Antioxidant and Neuroprotective Potential of Some Edible Fruits and Vegetable Extracts Based on Comparative Phytochemical Profiling and Bioactivity

Abstract

Polyphenols are key dietary bioactive compounds, reducing oxidative stress and neurodegeneration. This study investigated the in vitro antioxidant and neuroprotective potential of some edible fruits (apricots, plums, figs) and vegetable (parsley) extracts related to their phytochemical profile. Plum extract exhibited the strongest antioxidant capacity (ABTS IC₅₀ 1.733 ± 0.079 mg/g; DPPH IC₅₀ 1.593 ± 0.069 mg/g; FRAP 23.161 ± 1.094 mM Fe²⁺), linked to its high chlorogenic and caffeic acids content. Parsley displayed the most potent AChE inhibition (IC₅₀ 0.825 ± 0.026 mg/g), associated with an elevated flavonoids level (TFC 12.874 ± 0.534 mg QE/g) and the presence of ferulic and vanillic acids. Apricot was characterized by notable gallic, syringic, and chlorogenic acids, supporting moderate neuroprotective potential. Figs showed weaker radical scavenging ability but provided a balanced profile of protocatechuic, caffeic, and syringic acids. Correlation analysis revealed specific compound–activity associations, including syringic and vanillic acids with DPPH scavenging capacity, p-coumaric acid with TPC, and gallic/ferulic acids with AChE inhibition. Effect-directed HPTLC confirmed chlorogenic acid as a major contributor to the antioxidant capacity. To our knowledge, this is the first study to comparatively integrate spectrophotometric antioxidant assays, UHPLC-based quantitative phenolic profiling, effect-directed HPTLC bioautography, and AChE inhibition analysis across three edible fruits and one vegetable frequently co-consumed in Mediterranean-type diets, enabling a cross-species compound–activity correlation framework. These species exhibit distinct but complementary phytochemical and biofunctional profiles. Their combined use may support the formulation of functional foods with synergistic antioxidant and neuroprotective benefits.

1. Introduction

Neurodegenerative diseases, such as Parkinson and Alzheimer, represent a major global public health concern due to their increasing prevalence and their social and economic impact [1,2]. In this context, medicinal plants have attracted growing interest because of their phytochemicals, which may support health or serve as sources for the development of long-lasting therapeutic agents [3,4,5]. The literature highlights the demonstrated efficacy of plants and natural products in the treatment of various neurological and psychological disorders [4].

Nutritional neuroscience is an emerging research field that investigates how dietary components, including proteins, carbohydrates, lipids, supplements and phytonutrients, affect the central and peripheral nervous systems, neurochemical and neurobiological processes, as well as behavior and cognitive functions [6]. The human diet provides bioactive compounds, such as polyphenols (e.g., flavonoids), which can modulate oxidative stress and neurodegenerative processes by neutralizing free radicals, protecting cellular structures, and influencing enzymatic activities implicated in neurodegenerative diseases. Oxidative stress arises from an imbalance between oxidants and antioxidants within a biological system, typically due to excessive reactive oxygen species (ROS) or impaired antioxidant defenses [7]. Through free radicals and ROS, oxidative stress affects the cholinergic system and damages acetylcholine-producing cells, contributing to cognitive dysfunction and neuroinflammation [7,8]. The neurobiological activity of plant-derived polyphenols depends on their bioavailability in brain tissue. Studies indicate that certain dietary polyphenols can cross the blood–brain barrier and reach neuronal cells. Plant polyphenols exert beneficial effects on the brain, supporting neuronal plasticity and enhancing cognitive functions [9].

Plums (∗ Prunus domestica L.), belonging to the Rosaceae family, are fruits valued for their sweet-tart flavor and diverse colors (red, purple, yellow, green, black). Native to Western Asia and the Caucasus, plums have been consumed for centuries, either fresh, dried, or processed into jams, juices, and sauces [10]. Plums contain a variety of phenolic compounds, such as phenolic acids (p-coumaric acid, vanillic acid, protocatechuic acid, caffeic acid, caffeoylquinic acid isomers) and tannins, as well as fibers and essential minerals [11,12]. The traditional and medicinal uses of plums are well documented, including antioxidant, antimicrobial, antihemolytic, cytotoxic, hepatoprotective, laxative, and anti-inflammatory activities [13]. Plum extracts have demonstrated significant antioxidant and free radical-scavenging activity, comparable to ascorbic acid, in a dose-dependent manner. Additionally, polyphenols exhibit antimicrobial and immunomodulatory properties, highlighting their broad therapeutic potential [14,15].

Apricots (∗ Armeniaca vulgaris Lam. sin. Prunus armeniaca L.), also members of the Rosaceae family, are widely cultivated in temperate regions, Mediterranean countries, and Central Asia [16]. Apricots are associated with a high polyphenol content, contributing to their antioxidant activity. Characterized polyphenols include gallic acid, chlorogenic acid, p-coumaric acid, and rutin. In addition, apricots provide fatty acids, amino acids, vitamins A and C, and minerals such as potassium and iron, contributing to their significant nutritional value [16,17,18,19]. Other studies report that apricots are rich in amino acids, proteins, monosaccharides, polysaccharides, lipids, simple organic acids, and dietary fibers [19]. Apricots consumption may contribute to the prevention of degenerative diseases. Preclinical studies have demonstrated potential therapeutic effects, including hepatoprotective, anticancer, anti-inflammatory, and antimicrobial activities [16,17,18,19].

