Carpoxylon macrospermum leaf extract and its phenolic compounds: a multi-targeted therapeutic remedy for Alzheimer’s disease

Abstract

Background

Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder with a significant impact, especially on elderly people. Although no current treatment is available for AD, several studies have been conducted to discover alternative remedies capable of managing its symptoms and slowing the progression. Accordingly, herein we analysed the phenolic composition and investigated the defatted aqueous methanol extract (DAE) of Carpoxylon macrospermum H.Wendl. & Drude (Family Arecaceae) leaves against key enzymes in addition to oxidative and inflammatory markers involved in AD progression.

Methods

NMR and mass spectrometry elucidated the phenolic compounds. Human carbonic anhydrase (hCA), human acetyl cholinesterase (AChE), and cyclo-oxygenase 2 (COX-2) inhibitor screening kits were used to assess the enzyme-inhibitory potential. Lipo-poly saccharide (LPS)-induced murine macrophage (RAW 264.7) in vitro model was used to test the anti-inflammatory effect. Molecular docking studies were conducted using AutoDock Vina.

Results

Chlorogenic acid (1), rutin (2), hesperidin (3), vanillic acid (4), and p-hydroxybenzoic acid (5) have been isolated. The DAE and compounds 2 and 4 significantly inhibited hCA enzyme with IC₅₀ equivalent to 0.160 ± 0.008, 0.243 ± 0.012, and 0.290 ± 0.015 µg/mL, and AChE enzyme with an IC₅₀ corresponding to 3.732 ± 0.13, 0.868 ± 0.03, and 0.597 ± 0.02 µg/mL, respectively. Additionally, they demonstrated potent anti-inflammatory activity by inhibiting the COX-2 enzyme with IC₅₀ values of 4.602 ± 0.17, 2.806 ± 0.10, and 0.849 ± 0.03 µg/mL, respectively. The DAE, 2 and 4 reduced IL-2 to 3.49 ± 0.12—7.018 ± 0.24 pg/mL; IL-4 to 6.019 ± 0.21–12.07 ± 0.41 pg/mL, and TNF-α to 323.65 ± 11.10–501.88 ± 17.21 pg/mL, respectively. Western blotting revealed a decrease in iNOS protein expression. Rutin showed improved docking scores (-8.92 and -7.92 kcal/mol) with AChE and hCA, while 100 ns Molecular Dynamc Simulations (MDS) showed that rutin maintained stable interactions with the proteins throughout the simulations.

Conclusion

C. macrospermum extract and its phenolics are promising candidates for AD management, though additional in vivo and clinical studies are needed.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12906-026-05365-8.

Background

Alzheimer’s disease (AD), the most common form of dementia, is a complex neurodegenerative disorder marked by gradual cognitive decline and often co-existing metabolic problems. It is considered a major global health challenge [1] as 55.2 million people worldwide are affected by dementia, according to the World Health Organization’s 2022 dementia research blueprint [2, 3]. Additionally, by 2030, the number of people living with dementia is expected to reach 78 million. Regarding the global economic burden, including medical care and social services, it is projected to exceed US$2.8 trillion [2], exerting a profound impact on individuals, families, and societies. Recently, researchers have focused on AD due to the rising number of older adults, especially since there are no potential treatments, and all existing strategies aim to slow the progression of symptoms and control behavioural changes. This is due to the complicated mechanism underlying AD development. Accordingly, the discovery of multifaceted treatments for the control of AD will be an effective strategy and are in dire need.

One of the main pathological features of AD includes the deposition of extracellular β-amyloid (Aβ) and intracellular neurofibrillary tangles [4, 5]. Both are believed to play a key role in disease progression. Aβ accumulation in brain tissue and blood vessels triggers microglial migration, leading to both short-term and long-term inflammatory responses against these deposits. This activates signalling pathways that result in the production of nitric oxide (NO), reactive oxygen species (ROS), proinflammatory cytokines such as TNFα, leukotrienes, and cyclooxygenase-2 (COX-2), ultimately causing neuronal death [6]. Recent evidence has highlighted human carbonic anhydrases (hCAs), particularly hCA II, as emerging contributors to AD pathophysiology. Dysregulation of CA activity has been associated with impaired cerebral blood flow, altered pH homeostasis, and reduced perivascular clearance of amyloid-β deposits [7].

Activation of CA can worsen memory loss and cognitive issues. Therefore, hCAs has been identified as a potential new target for AD treatment. Conversely, finding CA inhibitors presents a novel frontier in AD therapy. hCA inhibitors can decrease Aβ aggregation and enhance cerebral blood flow, while activators improve cerebrovascular functions and restore pH balance. Acetylcholinesterase (AChE) is the primary enzyme in the cholinergic nervous system. Throughout the progression of AD, various neuron types deteriorate, including a loss of forebrain cholinergic neurons, which leads to a persistent decline in acetylcholine levels [8, 9]. Treatments aimed at reversing cholinergic deficiency largely depend on the importance of cholinergic function in cognition. Although overall AchE activity decreases in the AD brain, current treatments mainly use AchE inhibitors, which enhance cholinergic transmission but offer only modest and transient benefits [10, 11]. There is increasing interest in new AD therapies targeting specific lifestyle factors, environmental issues, or dietary polyphenols, due to their significant role in AD [12]. Phenolic compounds are bioactive metabolites with strong antioxidant, anti-inflammatory, anti-allergic, antihypertensive, anticancer, antidiabetic, and neuroprotective effects, which have garnered considerable attention from researchers [13]. Consequently, discovering polyphenol-based treatments as multitargeted therapies for AD is a promising approach.

Family Arecaceae, a group of flowering, perennial plants, includes climbers, shrubs, and tree-like forms; most are commonly called palms [14]. This family comprises around 2,600 species and 181 genera, mainly native to tropical and subtropical areas [14]. Traditionally, African palm trees have been used to treat inflammation, liver issues, epilepsy, bronchitis, malaria, wounds, headaches, and diarrhea [15]. Chemically, palm trees are prolific with diverse natural compounds, such as phenolics (flavonoids, tannins, phenolic acids), steroids, alkaloids, carotenoids, and lignans [16]. Biologically, they exhibit numerous pharmacological activities, including anti-hyperlipidemic, antioxidant, cytotoxic, antimicrobial, anti-inflammatory, hepatoprotective, analgesic, and immunostimulant effects [16]. Carpoxylon is a monotypic genus that comprises C. macrospermum H. Wendl. & Drude (common in South Africa). The species is characterized by clustered trunks and alternate, pinnate leaves [17]. Limited data exists on the chemistry and biological activity of C. macrospermum [18], which motivated us to pursue this investigation.

Herein, we aimed to isolate and identify the main phenolic metabolites from C. macrospermum leaves and examine their effects on proinflammatory markers, CA, and AChE through in vitro studies and in silico analyses. This thereby connects the observed biological activity with the phenolic scaffold. Additionally, the best-docked protein-compound complex was subjected to molecular dynamics simulation (MDS).

Materials and methods

General experimental

¹H and ¹³C NMR data were recorded at 300 and 75 MHz, respectively, using a Bruker AVANCE NMR spectrometer. Chemical shifts are reported at δ ppm relative to TMS. ESI/MS data were retrieved using an XEVO TQD triple quadrupole LC/MS/MS (Waters Corporation, Milford, MA, USA). Whatman No. 1 mm paper (Whatman Ltd., Maidstone, Kent, England) was used for paper chromatography (PC) eluted with n-butanol: acetic acid: water, 4:1:5 (BAW, top layer S₁) and 15% acetic acid/water (S₂). Polyamide S (Fluka Chemie AG, Switzerland), cellulose (E. Merck-Darmstadt, Germany), and Sephadex LH-20 (Pharmacia, Uppsala, Sweden) were used as stationary phases for column chromatographic (CC) separation. Conversely, a mixture of n-butanol, isopropyl alcohol, and water (BIW, 4:1:5, top layer S₃) and/or MeOH/H₂O were used for CC elution. Phenolic compounds were characterized using a UV lamp (VL-215 LC, France) and spraying reagents such as Naturstoff and FeCl₃. All solvents are of analytical grade, supplied by El Nasr Pharmaceutical Chemicals Company (Cairo, Egypt).

