Neuroprotective Activities of New Monoterpenoid Indole Alkaloid from Nauclea officinalis

Abstract

Phytochemical investigation on the bark of Nauclea officinalis led to the isolation of a new monoterpenoid indole alkaloid, nauclediol. The structure of the compound was identified through extensive spectroscopic analysis. Nauclediol displayed cholinesterase-inhibitory activities towards AChE and BChE with IC₅₀ values of 15.429 and 8.756 µM, respectively. Statistical analysis revealed that the mode of inhibition of nauclediol was non-competitive inhibitor for both AChE and BChE. Molecular docking revealed that nauclediol interacts with the choline-binding site and the catalytic triad of TcAChE and hBChE. This study also demonstrated the neuroprotective potential of nauclediol against amyloid beta-induced cytotoxicity and LPS-induced neuroinflammation activity in a dose-dependent manner.

1. Introduction

Alzheimer’s disease (AD) is the most common type of dementia, and it is estimated to affect 131 million people globally by the year 2050. Over the years, several hypotheses have been proposed to slow down the progression of AD, including by improving type 2 diabetes mellitus [1] and the discovery of next-generation therapeutics for the management of AD. It includes cholinesterase, β-amyloid, tau, and neuroinflammation hypotheses. However, the current drugs available to slow down the progression of AD are cholinesterase inhibitors (Donepezil, Rivastigmine, and Galantamine) and Memantine (NMDA antagonist); they are associated with limitations such as adverse effects, low bioavailability, and inconsistent efficacy [2]. These drugs are based on a one drug-one target hypothesis, whilst the etiology of AD is multifactorial [3]. Therefore, this necessitates the need for novel agents with multi-targeted potential. Natural products are of particular interest due to their ability to reduce oxidative stress and anti-inflammatory activity, and could potentially slow down the progression of AD [4,5]. This further reinforces the possibility of discovering new agents derived from natural sources.

Natural products are also known as secondary phytometabolites. These organic chemicals do not directly affect cell growth and proliferation, but they enhance survival mechanisms in living organisms [6]. Investigations into molecular mechanisms and docking analysis have shed light on the molecular activities of these secondary phytometabolites [7]. Most secondary phytometabolites are known to interfere with inflammatory mediators such as cytokines and peptides and to inhibit macrophage activity [8]; indole alkaloids, topsentins, and dragmacidins [9] are some good examples. Nauclea officinalis (Pierre ex Pitard) Merr. and Chun, a traditional Chinese medicine from the Rubiaceae family, is widely used to treat exogenous fever, pink eye, pneumonia and acute jaundice in China. It has anti-inflammatory and anti-malarial properties [10,11,12]. In addition, Nauclea officinalis has been known to contain many indole alkaloids with biological activities. For example, 17-oxo-19-(Z)-naucline and naucleoffieine H displayed anti-inflammatory effects by decreasing the LPS-stimulated production of nitric oxide in RAW264.7 cells [13,14]. Thus, in the continuous search for bioactive indole alkaloids from the Rubiaceae family [15,16,17], a new monoterpenoid indole alkaloid, nauclediol, was isolated from Nauclea officinalis. In the present study, the isolation and structural elucidation of nauclediol is reported together with the neuroprotective potential of the compound.

2. Materials and Methods

2.1. General Procedures

The general procedures were the same as previously described [18].

2.2. Plant Materials

The barks of Nauclea officinalis from Hutan Simpan Mersing, Johor were collected by a phytochemical group of the Department of Chemistry, Faculty of Science, Universiti Malaya. Botanical identification was conducted by the botanist, Mr. Teo Leong Eng. The voucher specimen (KL 4745) of the plant was deposited at the Herbarium of the Department of Chemistry, Faculty of Science, Universiti Malaya, Kuala Lumpur, Malaysia.

2.3. Extraction, Isolation and Purification of Compound

The extraction procedure for 1.5 kg of dried and grounded bark of Nauclea officinalis was the same as previously described [18,19]. An amount of 6.2 g of CH₂Cl₂ extract was obtained. The CH₂Cl₂ extract was then subjected to column chromatography over silica gel using CH₂Cl₂ and MeOH solvent (100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 90:10, 83:17, and 75:25 v/v), and 20 fractions were finally obtained. Further purification from fraction 6 by PTLC yielded nauclediol (8.2 mg, CH₂Cl₂:MeOH; 97:3 v/v saturated with NH₄OH). The structure of nauclediol was elucidated using various spectroscopic methods, such as NMR, UV, IR, and LCMS-IT-TOF.

