.

Symbol #represents p < 0.05 relative to normal while symbol * represents p < 0.05 relative to diabetic control.

To further rule out the effect of ArA, ArB, ArC, ArD and ArE on inflammatory mediators and cytokines, we performed PCR and ELISA of TNF-α and IL-1β (Figure 4). Compared with the normal group, the diabetic control group revealed elevated levels of these mediators (^#p < 0.05). Treatment with ArA, ArB, ArC, ArD and ArE significantly downregulated the level of these proteins (*p < 0.05), suggesting the enhanced anti-inflammatory effect of these aurones.

Figure 4.

Compounds ArB, ArC, ArD and ArE ameliorated the release of inflammatory mediators. (A) TNF-α mRNA expression. The data was presented as mean ± SEM. ^#shows p < 0.05 as compared with normal, while * shows p < 0.05 relative to disease, n = 5/group. (B) ELISA analysis of TNF-α. The effect of ArA was more significant in lowering the TNF-α expression than other aurone derivatives (C) ELISA analysis of IL-1β. The effect of ArE was more significant in lowering the IL-1β expression, n = 5/group.

IL: Interleukin-1; TNF: Tumor necrosis factor alpha.

6. Discussion

In the present study, the neuroprotective and anti-diabetic properties of aurone derivatives against STZ-induced neurodegeneration have been demonstrated. Our findings showed that these derivatives mitigated neurodegeneration by dampening inflammatory pathways triggered by oxidative stress. Treatment with aurone derivatives reduced proinflammatory cytokine levels while increasing anti-oxidant properties in vivo and in vitro; a similar feature was observed across these derivatives. Although numerous synthetic compounds have been synthesized and evaluated for their potential neuroprotective properties, aurone derivatives demonstrated significantly greater efficacy and safety in comparison to other compounds [57]. A significant number of small molecules that have been identified as potential therapeutics for amyloid aggregation have encountered challenges in clinical trials as a result of their insufficient efficacy and undesirable pharmacokinetic profiles. Pathological processes may also be influenced by a multitude of additional factors, including glutamate/calcium excess, oxidative stress, subacute neuroinflammation and ultimately, neuronal cell death. This emphasizes the critical need for new drug development strategies for AD, with a focus on improving safety and addressing multiple targets.

The management of hyperglycemia in diabetes can be achieved through various therapeutic protocols, one of which involves the inhibition of α-amylase and α-glucosidase metabolic enzymes [58]. α-amylase hydrolyses starch into simple monosaccharides and consists of salivary and pancreatic isoforms. On the other hand, α-glucosidase is an enzyme found in the small intestine where it causes the hydrolysis of α-(1,4) linkages among monosaccharide units. The inhibition of these enzymes is a pivotal therapeutic approach in managing hyperglycemia in diabetes. In addition to the inhibition of these metabolic enzymes, other factors such as oxidative stress and lipid profile abnormalities have been associated with the pathogenesis of diabetes and its complications [59,60]. These effects may be attributed to the anti-oxidant properties of aurones, which are likely a result of their unique structure. Their ability to scavenge free radicals is largely attributed to the conjugated benzylidene group and the arrangement of polyhydroxy substitutions, which function via electron transfer and hydrogen atom donation [61,62]. Consistent studies highlight aurones and analogs for ameliorating impaired glucose metabolism and targeting diabetic molecular signaling pathways. Notably, sulfuretin 3 demonstrates promising anti-diabetic effects by modulating chronic metabolic signaling pathways. C-geranyl aurones like Altilisin H-I exhibit significant α-glucosidase inhibition, suggesting their potential as anti-diabetic drug candidates. Aurones serve as crucial scaffolds in delaying glucose digestion and absorption, presenting a key strategy for managing postprandial hyperglycemia [63]. The presence of ROS can cause harm to essential cellular components and has long been implicated in the development of various neurodegenerative disorders, including AD [64,65]. Moreover, increased oxidative stress leads to lipid peroxidation which triggers the activation of numerous inflammatory signaling pathways [66].

