Natural Corynanthe-Type Cholinesterase Inhibitors from Malaysian Uncaria attenuata Korth.: Isolation, Characterization, In Vitro and In Silico Studies

The Uncaria genus is notable for its therapeutic potential in treating age-related dementia, such as Alzheimer’s disease. A phytochemical study of the leaves of Malaysian Uncaria attenuata Korth., afforded an undescribed natural corynanthe-type oxindole alkaloid, isovillocarine D (1) together with two known indole alkaloids, villocarine A (2) and geissoschizine methyl ether (3), and their structural identification was performed with extensive mono- and bidimensional NMR and MS spectroscopic methods. The isolated alkaloids were evaluated for their acetylcholinesterase (AChE)- and butyrylcholinesterase (BChE)-inhibitory activity. The results indicated that compound (2) was the most potent inhibitor against both AChE and BChE, with IC50 values of 14.45 and 13.95 µM, respectively, whereas compounds (1) and (3) were selective BChE inhibitors with IC50 values of 35.28 and 17.65 µM, respectively. In addition, molecular docking studies revealed that compound (2) interacts with the five main regions of AChE via both hydrogen and hydrophobic bonding. In contrast to AChE, the interactions of (2) with the enzymatic site of BChE are established only through hydrophobic bonding. The current finding suggests that U. attenuata could be a good source of bioactive alkaloids for treating age-related dementia.


Introduction
Alzheimer's disease (AD) is the most prevalent type of age-related dementia among the elderly, which is characterized by a progressive decline in cognitive function and memory [1,2]. The classical hallmarks of AD pathogenesis are the formation of extracellular amyloid-β plaques, the accumulation of abnormally phosphorylated tau at the intracellular level, and the progressive loss of cholinergic neurons [3][4][5]. Unfortunately, most clinical trials targeting a single protein target, such as Aβ and tau, have often failed [6]. Current clinically approved anti-AD drugs are mainly cholinesterase inhibitors, such as rivastigmine, donepezil, and galantamine (Figure S1) [7,8]. Cholinesterase inhibitors block the action of cholinesterase, increasing acetylcholine availability and, subsequently, its duration of action in the brains of AD patients, which is essential for arousal, attention, learning, memory, muscle activation, etc. [9,10]. Nonetheless, cholinesterase inhibitors Metabolites 2023, 13, 390 2 of 12 are symptomatic treatments that neither halt the disease course nor reverse the disease progression [11,12]. Due to the limited cholinesterase inhibitors available clinically, the search for more effective ones is ongoing, including from plant sources.
In our continuous effort to search for novel cholinesterase inhibitors from Malaysian Rubiaceous plants, the methanolic leaf extract of Uncaria attenuata Korth. has shown promising cholinesterase-inhibitory activity. Uncaria attenuata (also known as Uncaria salaccinesis or Uncaria bulusanensis) is a rare Uncaria species native to the Malay Archipelago (Peninsular Thailand, Malaysia, Indonesia, and the Philippines) [17,18]. A chemical investigation of U. attenuata was carried out between 1970 and 1997, of which the plant materials were collected from different localities in Thailand and Indonesia. Several common corynantheand heteroyohimbine-type alkaloids have been reported from the leaves of U. attenuata, including hirsuteine, hirsutine, rhynchophylline, isorhynchopylline, corynoxine B, mitraphylline, isomitraphylline, 3-isoajmalicine, tetrahydroalstonine, and rauniticine. However, the alkaloid profiles were greatly influenced by geographical origins [17][18][19]. In addition, four unusual D -secocorynanthe-type oxindole alkaloids-salacin, 3-oxo-7-hydroxy-3,7secorhynchophylline, Us-7, and Us-8-were isolated from the stem and hook of Thai U. attenuata, which are exclusive to this species [20,21] (Figure S2b). These alkaloids with an opened D-ring were formed by an oxidative cleavage at the enamine double bond (C20-C21) of strictosidine aglycone intermediate during the biosynthesis process [21]. Yet, the potential neuroprotective activities of U. attenuata and its alkaloids are unknown. All these have sparked our interest in isolating and evaluating the cholinesterase-inhibitory activity of the alkaloid constituents from the leaves of Malaysian U. attenuata.