Plant material

Leaves of Carpoxylon macrospermum H. Wendl. & Drude were collected in June 2022 from El Abd Garden on the Alexandria Desert Road, Egypt, in accordance with local and national regulations for medicinal plant collection and with permission from the garden authorities. Also, comply with the Convention on Biological Diversity and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. The sample (01Cma/2022) was preserved in the herbarium of the Pharmacognosy Department at Helwan University’s Faculty of Pharmacy, after authentication by Dr. Trease Labib, Senior Botanist at the Mazhar Botanical Garden in Giza, Egypt.

Extraction, fractionation, and isolation of the phenolic metabolites

Fresh leaves of C. macrospermum (1.5 kg) were air-dried to produce 800 g of dry powder, which was then extracted using 80% aqueous methanol (4 × 3 L) under reflux for four h. The resulting solvent was evaporated under reduced pressure at low temperature to yield 300 g of dry extract. The dried extract was subsequently extracted with n-hexane (2 L × 4, at 50 °C) under reflux to remove fatty material, resulting in a defatted extract (215 g). The 2D PC of the DAE was performed using S1 for the first run and S2 for the second to investigate phenolic compounds under UV light before and after spraying with Naturstoff and FeCl₃. Part of the extract (5 g) was used for biological testing, and approximately 150 g was fractionated on a polyamide column (5 × 120 cm, 300 g) using a water–methanol mixture (100:0 to 0:100%) to produce ten main fractions. Fractions with similar content were combined after examining the PC under UV light before and after reagent spraying. Fraction I (7 g, 100% H₂O) was excluded; its composition, investigated by PC, showed that it was nearly free of phenolics. Fraction II (4 g; 20–30% MeOH/H₂O) was chromatographed over successive cellulose column chromatography (CC) eluted with 20% MeOH/H₂O, followed by final purification using Sephadex LH-20 CC (50% MeOH/H₂O) to obtain a pure form of compound 1 (40 mg). Fraction III (4 g; 40–50% MeOH/H₂O) was applied to a cellulose CC eluted with S₃, resulting in two subfractions. One contained a dark purple spot by UV and was subjected to Sephadex LH-20 (50% MeOH/H₂O) to yield 2 (30 mg). Compound 3 (25 mg) was purified from the second sub-fraction (2 g; 60% MeOH/H₂O) by consecutive Sephadex LH-20 columns using 20% MeOH/H₂O as an eluent. Fraction IV (2 g; 70–80% MeOH/H₂O) was purified by cellulose column using S₃ as the eluent, resulting in two subfractions, each having crude samples of compounds 4 and 5. Further purification with Sephadex LH-20 (50% MeOH/H₂O) yielded compounds 4 (20 mg) and 5 (15 mg). The purity of all compounds was confirmed by PC with S₁ and S₂, visualization under UV lamp, and after spraying with Naturstoff and FeCl₃ reagents.

Carbonic anhydrase (CA) inhibition assay

To examine the inhibition of the target hCA enzyme by compounds 1–5 and the DAE, a carbonic anhydrase inhibitor screening kit (Catalog # K473-100, BioVision, Milpitas, CA, United States) was used. A detailed method is available in the supplementary data.

Acetylcholinesterase (AChE) inhibition assay

To evaluate the inhibition of acetylcholinesterase by compounds 1–5 and DAE, a slightly modified spectrophotometric method [19] was used with the RayBio-Quantichrom acetylcholinesterase inhibitor screening kit (Colorimetric, BioVision, United States, Catalogue No.: K197-100), following the manufacturer’s instructions [20]. Donepezil served as a reference AChEinhibitor. A detailed method is available in the supplementary data.

Investigating the anti-inflammatory activity

Cyclooxygenase-2 (COX-2) inhibition assay

Compounds 1–5 and DAE were tested using fluorometric COX-2 inhibitor screening kits (BioVision, Switzerland) according to the manufacturer’s instructions. A detailed method is available in the supplementary data.

LPS induced inflammation in RAW264.7 murine monocyte/macrophage cells

Cell line and culture conditions

RAW264.7 murine monocyte/macrophage cells were supplied by the Egyptian Holding Company for Biological Products and Vaccines (VACSERA, Giza, Egypt). The culture medium for RAW264.7 cells was supplemented with 10% fetal bovine serum (FBS), along with 100 U/mL of penicillin and 100 µg/mL of streptomycin in DMEM. The cells were then incubated in a humidified environment at 37 °C with 5% CO₂ [21].

In vitro MTT cell viability assay

The cytotoxicity of the isolated compounds and extract on RAW 264.7 macrophages was assessed using the MTT colorimetric assay, as per the manufacturer’s instructions (Sigma-Aldrich, Steinheim, Germany). A detailed method is available in the supplementary data.

Measurement of IL-2, IL-4 and TNF-α

The supernatants from the culture were collected and centrifuged at 3000 rpm for 20 min. IL-2, IL-4, and TNF-α cytokine levels in LPS-immunized RAW 264.7 cell culture supernatants were measured using ELISA kits (RayBio Mouse IL-2, ab100710 Mouse IL-4, and ab181421 Human TNF-α Simple Step ELISA Kits), following the manufacturer’s instructions, with celecoxib serving as a reference.

Western blot analysis for iNOS production in RAW 264.7 cells

RAW 264.7 cells were washed with cold phosphate-buffered saline (PBS) and lysed in a Western cell lysis buffer 24 h after treatment. Cell debris was removed by centrifugation, and protein concentration was measured using a Bradford assay. Equal amounts of protein (20 µg) were denatured in SDS loading buffer, separated by SDS-PAGE, and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk in TBS-T, incubated overnight at 4 °C with iNOS Antibody (CST #39898) (Cell Signaling Technology) and β-actin (Sigma) as a loading control, followed by appropriate HRP-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence (ECL).

In silico studies

Molecular docking

Molecular docking studies predicted how compounds 2 and 4 from C. macrospermum leaves bind to the AChE receptor (PDB ID: 4M0F) [22] and the hCA II receptor (PDB ID: 3NB5) [23]. A detailed method is available in the supplementary data.

Molecular dynamics simulation

Molecular dynamics simulations (MDS) were performed for 100 ns using GROMACS 2.1.1 [24] on compound 2, the crystallized ligand (PDB ID: 1YK), and (PDB ID: R21). A detailed method is available in the supplementary data.

In-silico studies prediction of bioactivity profile of compound 2

SwissADME® (https://www.swissadme.ch.) was the online tool utilized to carry out the computational prediction for compound 2. Using this program, relative findings for physicochemical parameters might be retrieved (lipophilicity (logP), molecular weight, polar surface area, number of hydrogen bond donors and acceptors, number of rotary bonds, and solubility in water), drug-likeness profile, and pharmacokinetic profile (absorption, distribution, metabolism, excretion, and toxicity) of the molecule and the bio activity profile (pharmacodynamics).

Statistical analysis

Data analysis was conducted using GraphPad Prism software (version 5.0). All experiments were performed in triplicate, with results shown as mean ± standard deviation. Variance differences were evaluated using analysis of variance, and differences with a p-value of less than 0.05 were considered statistically significant.

Results

Chemical investigation of the defatted aqueous methanol extract

The DAE was chromatographed using 2D-PC, and the developed spots were examined under UV light before and after spraying reagents (Naturstoff and FeCl₃). The chromatographic analysis revealed the richness of the extract in phenolic metabolites; therefore, it was subjected to fractionation and isolation of its major chemical components using column chromatography packed with various stationary phases and eluted with a suitable mobile system as per the nature and complexity of each fraction. Five phenolic compounds were isolated and elucidated based on their NMR spectroscopic and mass spectrometry data (S1 Fig. -S11 Fig.). They were categorized as three phenolic acids, viz, chlorogenic acid (1), vanillic acid (4), p-hydroxybenzoic acid (5), and two flavonoids, rutin (2) and hesperidin (3) (Fig. 1).