Figs (∗ Ficus carica L.), belonging to the Moraceae family, have been used for thousands of years and are considered among the earliest cultivated plants. Their origin is attributed to Southwestern Asia and the Middle East [20]. Phytochemical analyses have revealed the presence of flavonoids, tannins, saponins, coumarins, sterols, carbohydrates, and proteins. F. carica is particularly rich in two main categories of phenolic compounds: phenolic acids (such as gallic, chlorogenic, and syringic acids) and flavonoids (including catechin, epicatechin, and anthocyanins). The antioxidant activity of F. carica fruit extracts has been evaluated at 87% compared with ascorbic acid [21,22]. In recent decades, figs have attracted considerable interest as a functional food and potential therapeutic agent due to their palatable flavor and broad range of biological and pharmacological activities, including antioxidant, antidiabetic, anticancer, neuroprotective, anti-inflammatory, hepatoprotective, dermoprotective, and antiviral effects [20,23].

Parsley (∗ Petroselinum crispum (Mill.) A.W. Hill) is a bright green medicinal and aromatic plant of the Apiaceae family, native to the Mediterranean region and now cultivated worldwide [24]. The most important secondary metabolites in parsley are flavonoids, particularly flavones and flavonols, and their glycosides. Coumarins, especially furanocoumarins, have also been identified, along with other phenolic compounds, carotenoids, simple organic acids, carbohydrates, and fatty acids [24,25]. The plant exhibits a wide range of biological and pharmacological activities, including antioxidant, antibacterial, antifungal, antidiabetic, antihypertensive, antiplatelet, analgesic, anti-inflammatory, antihyperuricemic, hepatoprotective, nephroprotective, anticancer, wound healing, anti-obesity, estrogenic, and neuroprotective effects [24,26].

The selection of these four species was based on their frequent dietary co-consumption, complementary phytochemical compositions (hydroxycinnamic acid-rich fruits vs. flavonoid-dominant herb), and documented but separately investigated bioactivities, allowing the development of a comparative framework to explore convergent and divergent compound–activity relationships.

The aim of the present study was to compare plum, apricot, fig, and parsley ethanolic extracts in terms of their phenolic and flavonoid content, antioxidant capacity, and acetylcholinesterase (AChE) inhibition, thereby providing an integrated perspective on their nutraceutical and neuroprotective potential. Although numerous studies have investigated individual species or isolated bioactivities, comparative analyses integrating quantitative phenolic profiling with both antioxidant and neuroprotective endpoints across commonly co-consumed edible plants remain limited. Thus, the present work addresses the need for a cross-species, multi-assay evaluation linking specific phytochemicals to functional outcomes.

Unlike previous studies focusing on individual species or single bioactivity assays, the novelty of the present work lies in the integrated and comparative evaluation of antioxidant and neuroprotective potential using complementary spectrophotometric assays, ultra-high-performance liquid chromatography (UHPLC)-based quantitative phenolic profiling, effect-directed high-performance thin-layer chromatography (HPTLC) bioautography, and correlation analysis. Furthermore, the simultaneous investigation of three edible fruits and one culinary herb, frequently co-consumed in Mediterranean-type diets, provides a comparative framework that highlights both shared and species-specific phytochemical–bioactivity relationships. This approach allows the identification of complementary biofunctional profiles that may be exploited in the design of functional food formulations.

2. Results

2.1. Antioxidant Capacity

The antioxidant capacity of plum, apricot, fig, and parsley extracts was assessed using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), both expressed as half-maximal inhibitory concentration (IC₅₀), and ferric-reducing antioxidant power (FRAP) and clear differences emerged among the species, the results obtained being summarized in

Table 1. Antioxidant capacity of apricot, parsley, plum, and fig extracts (mean ± SD, n = 3).

Sample	ABTS IC50 (mg/g)	DPPH IC50 (mg/g)	FRAP (mM Fe2+)
apricot	23.850 ± 1.126	12.670 ± 0.365	1.021 ± 0.041
parsley	13.740 ± 0.672	15.750 ± 0.766	4.880 ± 0.110
plum	1.733 ± 0.079	1.593 ± 0.069	23.161 ± 1.094
fig	29.190 ± 1.358	14.630 ± 0.383	0.822 ± 0.030

ABTS: 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH: 2,2-Diphenyl-1-picrylhydrazyl; FRAP: Ferric-reducing antioxidant power; IC₅₀: Half-maximal inhibitory concentration; SD: Standard deviation.

.