The cholinesterase such as AChE and BChE enzymes are the main targets for AD therapeutic development. Previously, Mughal et al. carried out screening of aurones and thio-aurones using an in vitro enzyme inhibition assay method. They reported that (Z)-2-(4-isobutylbenzylidene) benzofuran-3(2H)-one inhibit both BChE (IC₅₀ = 1.02 μM) and AChE (IC₅₀ = 0.98 μM), while (Z)-2-(4-methoxybenzylidene) benzofuran-3(2H)-one(ArC) was only potent inhibitor of AChE [66]. Similarly, Sheng et al. reported in vitro and in vivo AChE inhibitory activity of some aurone derivatives using the Ellman method. They reported that compounds with amino methyl present at the para position in the benzene ring showed more choline esterase inhibitory effect than those substituted at the meta-position [67]. Keeping in view the results of Mughal et al. and Sheng et al. our synthesized compounds (ArC and ArE) correlate with their results. They performed anticholinesterase activity of aurone derivatives by performing only in vitro studies. We performed both in vitro and in vivo studies using STZ-induced memory impairment model. BChE acts as an endogenous bio scavenger and it is also the first line of defense against toxic substances that might block the activity of AchE [67]. Moreover, in AChE-deficient mice, BChE can counterbalance AchE [68]. Evidence suggests that BChE may have a role in the development of AD due to its presence in amyloid plaques, glia, white matter and NFTs inside the brain [69]. Significant AChE activity dysfunction is observed in AD, accompanied by cholinoceptive neurons and cortical cholinergic axons [70]. The in vitro inhibitory effect of aurone analogs against AChE and BChE is presented in Table 3. It can be observed that the compound ArE having dimethyl amino group at para position showed a better inhibitory effect against both AChE and BChE with IC₅₀ values of 33 and 70 ± 1 μg/ml respectively in comparison to standard drug galantamine. ArD having chlorine group at para position showed relatively good inhibitory effect against both AChE and BChE with IC₅₀ value of 51 ± 1 and 103 ± 1 μg/ml respectively, while ArC having methoxy group at para position showed excellent inhibitory effect only against AChE with IC₅₀ of 55 ± 1 μg/ml in comparison to standard drug galantamine with IC₅₀ of 27 μg/ml. ArA having hydrogen and ArB having a methyl group at the para position did not show an obvious inhibitory effect against both AChE and BChE.

The anti-diabetic potential of the synthesized aurones Ar (A–E) was evaluated by measuring blood glucose levels (Table 4). In comparison with the standard drug, aurones showed potential glucose-lowering properties consistent with previous studies [71]. Additionally, the research demonstrated that sulfuretin inhibits advanced glycation end products (AGE) significantly, with an IC₅₀ of 124.7 μM, which is ten-times lower than the reference aminoguanidine (1231.0 μM). Furthermore, it has been observed that these aurones may possess anti-inflammatory properties that can mitigate diabetic conditions [72]. Similarly, another research demonstrated that aurones are anti-diabetic because they tend to neutralize Maillard reactions, which is a non-enzymatic interaction of glucose with protein resulting in the creation of reversible Schiff's base adducts [73].

Brain tissues are predominantly affected by elevated oxidative stress due to their increased oxygen demands and confined capacity for anti-oxidant enzymes. GSH and GST, being essential endogenous anti-oxidants, participate in the process of free radical neutralization. Several aurones derivatives have been shown to decrease ROS by activating pathways like GSH, Nrf2/HO-1, sirtuins (SIRTs) and superoxide dismutase (SODs) [74,75]. Our results demonstrated a significant reduction in GSH and GST levels in the brain cortex of the STZ-intoxicated animal group compared with the normal group, signifying an increased production of ROS. To further rule out the effect of ArA, ArB, ArC, ArD and ArE on mediators of inflammation and cytokines, PCR and ELISA of IL-1β and TNF-α were carried out (Figure 4). Previously, numerous neuroprotectants aiming at reducing inflammation have fallen short due to their single-target approach, limited penetration into the central nervous system(CNS), and heightened side effects [75]. The findings from our current research suggest that our synthesized compounds, featuring fewer hydrogen bond donors and improved CNS permeability, exhibit promise as multi-functional neuroprotective agents. Compounds with this potential can target multiple pathways in AD, thereby effectively combating inflammation and oxidative stress.

7. Conclusion

In conclusion, our study demonstrated the anti-diabetic and neuroprotective effects of aurones with varied electron-donating capacity groups attached at the para position. Further, the synthetic compounds (ArC and ArE) demonstrated comparatively good anti-diabetic effects possibly having a methoxy group and dimethyl amine group attached to the aurone ring. Aurones with dimethylamine side chain at para position demonstrated significant effects against neurodegeneration. Multiple proinflammatory cytokines are secreted in response to neuronal damage induced by STZ, which triggers oxidative stress. The Ar (A–E), exhibited promise in reducing the oxidative stress and inflammatory response induced by STZ possibly by interrupting ROS/TNF-α/1L-1β cascade. Particularly, treatment with ArE significantly reversed cognitive impairment, oxidative stress and neuroinflammation. Furthermore, the mechanisms and therapeutic benefits of these novel compounds must be investigated in models of additional neurodegenerative disorders.

Funding Statement

This work was supported by the Prince Sattam bin Abdulaziz University (Grant No. PSAU/2024/R/1445).

Supplemental material

Supplementary data for this article can be accessed at https://doi.org/10.1080/17568919.2024.2363713

Financial disclosure

This work was supported by the Prince Sattam bin Abdulaziz University (Grant No. PSAU/2024/R/1445). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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No writing assistance was utilized in the production of this manuscript.