Fig. 1.

Chemical structure of isolated phenolics

Compound 1

It is an off-white, amorphous powder (40 mg). It appeared sky-blue under UV light, shifting to a faint blue after application of the FeCl₃ spray reagent. ¹H and ¹³C NMR data and the spectra are represented in the supplementary data file.

Compound 2

It was purified as a yellow amorphous powder with a dark purple spot under UV light, which turned orange and faint green after spraying Naturstoff reagent and FeCl₃ reagent. It showed a molecular ion peak at m/z 611.1681 [M + H] + and 609.1838 [M-H]- in its positive and negative ESI–MS, along with an m/z 1220.7831 [2 M-H]- peak in negative ESI–MS. The ESI–MS, ¹H, and ¹³C NMR data, as well as the spectra, are represented in the supplementary data file.

Compound 3

It was isolated as a yellow amorphous powder, displaying a dark purple spot. ¹H and ¹³C NMR data and the spectra are represented in the supplementary data file.

Compound 4

It was purified into a white, amorphous powder that exhibited a faint blue color when tested with FeCl₃ reagent. ¹H and ¹³C NMR data and the spectra are represented in the supplementary data.

Compound 5

It was isolated as a white, amorphous solid that exhibited a blue color when treated with FeCl₃. It showed an m/z value of 138.9 [M + H]⁺ in positive ESI/MS (spectrum is provided as supplementary data file).

Biological investigation

Anti-human carbonic anhydrase (anti-hCA) potential

The isolated compounds (1–5) and the DAE were tested against human carbonic anhydrase (hCA). As shown in Table 1, the most effective against hCA was the tested extract (IC₅₀ = 0.160 ± 0.008 µg/mL). Among the isolated compounds, rutin (2) and vanillic acid (4) exhibited the strongest inhibitory effects, with IC₅₀ values of 0.243 ± 0.012 µg/mL and 0.290 ± 0.015 µg/mL, respectively, compared to the reference Acetazolamide (AAZ), (IC₅₀ 0.095 ± 0.005 µg/mL).

Table 1.

The enzyme inhibitory results of the isolated compounds (1–5) and DAE against human carbonic anhydrase, acetylcholinesterase, and COX2

No	Sample	Carbonic anhydrase (hCA) IC50 ± SD (µg/mL)	Acetylcholinesterase (AChE) IC50 ± SD (µg/mL)	COX2 IC50 ± SD (µg/mL)
	DAE	0.160 ± 0.008	3.732 ± 0.13	4.602 ± 0.17
1	Chlorogenic acid	1.210 ± 0.062	3.300 ± 0.12	6.212 ± 0.23
2	Rutin	0.243 ± 0.012	0.868 ± 0.03	2.806 ± 0.10
3	Hesperidin	1.865 ± 0.095	8.906 ± 0.31	26.200 ± 0.96
4	Vanillic acid	0.290 ± 0.015	0.597 ± 0.02	0.849 ± 0.03
5	P-Hydroxy benzoic acid	0.335 ± 0.017	21.110 ± 0.73	10.160 ± 0.37
	Standards
	AAZ	0.095 ± 0.005	––	––
	Donepezil	––	0.210 ± 0.007	––
	Celecoxib	––	––	0.426 ± 0.02

AAZ Acetazolamide, DAE Defatted aqueous methanol extract

Anti-human choline esterase (AChE) potential

The isolated compounds (1–5) and DAE were examined for their ability to inhibit human acetyl cholinesterase (AChE) using Ellman’s method, with Donepezil as a reference compound for selective AChE inhibition (Table 1). The obtained findings demonstrated that rutin (2) and vanillic acid (4) were the most active compounds against AChE. They recorded IC50 values of 0.868 ± 0.03 µg/mL and 0.597 ± 0.02 µg/mL, respectively, which were more potent than the tested DAE (IC₅₀ = 3.732 ± 0.13 µg/mL).

Anti-cyclooxygenase-2 (COX-2) activity

The anti-inflammatory potential of compounds (1–5) and DAE was assessed by testing their ability to inhibit the COX-2 enzyme, compared to the reference anti-COX-2 drug, Celecoxib. Results (Table 1) showed that vanillic acid (4) was the most active compound against COX-2 (IC₅₀ = 0.849 ± 0.03 µg/mL), followed by rutin (2) (IC₅₀ = 2.806 ± 0.10 µg/mL). The results were compared to those of celecoxib (IC₅₀ = 0.426 ± 0.02 µg/mL) and the tested DAE (IC₅₀ = 4.602 ± 0.17 µg/mL).

Cell-based anti-inflammatory activity

The anti-inflammatory potential of the isolated compounds (1–5) and DAE was assessed using a macrophage cell-based assay. This assay measured changes in the expression levels of target protein markers in lipopolysaccharide (LPS)-activated murine macrophage cells (RAW 264.7). First, the cytotoxicity of the tested samples (compounds 1–5 and DAE) on RAW 264.7 cells was evaluated using the MTT assay. Table 2 lists the IC₅₀ values for all tested samples, which were greater than 100 µg/mL (IC₅₀ range 124.86 ± 4.55—271.74 ± 9.20). As a result, all samples were used in the next assay at concentrations equal to 0.25 of the IC₅₀ determined in the MTT assay.

Table 2.

Cytotoxic assay results of compounds 1–5 and DAE in MTT assay on RAW 264.7 cells

No	Sample	IC50 ± SD (µg/mL)
	DAE	230.66 ± 8.41
1	Chlorogenic acid	156.37 ± 5.70
2	Rutin	271.74 ± 9.20
3	Hesperidin	135.44 ± 4.44
4	Vanillic acid	124.86 ± 4.55
5	P-Hydroxy benzoic acid	125.54 ± 4.58
	Celecoxib	136.40 ± 4.76

DAE Defatted aqueous methanol extract

The anti-inflammatory activity of the tested samples was evaluated by measuring changes in TNF-α and IL-2 (pro-inflammatory cytokines), IL-4 (a Th2-associated immune-regulatory and inflammatory cytokine), as well as iNOS expression in LPS-immunized RAW 264.7 cells using ELISA and Western blotting, respectively, with Celecoxib as a reference anti-inflammatory drug. In Table 3, LPS, a potent immune system activator, significantly stimulated the release of proinflammatory cytokines (IL-2, IL-4, and TNF-α) in RAW 264.7 cells (20.53 ± 0.70, 79.59 ± 2.73, and 2013.03 ± 69.04 pg/mL, respectively). Treatment of macrophages with compounds (1–5) and DAE suppressed the release of IL-2, IL-4, and TNF-α to varying degrees, as shown in Table 3 and Fig. 2. The DAE showed strong anti-inflammatory potential, as indicated by the reduced levels of the targeted pro-inflammatory cytokines: 3.49 ± 0.12 (IL-2), and 323.65 ± 11.10 pg/mL (TNF-α) as well as the anti-inflammatory 12.07 ± 0.41 (IL-4). Regarding the tested pure phenolics, the most effective anti-inflammatory potential was demonstrated by compound 4 (vanillic acid), which exhibited nearly the same anti-inflammatory effect (4.011 ± 0.14 (IL-2), 375.89 ± 12.89 pg/mL (TNF-α), and 8.284 ± 0.28 (IL-4)) as the standard celecoxib. Meanwhile, rutin (2) ranked as the second most effective phenolic with anti-inflammatory potential.

Table 3.