For the ABTS assay, plum extract exhibited the most potent radical scavenging capacity, requiring only 1.733 ± 0.079 mg/g to inhibit 50% of radicals, a value significantly lower than parsley (13.740 ± 0.672 mg/g), apricot (23.850 ± 1.126 mg/g), and fig (29.190 ± 1.358 mg/g) (p < 0.0001) (Figure 1a). Similarly, in the DPPH assay, plum again showed the strongest activity (1.593 ± 0.069 mg/g), while parsley (15.750 ± 0.766 mg/g), fig (14.630 ± 0.383 mg/g), and apricot (12.670 ± 0.365 mg/g) were significantly weaker (p < 0.0001) (Figure 1a).

The FRAP assay revealed strikingly high reducing capacity in plum extract (23.161 ± 1.094 mM Fe²⁺ equivalents), which was more than fourfold higher than parsley (4.880 ± 0.110 mM Fe²⁺) and over twentyfold greater than apricot (1.021 ± 0.041 mM Fe²⁺) and fig (0.822 ± 0.030 mM Fe²⁺). These results establish plums as the most powerful antioxidant among the four extracts (Figure 1b).

2.2. Neuroprotective Potential Evaluated as Anti-Acetylcholinesterase Activity

AChE inhibition assays highlighted parsley as the most effective neuroprotective agent, with an IC₅₀ of 0.825 ± 0.026 mg/g, significantly lower than both apricot (7.359 ± 0.197 mg/g) and plum (5.401 ± 0.120 mg/g) (p < 0.0001) (

Table 2. Anti-AChE activity, TPC and TFC of apricot, parsley, plum, and fig extracts (mean ± SD, n = 3).

Sample	Anti-AChE IC50 (mg/g)	TPC (mg GAE/g)	TFC (mg QE/g)
apricot	7.359 ± 0.197	0.679 ± 0.019	0.790 ± 0.035
parsley	0.825 ± 0.026	2.966 ± 0.066	12.874 ± 0.534
plum	5.401 ± 0.120	5.443 ± 0.198	0.000 ± 0.000
fig	ND	0.763 ± 0.018	0.263 ± 0.007

AChE: Acetylcholinesterase; GAE: Gallic acid equivalents; IC₅₀: Half-maximal inhibitory concentration; QE: Quercetin equivalents; SD: Standard deviation; TFC: Total flavonoid content; TPC: Total phenolic content; ND: Not determined.

). Fig extract showed no measurable inhibitory activity within the tested concentration range; therefore, an IC₅₀ value could not be determined (Figure 2). These findings demonstrate that parsley’s phenolic profile confers exceptional AChE inhibitory activity compared to the edible fruits. However, among the fruits, plum revealed the highest anti-AChE activity.

2.3. Total Phenolic and Flavonoid Content

The spectrophotometric analyses revealed major differences in phytochemical richness among the four extracts (Table 2). Plum extract exhibited the highest total phenolic content (TPC) (5.443 ± 0.198 mg gallic acid equivalents (GAE)/g), consistent with its superior antioxidant performance. Parsley extract, on the other hand, contained by far the greatest amount of flavonoids (12.874 ± 0.534 mg quercetin equivalents (QE)/g total flavonoid content (TFC)). Apricot (0.679 ± 0.019 mg GAE/g TPC; 0.790 ± 0.035 mg QE/g TFC) and fig (0.763 ± 0.018 mg GAE/g TPC; 0.263 ± 0.007 mg QE/g TFC) extracts were significantly lower (p < 0.0001) (Figure 3).

A Wilcoxon test comparing FRAP with TFC revealed no significant association, indicating that reducing power in these extracts is not solely driven by flavonoid content but also by non-flavonoid phenolics.

2.4. Phenolic Acid Profiles

UHPLC analysis identified eight phenolic acids across the four extracts, with distinct species-specific distributions (

Table 3. Phenolic acid composition of apricot, parsley, plum, and fig extracts (mg/g dry matter, mean ± SD, n = 3).

Phenolic Acid	Sample				Wavelength (nm)	tR (min)
	Apricot	Parsley	Plum	Fig
p-coumaric acid	2.088 ± 0.090	3.213 ± 0.134	3.854 ± 0.168	1.191 ± 0.550	325	7.8
ferulic acid	3.873 ± 0.183	7.154 ± 0.321	2.433 ± 0.114	0.891 ± 0.032	325	8.5
protocatechuic acid	3.965 ± 0.171	1.407 ± 0.041	3.378 ± 0.084	1.814 ± 0.067	265	3.6
syringic acid	6.804 ± 0.333	2.695 ± 0.061	ND	2.550 ± 0.077	325	6.6
caffeic acid	1.141 ± 0.025	1.078 ± 0.048	3.603 ± 0.109	2.448 ± 0.057	325	6.3
chlorogenic acid	6.768 ± 0.252	0.861 ± 0.023	8.059 ± 0.332	1.153 ± 0.056	325	5.8
vanillic acid	0.847 ± 0.201	2.684 ± 0.132	ND	1.009 ± 0.045	265	6.1
gallic acid	1.118 ± 0.446	ND	ND	ND	265	1.9

ND: Not detected; SD: Standard deviation; t_R: Retention time.

; Figure 4 and Figure 5).