The General Experimental Procedures
Column chromatography was carried out on silica gel (230-400 mesh; Merck, Darmstadt, Germany). TLC (silica gel 60 F 254 , Merck, Darmstadt, Germany) was used to monitor fractions from column chromatography. Preparative TLC was performed on silica gel 60 F 254 (20 cm × 20 cm, 0.5 mm; Merck, Darmstadt, Germany). Visualization of the TLC plates was achieved with a UV lamp (λ = 254 and 365 nm). Optical rotations were obtained utilizing a JASCO P-1010 polarimeter. Circular dichroism (CD) absorption spectra were recorded using a Jasco J-815 spectropolarimeter. 1 H and 13 C NMR spectra were obtained on Bruker model AMX 500 NMR spectrometers with standard pulse sequences, operating at 500 MHz in 1 H and 125 MHz in 13 C. The chemical shift values were reported in parts per million units (ppm) from trimethylsilane (TMS) using known solvent chemical shifts. Coupling constants were recorded in hertz (Hz). Standard pulse sequences were used for COSY, HMQC, HMBC, NOESY, and DEPT. High-resolution mass spectrum (HR-MS) was measured on a Waters Xevo G2-XS QTof quadrupole time-of-flight mass spectrometer (Waltham, MA, USA). Gas chromatography-mass spectra (GC-MS) were measured on an Agilent 6890 N Network GC system coupled to an Agilent 5973i mass selective detector (Agilent Technologies, Waldbronn, Germany). IR spectra were recorded by KBr using Perkin Elmer (Waltham, MA, USA) 2000 FT-IR spectrophotometer. Melting points were determined using a Stuart Scientific Melting Point SMP 1 (Staffordshire, UK) and were uncorrected. UV spectra were recorded on a Shimadzu UV-1800 spectrophotometer (Kyoto, Japan). The absorbance for AChE and BChE inhibitory assay was recorded on a Thermo Scientific Multiskan Go microplate reader (Waltham, MA, USA).

Plant Material
The leaves of Uncaria attenuata Korth. (Rubiaceae) were collected from Bukit Kledang, Ipoh, Perak, Malaysia (4 • 34 18.7763 N, 101 • 1 39.4139 E). The taxonomical identity of the plant was authenticated by Dr. Ooi Im Hin, a botanist from Penang Botanic Gardens, Malaysia. A voucher specimen (no. TAF 1) was then deposited at the herbarium of Penang Botanic Gardens.

Extraction of Plant Material
The air-dried and powdered leaf material (300 g) was extracted with MeOH (1:15 w/v) using a 5 L Soxhlet extractor for 48 h. The obtained supernatant was filtered and evaporated to dryness in vacuo to yield a crude MeOH extract (70 g). The MeOH extract was then subjected to acid-base extraction to enrich the alkaloid constituents. Briefly, the MeOH extract was partitioned between hexane (3 × 1 L) and 10% CH 3 COOH. The acidic aqueous layer was then adjusted to pH 9.0 with 25% NH 4 OH and extracted with CHCl 3 (5 × 1 L). The CHCl 3 -soluble portion was washed with distilled water, dried over anhydrous sodium sulfate, and evaporated in vacuo to yield a crystalline alkaloid extract (2.5 g).

Cholinesterase Inhibitory Assay
The cholinesterase-inhibitory potential of the extracts and isolated compounds was determined using the spectrophotometric method described by Ellman et al. (1961) [24]. The assay procedure was the same as reported in our previous publications [25,26].

Molecular Docking
Molecular docking was performed only for the most active compound-villocarine A (2)-using Autodock 3.0.5 (La Jolla, CA, USA) along with AutoDockTools (ADT) to get insight into the molecular interactions and bonding affinities of the molecule in the active sites of the AChE and BChE enzymes following the method described in our previous publication [25]. Compound 2 was built using Hyperchem 8, and energy minimization was performed with a convergence criterion of 0.05 kcal/(mol A). The proper protein crystal structures of AChE from Torpedo californica in complex with galanthamine (PDB ID: 1W6R) and BChE from Homo sapiens (PDB ID: 2WIJ) were obtained from Protein Data Bank. For each docking experiment, one hundred independent dockings were carried out, and the lowest docked energy of each conformation in the most populated cluster was selected.