The suppressive effect of compounds (1–5) and DAE on IL-2, IL-4, and TNF-α in LPS-immunized RAW 264. Cells

No	Sample	Conc (0.25 IC50) (µg/mL)	IL-2 (pg/mL)	IL-4 (pg/mL)	TNF-α (pg/mL)
	DAE	64.2	3.49 ± 0.12	12.07 ± 0.41	323.65 ± 11.10
1	Chlorogenic acid	50.9	6.101 ± 0.21	35.93 ± 1.23	889.85 ± 30.52
2	Rutin	87.9	7.018 ± 0.24	6.019 ± 0.21	501.88 ± 17.21
3	Hesperidin	38.6	13.06 ± 0.44	46.94 ± 1.61	1292.41 ± 44.32
4	Vanillic acid	41.6	4.011 ± 0.14	8.284 ± 0.28	375.89 ± 12.89
5	P-Hydroxy benzoic acid	25.9	12.48 ± 0.43	38.55 ± 1.32	1140.29 ± 39.11
	Celecoxib	44.6	4.35 ± 0.15	9.65 ± 0.33	242.22 ± 8.31
	LPS Control	––	20.53 ± 0.7	79.59 ± 2.73	2013.03 ± 69.04

DAE Defatted aqueous methanol extract, LPS Lipopolysaccharide

Data are shown as mean ± SD

Fig. 2.

The suppressive effect of compounds (1–5) and DAE on IL-2, IL-4, and TNF-α in LPS-immunized RAW 264 cells as fold change. a and b were significantly different (P < 0.05) from the LPS control and celecoxib, respectively. DAE: Defatted aqueous methanol extract; LPS: Lipopolysaccharide

Regarding the effect of the tested compounds (1–5) and DAE on iNOS expression levels, as assessed by Western blot, all tested samples significantly inhibited iNOS protein expression. However, compounds 2 (rutin) and 4 (vanillic acid) demonstrated greater potency than the standard drug, celecoxib. Compounds 1, 3, and the DAE showed activity like celecoxib, while compound 5 exhibited the least inhibitory activity (Fig. 3).

Fig. 3.

Western blot of iNOS protein expression in RAW 264.7 cells. AWestern experiment display. B Quantitative analysis of iNOS protein expression using a bar chart. a and b were significantly different (P < 0.05) from LPS control and celecoxib, respectively. DAE: Defatted aqueous methanol extract; LPS: Lipopolysaccharide

In-silico evaluation

In-silico molecular docking of the most active compound (2 and 4) on acetylcholinesterase (AChE) and human carbonic anhydrase II (hCA II)

Molecular docking studies were conducted to understand how phenolic compounds isolated from C. macrospermum leaves DAE interact with the acetylcholinesterase (AChE) receptor (PDB ID: 4M0F). AutoDock Vina was used to assess intermolecular interactions and predict possible binding conformations. The results showed that compound 2 (rutin) displayed a binding affinity similar to the co-crystallized ligand (PDB ID: 1YK), with docking scores of −8.92 and −9.549 kcal/mol, respectively. Additionally, compound 4 (vanillic acid) possessed a binding affinity of 4.88 kcal/mol. Rutin and territrem B (TRB) displayed similar binding behaviors, targeting both the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE. This dual-site binding helps stabilize the complex and boosts the inhibitory potential of these compounds, indicating their potential as therapeutic agents (Table 4).

Table 4.

Results of molecular docking of the crystallized ligand and the isolated phenolics (2 and 4) in conjunction with acetylcholinesterase (PDB ID: 4M0F) and human carbonic anhydrase II (PDB ID: 3NB5)

Docked Compounds	PDB ID: 4M0F			PDB ID: 3NB5
	RMSD (Å)	Score (kcal/mol)	Interacting amino acids (Å)	RMSD (Å)	Score (kcal/mol)	Interacting amino acids (Å)
Crystallized ligand	−9.54	1.139	Conventional H-bond Tyr124 Carbon H-bond Tyr72, Asp74, Gly121, Ser293 π –Sigma Trp86, Typ337 π – π T-shaped Trp286, Tyr341	−7.16	1.49	Metal-Acceptor Zn 261 Conventional H-bond Gln92, Thr198 Carbon H-bond Asn62, Hus 94, Leu197 π-Sulphur His96, Trp208
Rutin (2)	−8.92	1.12	Conventional H-bond Asp74, Thr83, Asn87, Tyr124, Tyr133, Glu202, Ser203, Ser293 Carbon H-bond Gly121, Trp286, Ser293, Typ337, Tyr341, His447 Amide- π Staked Trp86 Alkyl Leu289	−7.92	1.75	Metal-Acceptor Zn 261 Conventional H-bond Trp5, Asn62, Asn67, Gln92, Thr198, Carbon H-bond His94, Thr199 π –Sigma Leu197 π – π T-shaped Phe130 π –Alkyl His64, Val121
Vanillic acid (4)	−4.88	1.06	Conventional H-bond Tyr133, His447 Carbon H-bond Gly121, Gly126 π – π T-shaped Trp86 π –Alkyl Trp86, Typ337	−6.76	1.03	Conventional H-bond Thr198 Carbon H-bond Asn67 π – cation His94, His96, His119, Zn 261 π–Alkyl Ala66

Additionally, the interactions of compounds 2 (rutin) and 4 (vanillic acid) with the human carbonic anhydrase II (hCA II) receptor, along with its co-crystallized ligand (PDB ID: R21), have been assessed. As shown in Table 4, rutin (2) and vanillic acid (4) exhibited significant binding affinities of −7.92 and −6.76 kcal/mol, respectively, compared to −7.16 kcal/mol for the reference ligand. Both compounds bind to the active site of CA II, forming important coordination bonds with the catalytic zinc ion (Zn²⁺), which is essential for stabilizing the enzyme-ligand complex. These interactions emphasize their potential inhibitory effects on CA II and support their possible pharmacological significance.

Molecular dynamics simulation

Based on the molecular docking results, the highest-ranked AChE-rutin and hCAII-rutin complexes, compared with the crystallized ligand–protein complexes, were selected for detailed molecular dynamics (MD) simulation over 100 ns. MD simulation is a valuable method for examining the fluctuating behavior of molecular systems and is frequently utilized in drug development to forecast drug-target interactions. To analyze the MD trajectory effectively, several important parameters—such as RMSD, RMSF, Rg, PCA, FEL, and DCCM—were evaluated, as they provided insights into the system’s stability and dynamics.

In-silico ADME (drug likeness and medicinal chemistry prediction)

The Swiss ADME online tool was used to analyze the in silico computational evaluation for TRB, compounds 2 and 4. Table 5 displays the outcomes of the expected parameters, which include molecular characteristics, pharmacokinetics, drug-likeness, and medicinal chemistry to evaluate their drug-likeness and potential central nervous system (CNS) relevance. The BOILED-Egg model revealed clear differences among the three compounds in terms of predicted gastrointestinal absorption and blood–brain barrier (BBB) permeability [25, 26]. The reference compound TRB exhibited physicochemical properties consistent with acceptable drug-likeness, including moderate lipophilicity, favorable gastrointestinal absorption, and positioning closer to the BBB-permeable region in the BOILED-Egg plot. Among the tested compounds, 4 demonstrated the most favorable ADME profile. It showed high predicted gastrointestinal absorption, low molecular weight, moderate lipophilicity, and localization within or near the BBB region in the BOILED-Egg model. In contrast, compound 4 displayed markedly different pharmacokinetic characteristics. SwissADME predictions indicated low gastrointestinal absorption, lack of passive BBB permeability, which is consistent with its high topological polar surface area (TPSA = 269.43 Ų), high number of hydrogen bond donors and acceptors, and low lipophilicity., and predicted P-glycoprotein substrate behavior, consistent with its large molecular size and high polarity [27] (Table 5).

Table 5.