Apricots contained high levels of syringic acid (6.804 ± 0.333 mg/g) and chlorogenic acid (6.768 ± 0.252 mg/g), both significantly higher than p-coumaric, ferulic, protocatechuic, and caffeic acids (p < 0.0001). Apricots were the only sample in which gallic acid was detected (1.118 ± 0.446 mg/g).

Parsley was dominated by ferulic acid (7.154 ± 0.321 mg/g), which was significantly higher than all other acids (p < 0.0001). Moderate amounts of p-coumaric acid (3.213 ± 0.134 mg/g), syringic acid (2.695 ± 0.061 mg/g), and vanillic acid (2.684 ± 0.132 mg/g) were also present, while chlorogenic acid was nearly absent (0.861 ± 0.023 mg/g).

Plums were rich in chlorogenic acid (8.059 ± 0.332 mg/g), significantly higher than p-coumaric acid (3.854 ± 0.168 mg/g), caffeic acid (3.603 ± 0.109 mg/g), and protocatechuic acid (3.378 ± 0.084 mg/g) (all p < 0.0001). Syringic and vanillic acids were not detected.

Figs presented a more balanced profile, with moderate concentrations of syringic acid (2.550 ± 0.077 mg/g), caffeic acid (2.448 ± 0.057 mg/g), and protocatechuic acid (1.814 ± 0.067 mg/g). Tukey’s analysis confirmed syringic acid as significantly higher than protocatechuic (p < 0.001) and p-coumaric (p < 0.0001) acids.

2.5. Correlation Analysis

Spearman’s rank correlations revealed notable trends. TPC showed a positive trend in relation to p-coumaric acid content (ρ = 1.0, p = 0.083), suggesting it is a major contributor to total phenolics. DPPH activity correlated positively with syringic and vanillic acids (ρ = 1.0, p = 0.083), supporting their role as radical scavengers. TFC was negatively correlated with caffeic, chlorogenic, and protocatechuic acids (ρ = −1.0, p = 0.083), reflecting an apparent trade-off between flavonoid and hydroxycinnamic accumulation. Anti-AChE activity correlated positively with gallic acid (ρ ≈ 0.775, p = 0.5) and ferulic acid (ρ = 0.4, p = 0.75), and with FRAP (ρ = 0.4, p = 0.75). Negative correlations were observed with ABTS, DPPH, syringic, and vanillic acids (ρ from −0.4 to −0.6, not significantly).

Although most associations did not reach statistical significance due to small sample size (n = 4), these patterns highlight distinct compound–activity relationships. Due to the small number of biological observations (n = 4 species), extreme Spearman’s coefficients (ρ = 1.0) are mathematically possible and should not be interpreted as evidence of deterministic biological relationships.

2.6. HPTLC Fingerprints and Effect-Directed Assays

HPTLC fingerprinting provided qualitative confirmation of UHPLC findings. Under ultraviolet (UV) light, parsley displayed the richest profile with multiple fluorescent bands, many of which co-migrated with caffeic acid and chlorogenic acid standards. Plum extracts also showed intense bands corresponding to these acids, whereas apricot extracts exhibited faint chlorogenic acid bands and fig extracts revealed fewer, weaker bands overall (Figure 6a,b).

Effect-directed DPPH bioautography localized antioxidant activity to the bands corresponding to chlorogenic acid, especially in plums, providing direct evidence that this compound is functionally responsible for radical scavenging activity. Apricots, parsley, and figs showed only weak activity zones in starting area (Figure 6c).

3. Discussion

This study provides a comparative assessment of the antioxidant and neuroprotective potential of apricot, parsley, plum, and fig extracts, demonstrating clear differences in activity that can be explained by their phenolic compositions.

The literature highlights the demonstrated efficacy of plants and natural products in the treatment of various neurological and psychological disorders [4].

Cognitive enhancement associated with plum consumption has not been extensively studied in humans, with most evidence coming from animal studies. This cognitive effect is primarily attributed to the antioxidant properties of plums, due to their high polyphenol content [27]. One study demonstrated that oral administration of Prunus fruit extracts (75, 100, 150 mg/kg) to male mice significantly improved learning capacity and memory [28].

Plums consistently emerged as the most potent antioxidant source. Their remarkably low IC₅₀ values in ABTS and DPPH assays, together with their outstanding FRAP activity, reflect the abundance of hydroxycinnamic acids, particularly chlorogenic acid. These compounds are well-established for their radical scavenging and electron-donating abilities, and their dominance in plum extracts explains the superior performance. However, plums are also known to contain flavan-3-ols such as catechins and condensed tannins, which possess strong hydrogen-donating and metal-chelating properties. Although these compounds were not individually quantified in the present UHPLC analysis, their presence may additionally contribute to the overall antioxidant capacity observed, suggesting that hydroxycinnamic acids are likely part of a broader polyphenolic network underlying plum bioactivity. The HPTLC–DPPH bioautography directly confirmed this relationship, as strong antioxidant activity localized at bands corresponding to these acids. Also, anthocyanins could also contribute indirectly to the observed antioxidant effect.