Cholinesterase-Inhibitory Activity
The total alkaloid extract of U. attenuata and the two major alkaloids, isovillocarine D (1) and villocarine A (2), and the minor alkaloid, geissoschizine methyl ether (3), were evaluated for their inhibition against AChE and BChE enzymes. The total alkaloid extract inhibited both enzymes with IC 50 values of 8.90 and 21.74 µg/mL, respectively, which were about two times more potent than the origin methanolic extract (Table 2). Among the tested alkaloids, compound (2) displayed the most potent inhibitory effect against both AChE and BChE with IC 50 values of 14.45 and 13.95 µM, respectively, regarded as a dual cholinesterase inhibitor (selectivity index ≈ 1). On the other side, compounds (3) and (1) were found to be moderate but selective BChE inhibitors (IC 50 17.65 and 35.28 µM, respectively). The dose-dependent curve of extracts and individual alkaloids against AChE and BChE are provided in the Supplementary Materials (Supplementary Figures S22 and S23). Surprisingly, all the isolated compounds showed better selectivity toward BChE than AChE, which differed from the methanolic and alkaloid-enriched extracts. This suggested that the anti-AChE activity of U. attenuata extracts was possibly due to the synergistic effects among the alkaloid constituents.
A closer study of their structure-inhibition correlations revealed that the H-3β position at the indole C/D ring of (2) is critical for AChE inhibition, as evidenced by the higher IC 50 value observed for its 3S-epimer (3), which was about three times higher than (2). Remarkably, a loss of anti-AChE activity was observed when the indole ring (3) was oxidized to form the spirocyclic oxindole (1), suggesting that the indole moiety is essential for inhibiting the enzyme. For BChE, both compounds (2) and (3) inhibited the enzyme with similar IC 50 values, demonstrating that the chirality at C-3 has little to no effect on BChE inhibition. Like the AChE enzyme, the anti-BChE activity of (1) was cut in half compared to the IC 50 value of (3). This showed that indole moiety is also preferable for BChE inhibition. The lack of anticholinesterase activity in corynanthe-type oxindole alkaloids was supported by other published data, in which rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine had no inhibition on AChE and BChE enzymes, even when tested at 100 µg/mL [36][37][38].

Molecular Docking Study
The binding interactions between the most potent compound-compound (2)-and both AChE and BChE enzymes were evaluated in silico. The free energy of binding (FEB) of (2) with AChE was slightly higher than BChE, which correlated with the IC 50 values obtained (Table 3). In silico analysis showed that the β-methoxy acrylate moiety of (2) interacted with key amino acids of AChE at the oxyanion hole (Gly 118 and Ala 201) via hydrogen bonding. Further, (2) was predicted to interact with the key residues of the choline-binding site and acyl-binding pocket of AChE through hydrophobic interactions. Multiple π-alkyl interactions were observed between Trp 84 with indole C-ring, D-ring, and the C 20 -ethylidene chain of (2), which further substantiates the importance of the indole moiety and C 20 -ethylidene group in inhibiting AChE (Figure 3a). On the other hand, only the hydrophobic interaction of (2) with the choline-binding site, catalytic side, and oxyanion hole may explain the higher FEB with BChE. Similar to AChE, the interactions of (2) with the key amino acids within the BChE pocket were mainly established through its indole moiety and C 20 -ethylidene group (Figure 3b).

Conclusions
The present study reported the cholinesterase-inhibitory activity of U. attenuata and its alkaloid constituents for the first time. A new corynanthe-type oxindole alkaloid and two known indole alkaloids were isolated and characterized from the leaves of Malaysian U. attenuata. The new oxindole alkaloid was identified as isovillocarine D (3S,7S,15S) (1) using various modern spectroscopic analyses. All the isolated compounds (1-3) showed moderate to weak cholinesterase inhibition with IC 50 values lower than 50 µM. Compound (2) had the most potent inhibitory activity against both AChE and BChE, with IC 50 values of 14.45 and 13.94 µM, respectively, followed by compounds (3) and (1). In terms of selectivity, compound (2) is a dual inhibitor (selectivity ≈ 1), whereas (1) and (3) are selective BChE inhibitors. Molecular docking studies showed that (2) interacted with the active site of AChE and BChE mainly by forming hydrogen and hydrophobic bonds between the key amino acids and the indole moiety and ethylidene side chain. Overall, corynanthe-type indole alkaloids from U. attenuata showed potential inhibitory activity on AChE and BChE. The plant could be a good source for searching for new cholinesterase inhibitors.