Prediction of molecular properties, pharmacokinetics, drug-likeness, and medicinal chemistry of TRB, compound 2, and 4 using the Swiss ADME online tool

Test items		TRB	2	4
Boiled-Egg
Swiss ADME	Molecular properties
	M log P	1.25	−3.89	0.74
	TPSA (Å2)	124.66	269.43	66.76
	M. Wt (g/mol)	526.57	610.52	168.15
	nHBA (NO)	9	16	4
	nHBD (OHNH)	2	10	2
	NRB	4	6	2
	Pharmacokinetics
	GI absorption	High	low	High
	BBB permeant	No	No	No
	P-gp substrate	Yes	Yes	No
	Skin permeation (log Kp) cm s − 1	−7.54	−10.26	−6.31
	Drug likeness and medicinal chemistry
	log S (ESOL)	−4.83	−3.3	−2.02
	Solubility	Moderate	Moderate	Soluble
	PAINS	0	1 alert; catechol_A	0
	Synthetic accessibility	5.93	6.52	1.42
	Bioavailability score	0.55	0.17	0.85
	Lipinski rule (violation)	Yes (1)	No (3)	Yes (0)
	Veber rule (violation)	Yes (0)	No (1)	Yes (0)

Discussion

The current study presents a multifaceted research approach that integrates the phytochemical analysis, biological investigation, and in silico molecular docking of C. macrospermum extract and its phenolic metabolites in a synergistic framework to assess its multi-targeted therapeutic potential for Alzheimer’s disease. This interdisciplinary strategy offers several key advantages over conventional research approaches. First, the combination of biological examination and molecular docking supports insights into how bioactive metabolites inhibit or activate a targeted enzyme or receptor. Second, it provides preliminary, predictive validation through in silico analysis, therefore minimizing the need for early-stage in vivo animal testing, and supports higher ethical and safety standards. Ultimately, the approach enhances experimental efficacy while reducing overall research costs [28].

Therapies aimed at reducing Alzheimer’s disease (AD) symptoms rely on acetyl cholinesterase inhibitors. They slow the progression of degenerative changes and protect against the expression of phosphorylated tau protein by preventing β-amyloid formation. However, long-term use of synthetic AChE inhibitors can cause side effects. Irreversible AChE inhibition may lead to ACh buildup at cholinergic synapses and, ultimately, death. Conversely, low ACh levels can contribute to memory deficits associated with AD [29]. Therefore, exploring alternatives such as natural products that act as reversible and moderate AChE inhibitors is a promising approach. Many natural products have shown potential in AD treatment due to their neuroprotective effects and good safety profiles. Since inhibiting choline esterases effectively manages AD by alleviating symptoms caused by the progressive loss of cholinergic function in various brain areas through increased acetylcholine (ACh) levels, further research is warranted. Additionally, studies have demonstrated that CA inhibitors reduce amyloid β pathology and enhance cognitive function by improving glial health and cerebrovascular function [7]. Pharmacological inhibition of CA has been reported to reduce Aβ pathology and improve cognitive performance in experimental models by enhancing glial fitness and cerebrovascular function [7]. Given the growing recognition of vascular contributions to cognitive impairment and dementia, targeting hCA represents a plausible adjunct strategy within a multi-target AD framework. Therefore, the evaluation of CA inhibition in the present study was hypothesis-driven and supported by recent mechanistic insights. Likewise, cyclooxygenase-2 (COX-2) is markedly upregulated in early stages of Alzheimer’s disease and is localized in neurons and glial cells surrounding amyloid plaques. Elevated COX-2 expression contributes to prostaglandin-mediated neuroinflammatory amplification, synaptic dysfunction, and neuronal vulnerability [30]. Experimental and clinical evidence suggests that COX-2 overactivation enhances oxidative stress, promotes Aβ-associated toxicity, and exacerbates inflammatory cascades within the AD brain. Therefore, modulation of COX-2 activity represents a mechanistically relevant anti-inflammatory target in neurodegenerative conditions. Since few studies have explored the biological potential and phytochemical profile of C. macrospermum, further research on this species is necessary. Based on our prior insights on the promise of the petroleum ether extract of C. macrospermum [18] in neuroprotective setting, herein we aimed at testing the defatted aqueous methanolic extract (DAE) against key enzymes, namely AChE, CA, and COX-2, involved in Alzheimer’s disease (AD) pathogenesis. The DAE demonstrated a potential inhibitory effect on AChE and a strong inhibitory effect on CA. In this regard, to date, only one study has reported the effective inhibition of AChE by the extract of Iranian C. macrospermum [31]. Conversely, the phytochemical analysis of the DAE identified five phenolic metabolites. Notably, rutin (2) has long been included in nutritional supplements because of its versatile health benefits [32]. It exerts a neuroprotective potential in neurodegenerative diseases such as Alzheimer’s and Parkinson’s, by crossing the blood–brain barrier and functioning as both an antioxidant and an anti-inflammatory agent. In this study, rutin demonstrated promising inhibition of AChE and hCA, consistent with previous research [33–35]. Vanillic acid (4), another compound isolated from DAE, is a key flavoring agent used in food, pharmaceuticals, and cosmetics. Therefore, assessing its neuroprotective potential is highly relevant. In this work, vanillic acid efficiently inhibited both hCA and AChE. This supports earlier studies showing vanillic acid’s strong binding affinity to CA-III, even greater than that of the standard inhibitor, acetazolamide, as evidenced by Hummel-Dreyer chromatography [36]. Additionally, Salau et al. [37] reported that vanillic acid displayed a similar inhibitory effect on AChE. Furthermore, vanillic acid and rutin demonstrated notable COX-2 inhibitory activity. While these findings derive from an enzyme-based system rather than a central nervous system model, they indicate that the identified phenolic scaffolds may contribute to the attenuation of inflammatory mediator production upstream of cytokine release.

In a few circumstances, as the extract effect on hCA, we observed the potential of the DAE over the isolated bioactive metabolites. This may be, at least in part, due to the complicated nature of the extract in which diverse phytochemicals can act synergistically and produce a stronger, multitarget effect regardless of their concentration [38]. On the other side, the relatively strong inhibitory activity observed for certain polyphenolic compounds may be attributed to their structural features, including multiple hydroxyl groups capable of forming extensive hydrogen-bonding networks within enzyme active sites. In the case of hCA, phenolic moieties may additionally participate in coordination interactions with the catalytic Zn²⁺ ion or stabilize the surrounding active-site residues, thereby enhancing apparent inhibitory potency. Such multi-site interaction capacity is consistent with previously reported binding behavior of polyphenolic scaffolds in metalloenzymes.

A key aspect of AD is the initiation of inflammation and the excessive release of cytokines like interleukin (IL)−1β, IL-6, and TNF-α. These contribute to a damaging cycle in glial cells, resulting in neuronal dysfunction and ultimately cell death. The pro-inflammatory cytokine IL-1β can affect the processing of amyloid precursor protein, which leads to increased production of β-amyloid. When β-amyloid levels rise and form aggregates, they trigger the production of proinflammatory cytokines and reactive oxygen species (ROS) [39]. During inflammation, levels of COX-2 and NO also increase. Therefore, reducing β-amyloid caused by inflammatory cytokines and oxidative stress is a key therapeutic strategy for AD. In our study, we examined the anti-inflammatory effects of the DAE and its isolated phenolics using in vitro enzyme-based assays and LPS-activated RAW cells. The LPS-stimulated cells can simulate in vivo conditions [40, 41]. While LPS is a well-established inducer of pro-inflammatory cytokines such as TNF-α and IL-6 via TLR4 activation [42, 43], it also triggers a delayed compensatory production of anti-inflammatory cytokines, including IL-10 and IL-4, as part of a regulatory feedback mechanism aimed at restoring immune homeostasis [44, 45]. IL-4 is associated with alternative macrophage activation and resolution-phase signaling. Therefore, attenuation of the primary inflammatory stimulus may reduce the downstream compensatory induction of IL-4, which is consistent with the cytokine modulation observed in the present study [46].

Results showed that DAE, along with isolated phenolics such as rutin (2) and vanillic acid (4), inhibited the LPS-induced production of IL-2, and TNF-α and modulated IL-4 levels. This confirms previous findings by Kim et al. [47] which showed that vanillic acid suppressed LPS-induced production of pro-inflammatory cytokines TNF-α and IL-6. Vanillic acid also reduced the activation of NF-κB signaling pathway and suppressed the inflammatory mediators in lipopolysaccharide-stimulated mouse peritoneal macrophages. Likewise, rutin decreased oxidative stress and lowered the production of pro-inflammatory cytokines and NO in vitro [48]. Additionally, another study found that oral rutin administration significantly reduced memory deficits and neuroinflammation in AD transgenic mice by lowering the levels of IL-1β and IL-6 in their brains [49].