Apricots, though modest in antioxidant capacity, were notable for their high gallic acid content. This compound is known for both antioxidant and AChE inhibitory activity. The moderate positive correlation between gallic acid and anti-AChE activity observed in this study supports its role in apricot’s neuroprotective profile [18,29,30,31].

Figs, while weaker in ABTS and DPPH scavenging and lacking detectable anti-AChE activity, were distinguished by high levels of protocatechuic and syringic acids. These compounds are associated with anti-inflammatory and vascular protective activities, which may contribute to the ethnopharmacological use of figs even though they scored lower in antioxidant and neuroprotective assays in this study [32,33,34]. Nevertheless, figs are recognized as complex functional foods containing additional bioactive constituents, including dietary fibers, organic acids, sugars, minerals, and various secondary metabolites such as anthocyanins and sterols, which may contribute to metabolic, vascular, and gastrointestinal health. Therefore, the comparatively lower radical-scavenging performance observed here does not diminish their broader nutraceutical relevance but rather underscores the importance of multi-dimensional evaluation strategies.

Parsley has been shown to benefit spatial and recognition memory, modulate M1 receptor expression, regulate apoptosis and oxidative stress, and enhance AChE inhibitory activity [24,26]. Polyphenols from parsley promote neuroplasticity by supporting neuronal growth and survival, thereby improving mood and cognitive function [25].

Parsley, while less effective than plum in radical scavenging assays, demonstrated extraordinary AChE inhibition, with an IC₅₀ order of magnitude lower than apricot or plum. This can be attributed to its unique composition, dominated by vanillic, p-coumaric, and syringic acids, together with its exceptionally high flavonoid content. Syringic and vanillic acids are methoxylated phenolic acids previously reported to modulate oxidative stress and AChE activity, providing a mechanistic basis for parsley’s neuroprotective effect. The correlation between syringic and vanillic acids and DPPH scavenging further underscores their functional relevance [35,36].

The correlation analysis provided preliminary insight into potential compound–activity relationships. TPC was strongly driven by p-coumaric acid, particularly abundant in parsley. Conversely, flavonoid accumulation was inversely correlated with hydroxycinnamic acids, suggesting that species may allocate resources to different biosynthetic pathways. These findings emphasize the need to evaluate both total content and individual compound contributions.

HPTLC profiling revealed relatively simple phenolic patterns in the tested extracts. Under UV and after derivatization, a distinct band corresponding to chlorogenic acid was observed, confirming its presence as a major phenolic marker. In contrast, the other quantified phenolic acids (e.g., syringic, ferulic, protocatechuic acids) did not migrate clearly and probably remained at the application line, suggesting that they may either co-migrate with more polar, high-molecular-weight compounds or represent phenolics that are less mobile under the chosen solvent system. These stationary bands likely correspond to other classes of compounds, such as glycosides, polymeric tannins, or complex flavonoids, which commonly exhibit limited migration on silica gel.

Effect-directed DPPH bioautography confirmed antioxidant activity localized to the chlorogenic acid band, while the intense quenching observed at the origin indicated that immobile constituents also contributed to radical scavenging activity, even if they were not resolved on the plate. The intense DPPH scavenging observed at the application zone suggests the contribution of highly polar or polymeric constituents; however, this interpretation remains tentative and requires further fractionation or elution-based confirmation. These findings contribute to the emerging field of plant-based neuroprotection by illustrating how dietary diversity across phenolic classes may support multi-target modulation of oxidative stress and cholinergic dysfunction, both central mechanisms in neurodegenerative disorders.

3.1. Limitations

The present study has several limitations. The small number of biological replicates and the inclusion of only four species limited the statistical power of correlation analyses, which explains why several strong trends did not achieve significance. Due to the small dataset, the observed correlations primarily serve to generate hypotheses regarding compound–activity relationships rather than to establish statistically robust associations. Additionally, the assays used provide in vitro estimates of antioxidant and neuroprotective potential but do not account for bioavailability, metabolism, or synergistic interactions in vivo. Finally, while phenolic acids were confirmed as major contributors, the roles of minor compounds and complex polyphenols such as tannins or anthocyanins remain to be explored.

3.2. Future Perspectives

The integration of antioxidant and neuroprotective data provides a conceptual framework for future formulation design. While complementary phytochemical distributions suggest the possibility of synergistic interactions, no combination experiments were performed in the present study. Therefore, formulation development remains a hypothesis-driven research direction requiring mixture-design experiments and interaction modeling.

4. Materials and Methods

4.1. Plant Material

The plant material, edible fruits (apricots, figs, plums) and parsley leaves, respectively, were obtained from a certified local producer, in July 2025, from distinct ecological zones in southwestern Romania (

Table 4. Plant material used for analysis.