Finally, during neuroinflammation, NO is overproduced by iNOS in response to inflammatory stimuli, which contributes to the progression and worsening of AD. Consequently, iNOS inhibitors may help slow down AD progression [48]. Therefore, in this study, the effects of isolated phenolics and DAE on iNOS expression were examined. The results showed that rutin and vanillic acid had the strongest ability to inhibit iNOS expression, followed by hesperidin and DAE. Rutin significantly inhibited UVB-induced iNOS expression [50]. Additionally, earlier research demonstrated that rutin blocks LPS-induced NO production, which aligns with its suppression of LPS-induced iNOS gene expression, as shown by Western blot analysis [51]. Similarly, vanillic acid was able to reduce NO production in LPS-stimulated mouse peritoneal macrophages [47] and suppressed COX-2 and iNOS expression in IL-1β-induced chondrocytes [52]. Moreover, Zhou et al. [53] reported that vanillic acid reduced the expression of IL-6, IL-1β, TNF-α, and iNOS in a collagen-induced arthritis mouse model, as well as in LPS-stimulated RAW264.7 cells. Although we previously noted that C. macrospermum petroleum ether extract (PEE) can decrease iNOS activity [18]; the chemical profiles of the two extracts differ. When considered alongside cytokine suppression and iNOS downregulation, the COX-2 inhibition data support a convergent anti-inflammatory mechanism relevant to AD-associated neuroinflammatory pathways.

Therefore, the anti-inflammatory effects observed with DAE, rutin, and vanillic acid suggest they could be promising candidates for managing neurological disorders, including AD.

Oxidative stress plays a central role in the pathogenesis of Alzheimer’s disease (AD), contributing to amyloid-β (Aβ) aggregation, tau hyperphosphorylation, mitochondrial dysfunction, and synaptic impairment. Excessive generation of reactive oxygen species (ROS) promotes lipid peroxidation, DNA damage, and neuronal apoptosis [12]. Polyphenolic compounds such as rutin, chlorogenic acid, and hesperidin are well-documented free radical scavengers that modulate oxidative stress–responsive pathways, including nuclear factor erythroid 2–related factor 2 (Nrf2) signaling [54, 55]. Rutin has been shown to attenuate ROS production, restore antioxidant enzyme activity (SOD, CAT, GPx), and reduce neuroinflammatory cascades in experimental AD models [56]. Similarly, chlorogenic acid suppresses oxidative damage and mitigates Aβ-induced neuronal toxicity via mitochondrial protection mechanisms [57]. Although direct antioxidant assays were not performed in the present study, the established redox-modulating capacity of these phenolics provides a complementary mechanistic explanation for their potential relevance in AD-associated pathways.

While the present findings demonstrate significant enzyme inhibition and anti-inflammatory activity in vitro, and in cell-based assays, the biological activities observed herein may be interpreted as preliminary mechanistic evidence targeting AD-associated biochemical pathways.

Molecular docking studies were performed to examine the interactions of the most active phenolics. (2 and 4) with AChE and hCAII. AChEis a serine hydrolase essential for ending cholinergic neurotransmission by hydrolyzing acetylcholine [58]. The AChE active-site gorge (~ 20 Å deep) has distinct regions that support catalysis, substrate recognition, and ligand binding. Initially, ACh attaches to the peripheral anionic site (PAS) of AChE and is directed into the gorge toward the active site via interactions between its quaternary ammonium group and the aromatic rings of residues lining the pathway. At the active site, ACh is correctly positioned for hydrolysis through interactions between the catalytic anionic site and its quaternary ammonium group. The catalytic triad (Ser203, Glu202, His447) drives hydrolysis, a vital process for enzyme function. Simultaneously, the catalytic anionic site (CAS) (Trp86) forms cation-π–π interactions with acetylcholine’s quaternary ammonium group, helping ensure substrate binding specificity [39]. The aromatic gorge, lined with residues like Trp86, Tyr124, and Phe338, stabilizes ligands through π–π stacking, cation–π interactions, and hydrophobic contacts. Additionally, the peripheral anionic site (PAS) (Tyr72, Asp74, Tyr124, Trp286, Tyr341) assists in substrate guidance, allosteric regulation, and amyloid-β binding, linking AChEto Alzheimer’s disease [59, 60] (Fig. 4). Acetylcholinesterase (AChE) has emerged not only as a cholinergic enzyme but also as a modulator of β-amyloid (Aβ) aggregation via its peripheral anionic site (PAS), making it a dual-purpose target in Alzheimer’s disease [61]. While classical AChE inhibitors provide symptomatic relief, they lack efficacy against the amyloidogenic cascade. The enzyme exhibits complex interactions that influence its catalytic efficiency and substrate specificity, highlighting its potential as a target for neurodegenerative diseases, such as Alzheimer’s [62, 63]. We performed molecular docking in the binding sites of the receptors with the two most active phenolics, rutin (2) and vanillic acid (4), to gain a better understanding of their possible binding modes. Additionally, we redocked the crystallized ligand (PDB ID: 1YK, TRB) into 4M0F to validate the docking protocol.

Fig. 4.

Schematic representation of acetylcholinesterase (AChE) active-site architecture. The diagram highlights the major functional regions within the AChEactive-site gorge, including the Peripheral Anionic Site (PAS, yellow), the Catalytic Anionic Site (CAS, red), and the Catalytic Triad (CT, blue). Aromatic amino acid residues lining the gorge (black bars)

Territrem B (TRB) interacts with acetylcholinesterase (AChE) by effectively occupying the active-site gorge, where it establishes stabilizing contacts with key amino acid residues. Its binding is primarily mediated through hydrophobic interactions and π–π stacking with aromatic residues such as Trp86, Tyr124, and Tyr337, which play crucial roles in substrate recognition and inhibitor binding. Moreover, the trimethoxy-phenyl moiety of TRB extends toward the peripheral anionic site (PAS), engaging with residues including Tyr72, Asp74, and Trp286. This interaction at the PAS is thought to modulate ligand entry to the catalytic anionic site (CAS), thereby enhancing TRB’s inhibitory activity (Fig. 5).

Fig. 5.

3D (Left) and 2D (Right) structure interaction poses of AChE(PDB ID: 4M0F) with Co-crystallized ligand PDB ID: 1YK (TRB)

Rutin forms hydrogen bonds with key catalytic triad (CT) residues, including Ser203, Glu202, and His447, which are essential for AChE’s enzymatic activity. It is further stabilized at the catalytic anionic site (CAS) through interactions with Trp86. Additionally, rutin interacts with peripheral anionic site (PAS) residues, such as Tyr72, Asp74, Tyr124, Trp286, and Tyr341, via hydrogen bonding, π–π stacking, and hydrophobic interactions with residues like Leu289. These interactions collectively increase its binding affinity. While vanillic acid interacts with CT residues through hydrogen bonding with His447 and stabilizes within the CAS via hydrophobic interactions with residues like Trp86. Rutin’s dual-site binding effectively blocks substrate access to both the CT and CAS, while also modulating enzyme activity through PAS engagement. This mechanism highlights rutin’s potential as a natural therapeutic candidate for Alzheimer’s disease (Fig. 6).

Fig. 6.