Sample	Species/Vegetal Product	Date/Collection Site (Southwest Romania)	Voucher Specimen
apricots	∗ Armeniaca vulgaris Lam./fructus	16 July 2025/Râmnicu Vâlcea City, Vâlcea County	ARM-VLG-2025-07-16
figs	∗ Ficus carica L./fructus	21 July 2025/Râmnicu Vâlcea City, Vâlcea County	FCS-CAR-2025-07-21
plums	∗ Prunus domestica L./fructus	23 July 2025/Râmnicu Vâlcea City, Vâlcea County	PRN-DOM-2025-07-23
parsley	∗ Petroselinum crispum (Mill.) A.W. Hill/folium	20 July 2025/Râmnicu Vâlcea City, Vâlcea County	PSL-CSP-2025-07-20

∗: Cultivated plant.

). The plant material for analysis was stored in the Herbarium of the Department of Pharmaceutical Botany, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova. Our research did not involve endangered or protected plant species. Edible fruits, after separating the seeds, were frozen overnight and processed by freeze drying (Alpha 1-2 LSCbasic freeze dryer; Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany), ensuring longer shelf time without additives and preservatives. Parsley leaves were air-dried (20–22 °C) in the shade for one week and subsequently milled through a 0.5 mm sieve to obtain a homogeneous powdered material. To preserve thermolabile phenolic constituents until extraction, the final powdered herbal samples were stored in amber glass containers under cool, dry conditions.

4.2. Chemicals and Reagents

The solvents used in this study included ethanol, methanol, ethyl acetate, and acetonitrile (Merck Millipore, Darmstadt, Germany). Ultrapure water was produced using a HALIOS 6 laboratory water system (Neptec, Montabaur, Germany). To improve the performance of the mobile phases for UHPLC analysis, formic acid (Merck KGaA, Darmstadt, Germany) was added.

For TPC, TFC, antioxidant activity, and enzymatic assays, the following Sigma-Aldrich (Taufkirchen, Germany) chemicals and reagents were used: Folin–Ciocalteu reagent, sodium carbonate (Na₂CO₃), gallic acid, aluminum chloride (AlCl₃), quercetin, DPPH, ABTS, potassium persulfate (K₂S₂O₈), sodium acetate, acetic acid, 2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ), hydrochloric acid (HCl), ferric chloride (FeCl₃), ferrous sulphate heptahydrate (FeSO₄·7H₂O), natural products–polyethylene glycol (NP–PEG) reagent, 1-naphthyl acetate, AChE from Electrophorus electricus, Fast Blue B salt, and rivastigmine.

For the UHPLC analysis, a set of eight phenolic acid standards (caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, gallic acid, protocatechuic acid, syringic acid, and vanillic acid), supplied by Merck Millipore (Darmstadt, Germany), was used for both calibration and compound identification.

Silica gel 60 F₂₅₄ glass plates (20 × 10 cm), obtained from Merck Millipore (Darmstadt, Germany), were used for HPTLC analysis.

4.3. Extraction Procedure

The extraction of plant material was carried out using an ultrasound-assisted extraction method, with 70% ethanol as the solvent. Seventy percent ethanol was selected as a food-grade, environmentally acceptable solvent with documented efficiency for extracting both polar phenolic acids and moderately polar flavonoids. A sample of 1 g of finely ground plant material was combined with 10 mL of the ethanol solution. This mixture was then treated in a Bandelin Sonorex Digiplus DL 102H ultrasound bath (Bandelin Electronic GmbH & Co., KG, Berlin, Germany). The treatment lasted for 20 min. at a constant temperature of 50 °C. The extraction temperature of 50 °C was selected to enhance extraction efficiency while minimizing thermal degradation of phenolic compounds, as reported in previous ultrasound-assisted extraction studies. For UHPLC analysis, the sample was filtered through a 0.22 μm membrane filter into appropriate vials to obtain a clear extract suitable for chromatographic injection.

4.4. Standards Preparation

Caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, gallic acid, protocatechuic acid, syringic acid, and vanillic acid were used as standards for the UHPLC analysis. Stock solutions (1 mg/mL) were prepared from which dilutions were made, and 10 μL of each standard or sample was injected into the system.

4.5. Antioxidant Activity Assays

4.5.1. DPPH Antioxidant Assay

Using a 96-well microplate, the DPPH radical scavenging assay began by adding 50 μL of each sample, which was then serially diluted to achieve a gradient of decreasing concentrations. After this, 150 μL of a 0.2 mM DPPH solution in ethanol was added to each well. The mixtures were then incubated in the dark at room temperature (RT) for 30 min. The absorbance of each well was measured at 517 nm with a FLUOstar Optima microplate reader (BMG Labtech, Ortenberg, Germany). The antioxidant capacity was determined by calculating the IC₅₀, which corresponds to the concentration required to scavenge 50% of the DPPH radicals. To ensure accurate results, each sample was tested in triplicate [37].

4.5.2. ABTS Antioxidant Assay

In the ABTS radical scavenging assay, 50 μL of each sample was added to a 96-well microplate. The samples were then serially diluted, like in the DPPH assay, to obtain a concentration gradient. Next, 150 μL of an ABTS solution was added to each well. ABTS solution was made by combining 7.4 mM ABTS with 2.6 mM K₂S₂O₈. The absorbance was measured at 620 nm using a FLUOstar Optima microplate reader (BMG Labtech, Ortenberg, Germany). The IC₅₀ value was then calculated from a dose–response curve, which shows the sample concentration needed to inhibit 50% of the ABTS radicals. All samples were analyzed in triplicate [38].

4.5.3. FRAP Antioxidant Assay

The FRAP assay was performed using freshly prepared FRAP reagent, consisting of acetate buffer, 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl₃ solution. A calibration curve (y = 0.0006125x + 0.05583, R² = 0.9976) was created using Fe²⁺ standards ranging from 31.2 to 1000 μM. For each assay, 10 μL of sample or standard was added to a 96-well microplate, followed by 190 μL of freshly prepared FRAP reagent. The resulting mixtures were incubated at RT for 30 min before the absorbance was measured at 595 nm using the FLUOstar Optima microplate reader (BMG Labtech, Ortenberg, Germany). The final results were expressed as μM Fe²⁺ equivalents per mL of extract. All measurements were performed in triplicate to ensure accuracy and reproducibility [38].

4.6. Neuroprotective Activity Assay

AChE inhibitory activity was evaluated using a microplate-based assay. The samples were tested at various concentrations, with all analyses run in triplicate for reliability. A concentration gradient was created by serially diluting each sample on a 96-well microplate. The enzymatic reaction was initiated by adding 50 μL of 1-naphthyl acetate solution (3 mg/mL in ethanol) as the substrate, followed by 150 μL of AChE solution (3.33 U/mL) to catalyze the reaction. Subsequently, 50 μL of Fast Blue B salt solution (3 mg/mL in water) was added to each well, producing a colored product proportional to the enzymatic activity. Rivastigmine (1 mg/mL in methanol) was used as a positive control. Absorbance was recorded at 595 nm using a FLUOstar Optima microplate reader (BMG Labtech, Ortenberg, Germany), and IC₅₀ values were calculated to determine the concentration required to inhibit 50% of AChE activity [38]. Percent inhibition was calculated relative to enzyme control wells at a fixed incubation time, and IC₅₀ values were derived from nonlinear regression of concentration–response curves.

4.7. Total Phenolic Content Assay

The TPC of plant extracts was determined using the Folin–Ciocalteu method. Briefly, 20 μL of each extract was added to a 96-well microplate, followed by 100 μL of Folin–Ciocalteu reagent. After mixing for 3 min, 80 μL of 4% Na₂CO₃ solution was added, and the plate was mixed again for uniformity. The reaction mixtures were incubated in the dark for 2 h. Absorbance was measured at 620 nm using the FLUOstar Optima microplate reader (BMG Labtech, Ortenberg, Germany). To quantify the phenolic compounds, a gallic acid standard curve (y = 0.003052x − 0.06985, R² = 0.9914) prepared over a concentration range of 31.25 μg/mL to 1 mg/mL, and results were expressed as μg GAE per mL of extract. All measurements were performed in triplicate to ensure accuracy and reproducibility [37].

4.8. Total Flavonoid Content Assay

The TFC of plant extracts was determined using an AlCl₃ colorimetric assay. A quercetin standard curve (y = 0.008508x + 0.05495, R² = 0.9886) was prepared with concentrations from 3.125 to 100 μg/mL in 96% ethanol. For each assay, 50 μL of plant extract or a quercetin standard solution was added to a 96-well microplate, followed by 10 μL of 10% AlCl₃ solution. Subsequently, 150 μL of 96% ethanol and 10 μL of 1 M sodium acetate were added. A blank control was prepared by replacing the sample with 96% ethanol. The plate was thoroughly mixed and incubated in the dark at RT for 40 min. Absorbance was recorded at 410 nm using a FLUOstar Optima microplate reader (BMG Labtech, Ortenberg, Germany). The results were expressed as μg QE per mL of plant extract, and each sample was tested in triplicate to ensure reproducibility [37].

4.9. HPTLC Fingerprinting for Antioxidant and Neuroprotective Activity

HPTLC fingerprinting was used to evaluate the antioxidant capacity (DPPH assay) of the plant extracts, using caffeic acid and chlorogenic acid as reference standards. For sample application, a Linomat 5 (CAMAG, Muttenz, Switzerland) applicator was used to apply 2 μL of each extract and standard onto the HPTLC plates. Chromatographic separation was performed in a twin trough chamber with a mobile phase composed of ethyl acetate, formic acid, and water (90:6:9, v/v/v). The chamber was saturated for 20 min before development to ensure ideal separation, and the plates were developed until the solvent front reached 7 cm. The visualization of the HPTLC plates was initially performed at 254 nm and 366 nm without derivatization. After derivatization, plates were visualized with NP–PEG reagent at 366 nm, while the DPPH assay was observed under white light. This approach facilitated the identification of bioactive compounds based on their retention factor (R_f) values and characteristic color changes, indicative of their antioxidant properties [39].

4.10. UHPLC Analysis of Phenolic Acids

UHPLC analysis (

Table 5. Calibration equations, linearity, LOD and LOQ of phenolic acids determined by UHPLC analysis.