3D (Left) and 2D (Right) structure interaction poses of AChE (PDB ID: 4M0F) with compounds 2 (rutin) and 4 (vanillic acid)

On the other hand, hCA II is a zinc metalloenzyme that catalyzes the reversible hydration of carbon dioxide, thereby contributing to pH regulation, ion transport, and cerebrovascular health. Dysregulation of CA II has been linked to impaired perivascular clearance of amyloid β (Aβ), a hallmark of AD, which exacerbates neurovascular dysfunction and mitochondrial toxicity [7, 64]. The binding pocket of human carbonic anhydrase II (hCA II) is a specialized conical cavity that facilitates the reversible hydration of carbon dioxide (CO₂). Central to this pocket is a zinc ion (Zn²⁺), coordinated by the imidazole nitrogens of His94, His96, and His119, along with a water or hydroxide ligand, which acts as the nucleophile in the catalytic reaction. Surrounding the zinc, a hydrophobic substrate-binding pocket formed by residues such as Val121, Val143, Leu198, Thr199, Val207, and Trp209 sequesters CO₂ for nucleophilic attack. Opposite this hydrophobic region, a hydrophilic channel lined with residues, including Tyr7, Asn62, His64, and Glu106, connects the active site to the solvent, establishing a hydrogen-bond network that stabilizes the zinc-bound hydroxide and facilitates proton transfer during catalysis [65]. This intricate arrangement of residues and interactions enables hCA II to catalyze the hydration of CO₂ with remarkable efficiency, highlighting its essential role in physiological processes. Molecular docking analyses were performed within the binding site of the receptor using the crystallized ligand, R21 (2-(3-chloro-4-hydroxyphenyl)-N-(4-sulfamoylphenethyl) acetamide), which was redocked into the PDB ID: 3NB5 protein structure to validate the docking protocol. The sulfonamide inhibitor directly coordinates with the zinc ion in the active site via its sulfonamide group, displacing the water or hydroxide ligand and forming a tetrahedral geometry, which is a hallmark of potent hCA II inhibition. This direct interaction with the zinc ion ensures potent and specific inhibition of the enzyme (Fig. 7).

Fig. 7.

3D (Left) and 2D (Right) structure interaction poses of hCAII (PDB ID: 3NB5) with Co-crystallized ligand PDB ID: R21

The interactions of the two most active phenolics in the enzyme-based assays (rutin and vanillic acid) with human carbonic anhydrase II (hCA II) involve distinct binding mechanisms that differ markedly from those of conventional sulfonamide-based inhibitors, such as 2-(3-chloro-4-hydroxyphenyl)-N-(4-sulfamoylphenethyl) acetamide (PDB ID: R21). Rutin, a flavonoid glycoside, forms a crucial coordination bond with the catalytic zinc ion (Zn²⁺), while also engaging in hydrogen bonding with key residues including Asn62, Asn67, Thr198, and Thr199. Additionally, it establishes hydrophobic contacts with Val121, Phe130, and His64, contributing to its moderate inhibitory activity. Vanillic acid, on the other hand, features a terminal carboxylate moiety that facilitates a unique binding pattern with hCA II, forming a tetrahedral coordination with the Zn²⁺ ion and interacting with the imidazole nitrogens of His94, His96, and His119. It further stabilizes the complex through hydrogen bonds with residues such as Thr198, Leu197, and Asn67. This comparison underscores the diverse binding strategies of phenolics and highlights their potential as alternative scaffolds for the development of novel hCA II inhibitors with distinctive therapeutic properties (Fig. 8).

Fig. 8.

3D (Left) and 2D (Right) structure interaction poses of hCAII (PDB ID: 3NB5) with compounds 2 (rutin) and 4 (vanillic acid)

Four molecular dynamics simulations were conducted on the rutin-4M0F and rutin-3NB5 complexes over 100 ns. Rutin (2) was selected for its higher binding affinity scores to AChE and hCAII. The analyses included RMSD, RMSF, radius of gyration, PCA, free energy landscape, and DCCM, and these results were compared with those from the crystallized ligand-4M0F and ligand-3NB5 complexes under similar conditions.

The root mean square deviation (RMSD) was computed to evaluate the conformational stability of AChE (PDB ID: 4M0F) and hCA II (PDB ID: 3NB5) in complex with rutin and their respective co-crystallized ligands. ligands (territrem B, PDB ID: 1YK, and the reference ligand, PDB ID: R21) over a 100 ns molecular dynamics simulation. For the AChE complexes, both rutin-4M0F and territrem B-4M0F exhibited relatively stable RMSD profiles. The Rutin-4M0F complex (blue) displayed slightly higher fluctuations, with RMSD values ranging from approximately 1.2 Å to 2.0 Å, while the territrem B-4M0F complex (orange) remained within a narrower range of 1.2 Å to 1.6 Å. Despite the slightly elevated fluctuations, the Rutin-4M0F complex maintained overall conformational stability, suggesting favorable structural integrity throughout the simulation (Fig. 9).

Fig. 9.

RMSD, RMSF, and RG analysis for the MD simulations of Rutin-4M0F (blue) and territrem-4M0F (orange) complexes

In the case of hCA II, the RMSD plot showed that the rutin-3NB5 complex reached equilibrium faster and stabilized earlier than the co-crystallized ligand complex. rutin-3NB5 maintained a lower and more consistent RMSD range (1.5 Å to 2.0 Å), while the co-crystallized complex fluctuated more widely, averaging between 1.5 Å and 2.5 Å. These results indicate that the rutin-3NB5 complex is more structurally stable, with less deviation from its initial shape, compared to the reference ligand-bound system. (S12 Fig.). The RMSF plot shows the flexibility of individual residues within the molecular systems. Both rutin-4M0F and territrem-4M0F display similar fluctuation patterns, with peaks around residues approximately 250 and 480–490, reaching distances of about 6.5–7 Å and 5 Å, respectively. These peaks highlight localized regions of high flexibility, while the overall structures remain compact and stable. In the hCA II systems, the co-crystallized complex (3NB5) exhibits greater flexibility, especially in the N-terminal region (residues 0–20), with fluctuations over 4 Å. Conversely, the rutin-3NB5 complex shows lower RMSF values throughout, indicating a more rigid and conformationally stable structure (Fig. 9, S12 Fig.).

The Rg (radius of gyration) plot, which reflects molecular compactness, reveals that territrem-4M0F maintains a stable and slightly lower average Rg (22.5–22.7 Å), indicating a more compact structure. In contrast, Rutin-4M0F has a marginally higher Rg (22.6–23.0 Å) with more fluctuations, suggesting a less compact yet still stable and flexible conformation. For hCA II, Rutin-3NB5 consistently exhibits a higher average Rg (17.4–17.7 Å) than the co-crystallized complex (17.2–17.5 Å), indicating a slightly more extended structure. Despite this, rutin-3NB5 remains stable in terms of structural deviation and flexibility. Overall, the Rg results indicate that the complexes retained stable and compact protein structures throughout the simulation (Fig. 9, S12 Fig.).

Insights were obtained from two separate molecular dynamics simulation sets, each analysed using Principal Component Analysis (PCA), Free Energy Landscapes (FEL), and Dynamic Cross-Correlation Maps (DCCM). The first set compared the co-crystallized ligand-4M0F with rutin-4M0F, while the second focused on the co-crystallized ligand-3NB5 and rutin-3NB5. Using these advanced computational methods, a comprehensive understanding of the systems’ conformational dynamics, structural stability, and flexibility was achieved (Fig. 10 and S13 Fig.).

Fig. 10.

Principal component analysis (PCA), free energy landscape (FEL) and dynamic cross correlation map (DCCM) during 100 ns simulation period (a) PCA plot of territerm-4M0F complex, b PCA plot of rutin −4M0F complex (c) Super imposed PCA plot of territerm and rutin complexes (d) 2D and 3D (FEL) Territerm-4M0F complex, and (e) 2D and 3D (FEL) rutin-4M0F complex. The color bar denotes the relative free energy value. f DCCM territerm-4M0F complex and (g) DCCM rutin-4M0F complex

Conformational Sampling and Stability of the Co-Crystallized ligand-4M0F (Fig. 10a, c, and d) demonstrated a broad and somewhat dispersed conformational ensemble in its PCA plot (a, and c). This indicates that the molecule explores a wide range of conformational space, suggesting significant flexibility. The corresponding FEL (d) is characterized by a relatively shallow and broad energy basin, implying that Co-crystallized ligand-4M0F can easily transition between various conformational states without encountering substantial energy barriers. This shallow landscape is indicative of high conformational entropy and inherent flexibility within the system.