Phenolic Acid	Equation	R2	LOD (μg/mL)	LOQ (μg/mL)
p-coumaric acid	y = 1.51 × 103x + 1.76 × 103	0.9997	0.239	0.723
ferulic acid	y = 4.87 × 103x + 7.36 × 103	0.9998	0.255	0.774
protocatechuic acid	y = 8.03 × 102x + 1.46 × 103	0.9998	0.200	0.607
syringic acid	y = 2.44 × 103x + 3.54 × 103	0.9998	0.215	0.653
caffeic acid	y = 2.79 × 103x + 4.54 × 103	0.9998	0.226	0.685
chlorogenic acid	y = 2.21 × 103x + 3.51 × 103	0.9998	0.205	0.620
vanillic acid	y = 2.49 × 103x + 4.48 × 103	0.9996	0.217	0.657
gallic acid	y = 3.13 × 103x + 5.66 × 103	0.9998	0.067	0.204

LOD: Limit of detection; LOQ: Limit of quantification; R²: Coefficient of determination; UHPLC: Ultra-high-performance liquid chromatography.

) was performed using a Waters Acquity Arc system equipped with both a photodiode array (PDA) detector and a QDa mass detector (Waters, Milford, MA, USA). The compounds were separated using a CORTECS C18 column (4.6 × 50 mm, 2.7 μm particle size) kept at 28 °C. The mobile phase was a mixture of water with 0.01% formic acid (A) and acetonitrile with 0.01% formic acid (B). Gradient elution was applied, beginning with 99% A at a flow rate of 0.8 mL/min for 1 min, followed by a gradual decrease to 70% A over 1–13 min, which was maintained until 13.10 min. For column washing, the mobile phase was adjusted to 20% A from 13.60 to 17.60 min to elute strongly retained compounds. The system was then re-equilibrated by returning to 99% A at 18.10 min and holding it until 21.10 min. To ensure analytical stability, the column was equilibrated for 10 min between injections, and samples were stored at 8 °C. Compound quantification was performed using absorbance detection at two wavelengths: gallic acid, protocatechuic acid, vanillic acid, and syringic acid at 265 nm, and chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid at 325 nm. Mass spectrometry (MS) confirmation for definitive compound identification was performed in negative ion mode, targeting a specific mass-to-charge ratio (m/z): 153 for protocatechuic acid, 163 for p-coumaric acid, 167 for vanillic acid, 169 for gallic acid, 179 for caffeic acid, 193 for ferulic acid, 197 for syringic acid, and 353 for chlorogenic acid [38,39].

4.11. Statistical Analysis

All statistical analyses were performed using GraphPad Prism software, version 9.0.1 (GraphPad Software, San Diego, CA, USA). Data were first assessed for normality using the Shapiro–Wilk test. Normally distributed datasets (ABTS, DPPH, TPC, anti-AChE, and the phenolic acids p-coumaric, ferulic, protocatechuic, syringic, caffeic, chlorogenic and vanillic) were analyzed using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Homogeneity of variances was evaluated using the Brown–Forsythe test prior to ANOVA application. Non-normally distributed datasets (FRAP, TFC) were analyzed using the Wilcoxon test.

Correlation analyses among antioxidant, neuroprotective, and phytochemical parameters were performed using Spearman’s rank correlation coefficient (ρ), with corresponding p-values reported. Significance thresholds were set at p < 0.05. All experiments were conducted in triplicate and results are expressed as mean ± standard deviation (SD) unless otherwise specified. Given the limited number of biological groups (four species), statistical comparisons should be interpreted cautiously and primarily as descriptive indicators of interspecies trends rather than definitive inferential conclusions.

5. Conclusions

This study provides an integrated phytochemical and bioactivity comparison of apricots, parsley, plums, and figs, demonstrating that each species exhibits distinct antioxidant and neuroprotective signatures linked to specific phenolic profiles. Plums emerged as the most powerful reducing agents, owing to their high chlorogenic and caffeic acids content, while parsley displayed pronounced anti-AChE activity and moderate radical scavenging capacity linked to ferulic and vanillic acids, and flavonoids. Parsley exhibited the strongest anti-AChE activity, which can be attributed to its high flavonoid content and elevated levels of ferulic and vanillic acids. Figs, although lower in overall activity, provided a balanced profile of syringic, caffeic, and protocatechuic acids, contributing to moderate antioxidant capacity.

Correlation analyses indicated specific compound–activity relationships: syringic and vanillic acids were linked to DPPH scavenging, p-coumaric acid was linked to total phenolics, and gallic and ferulic acids were linked to anti-AChE activity. HPTLC fingerprinting and effect-directed assays corroborated these associations by directly localizing antioxidant activity to phenolic acid bands.

Together, these findings underline the complementary roles of these natural products. Their distinct phenolic compositions translate into specialized biological activities, suggesting that dietary diversity across these species can provide synergistic antioxidant and neuroprotective benefits.

By integrating chromatographic, spectrophotometric, enzymatic, and correlation-based analyses within a comparative framework, this work advances methodological approaches for evaluating plant-derived neuroprotective resources and supports future in vivo and combinatorial investigations in functional food research.