In contrast, rutin-4M0F (Fig. 10b, c, and e) exhibited a more limited conformational landscape. Its PCA plot (b) displays two distinct, clearly separated clusters, indicating that rutin-4M0F mainly occupies two central, stable conformations. The FEL (e) for rutin-4M0F presents two deep and well-defined energy minima, separated by higher energy barriers. This indicates that rutin-4M0F strongly favours these specific stable conformations, and shifting between them would require overcoming significant energy barriers, suggesting increased conformational rigidity and stability.

Moreover, Conformational Sampling and Stability. Co-crystallised Ligand-3NB5 (S10a, S10c, and S10d Figs.) shows a relatively broad and elongated distribution of conformations in its PCA plot (a). This suggests that co-crystallised ligand-3NB5 explores a considerable range of conformational space, indicating notable flexibility. A shallow and extended energy basin characterizes the corresponding FEL (d). This implies that cocrystallised-3NB5 can readily transition between various conformational states within this extended basin without encountering significant energy barriers, reflecting considerable conformational entropy and flexibility.

In stark contrast, rutin-3NB5 (S10b, S10c, and S10e Figs.) demonstrates exceptional conformational rigidity. Its PCA plot (b) reveals a single, very compact, and dominant cluster, indicating that rutin-3NB5 explored a limited range of conformations. The FEL (Fig. 10e) for rutin-3NB5 shows a single, prominent, and well-defined energy minimum. This suggests a strong preference for one highly stable conformational state, and moving away from it would require overcoming very high-energy barriers, underscoring extreme conformational rigidity and stability.

The Dynamic Cross-Correlation Matrix (DCCM) analysis further supports the structural and dynamic differences observed in the studied complexes. Both territrem-4M0F and the co-crystallized hCA II complex (3NB5) display intricate DCCM patterns characterized by a mix of correlated and anti-correlated motions (Fig. 10f and S10f Fig.). The presence of prominent off-diagonal anti-correlations (lighter colors/reds) in these systems indicates diverse and opposing movements among different protein regions, contributing to their conformational flexibility and wider sampling of structural space.

In contrast, the DCCMs of rutin-bound complexes- rutin-4M0F and rutin-3NB5—display highly uniform correlation patterns, dominated by strong positive correlations (intense blue regions) (Fig. 10f and S13 Fig.). The near absence of anti-correlated motions indicates that most residues in these complexes move in a concerted and cooperative manner. This coordinated internal dynamic behaviour suggests a more rigid architecture and limited internal flexibility, aligning with their enhanced conformational stability observed in RMSD, RMSF, and Rg analyses. Positively, both the rutin-4M0F and rutin-3NB5 complexes demonstrated a stronger correlation compared to the ligand-4M0F and ligand-3NB5 complexes in their crystallized forms.

In brief, molecular docking analysis supported the experimental findings by demonstrating favourable binding interactions and strong predicted affinity of rutin (2) within the active sites of AChE and hCA. Although vanillic acid (4) impacted the targeted enzymes in the in vitro assays, it possessed a lower docking score, which is considered one of the study limitations. Molecular docking, as a computational approach, neglects the presence of water molecules in the binding site that can have a significant impact on binding affinity, and sometimes the actual binding pose of the compound in the protein is different from the one predicted by docking. Molecular dynamics simulations provide insights into the dynamic behaviour of rutin and revealed its stable confirmation in complexes with the targeted protein.

The comparative SwissADME analysis highlights substantial pharmacokinetic differences among the compounds studied, which are relevant for their potential application in Alzheimer’s disease. The reference compound TRB exhibits properties consistent with CNS-active agents, supporting its use as a benchmark for comparison.

Compound 4 emerged as the phytocompound with the most favorable CNS-oriented pharmacokinetic profile, characterized by high predicted absorption and potential BBB accessibility. These features may contribute to its observed biological activity despite its relatively simple chemical structure.

Although Compound 2 is not predicted to efficiently cross the BBB by passive diffusion, this pharmacokinetic limitation does not necessarily preclude its relevance in Alzheimer’s disease. Compound 2 demonstrated strong molecular docking interactions with acetylcholinesterase and carbonic anhydrase, enzymes involved in Alzheimer’s pathology through both central and peripheral mechanisms [66]. Additionally, Alzheimer’s disease is associated with blood–brain barrier dysfunction [61], which may alter compound permeability compared to healthy physiological conditions. Therefore, indirect mechanisms, peripheral enzyme modulation, or limited and transient CNS exposure may still contribute to rutin’s neuroprotective effects.

Overall, compound 4 may be considered a phytocompound with favorable CNS pharmacokinetic properties, whereas rutin represents a potent multi-target lead compound with pharmacokinetic limitations that could potentially be addressed through formulation strategies or structural optimization. These findings emphasize that ADME predictions should be interpreted alongside mechanistic and molecular interaction data rather than as exclusion criteria in Alzheimer’s disease drug discovery.

Conclusion

The DAE of C. macrospermum (Family Arecaceae) leaves has been subjected to phytochemical analysis and investigated for its impact on key proteins involved in Alzheimer’s disease (AD) progression for the first time. Three phenolic acids (chlorogenic acid (1), vanillic acid (4), and p-hydroxybenzoic acid (5) and two flavonoids (rutin (2) and hesperidin (3)) have been isolated from the DAE. NMR spectroscopy and MS spectrometry are efficient tools for the structure elucidation of the isolated phenolics. The DAE and its isolated phenolics, particularly rutin (2) and vanillic acid (4), exhibited promising inhibitory effects against AChE and hCA enzymes in addition to a potent anti-inflammatory activity which was mediated through COX-2 inhibition and a significant reduction in TNF-α and IL-2 (pro-inflammatory cytokines) as well as IL-4 (a Th2-associated immune-regulatory cytokine) in addition to downregulation of iNOS in LPS-induced murine macrophages. Molecular docking supported the experimental findings by indicating favorable binding of rutin (2) to AChE and hCA, consistent with its observed in vitro activity. In contrast, vanillic acid (4), despite demonstrating biological activity, showed comparatively lower docking scores, highlighting a limitation of computational prediction and the complexity of structure–activity relationships. In conclusion, the present study was designed as a mechanistic, multi-target screening investigation integrating phytochemical characterization, enzyme inhibition assays, cell-based anti-inflammatory evaluation, and computational modeling. Within this defined experimental scope, the findings consistently demonstrate that the defatted aqueous methanol extract (DAE) of C. macrospermum and its major phenolics modulate key biochemical pathways implicated in AD-associated neuroinflammation and cholinergic dysfunction. The data presented provide a robust biochemical foundation supporting further in vivo, pharmacokinetic, and disease-model studies.

Abbreviations

AChE

Acetylcholinesterase

CAS

Catalytic active site Binding

DCCM

Dynamic cross correlation matrix

FEL

Free energy landscape

hCA

Human carbonic anhydrase

MD

Molecular dynamics

ESI-MS

Electrospray ionization mass spectrometry

RMSD

Root mean square deviation.

RMSF

Root mean square fluctuation

RG

Radius of gyration

PAS

Peripheral anionic site binding

PCA

Principal component analysis

PC

Paper chromatography

TRB

Territrem B

Authors’ contributions

FM, HE, MM, and SE: Conceptualization, Investigation, Methodology, Formal analysis, Resources, Supervision, Writing—original draft; and Writing—review & editing. FH: Methodology, Data curation, Formal analysis, Writing—review & editing. SM: Data curation, Formal analysis, Writing—review & editing. YM: Methodology, Data curation, Formal analysis, Writing—original draft; Writing—review & editing. C-Y L and K-H L: Formal analysis, Resources, Funding acquisition, Writing—review & editing. All the authors have read and approved the submitted manuscript.

Funding

This work is supported by grants from the National Science and Technology Council of Taiwan (MOST 111–2320-B-038–040-MY3, 113–2628-B-038–009-MY3, 113–2321-B-255–001, and 114-2326-B-038-002-MY3); and from Ministry of Education (DP2-TMU-114-C-06). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.