2.4. Cholinesterase Enzyme Inhibitory Activity

Nauclediol was evaluated for its enzyme inhibition potential against acetylcholinesterase from Electrophorus electricus (EeAChE) and butyrylcholinesterase from Equine serum (eBChE). Cholinesterase inhibition was conducted using Ellman’s calorimetry method as described previously [20]. In a 96-well plate, buffer solution, DTNB (Ellman’s reagent), ACh/BCh, and nauclediol were added in ascending concentrations across the row. Then, 1% DMSO was added as the control for this experiment. Lastly, the cholinesterase enzyme was added to the assigned enzymatic columns. The absorbance of the plate was then measured using a spectrophotometer at a wavelength of 412 nm for 30 min after the addition of enzymes. Each test was conducted in triplicate to ensure consistency of the results.

The enzyme kinetic mechanism of nauclediol was assessed with compound concentrations from 1.56 to 25 uM. The enzyme and DTNB concentrations were in accordance with the method described above. The statistical analysis was performed using GraphPad Prism v8.0e (Dotmatics, Boston, United Kingdom) and Excel [21].

2.5. Molecular Docking

Molecular docking was carried out following the method described by Abdul Wahab et al. [22]. Briefly, molecular docking of nauclediol was performed using Autodock 3.0.5 (The Scripps Research Institute, La Jolla, CA, USA) along with AutoDockTools (ADT) [23]. Hyperchem 8 was used to build the 3D-crystal structure of the nauclediol, and the structure was subjected to energy minimization with a convergence criterion of 0.05 kcal/(molA). The three-dimensional crystal structures of AChE from Torpedo californica (TcAChE) (PDB ID: 1W6R) [24] and BChE from Homo sapiens (hBChE) (PDB ID: 2WIJ) [25] were retrieved from the Protein Data Bank. The proteins were edited using ADT to add hydrogen atoms and remove all water molecules. Non-polar hydrogens and lone pairs were then merged, and each atom was assigned Gasteiger partial charges. A grid box was generated at the center of the active site gorge with 60 × 60 × 60 points and spacing of 0.375 Ǻ. In total, 100 independent dockings were carried out for each docking experiment, with a population size of 150 and 2,500,000 energy evaluations. The best conformation with the lowest docked energy in the most populated cluster was selected. Analysis and visualization of the conformations from the docking experiments were conducted using Acceryls Discovery Studio 2.5 (Accelrys Inc., San Diego, CA, USA).

2.6. Neuroprotective Potential of Nauclediol on Aβ-(1-42)-Induced Toxicity on SH-SY5Y Cells

2.6.1. Cell Culture and Cell Viability Assay

Human neuroblastoma cells (SH-SY5Y) were cultured in a complete culture medium containing DMEM nutrient mixture F-12 (1:1) supplemented with 10% fetal calf serum, glutamine, and 1% antibiotics (penicillin–streptomycin), and kept at 37 °C under 5% CO₂.

A cell viability assay was performed to determine the non-cytotoxic concentration of nauclediol in SH-SY5Y cells. Cells were seeded with a cell density of 25,000 cells/well in 96-well plates for 24 h. The cells were then treated with nauclediol at concentrations of 1, 2, 5, and 10 μM for 24 h. An MTT assay was used to assess cell viability after drug treatment. In brief, 20 μL of the MTT solution (5 mg/mL) was added to each well and incubated at 37 °C for 4 h. Then, 100 μL of DMSO was added to lyse the cells after the removal of the supernatant from each well. The amount of formazan generated was quantified using a microplate reader (SpectraMax ID3, Pennsylvania, USA) at an absorbance of 595 nm. All experiments were carried out 3 times.

2.6.2. Neuroprotective Assay against Beta Amyloid-Induced Toxicity

Aβ-(1-42) oligomers were prepared according to the protocol of Huang et al. [26], with some modifications. Briefly, lyophilized Aβ-(1-42) powder was initially dissolved to 1.0 mM in hexafluoro-2-isopropanol (HFIP) and incubated at room temperature for 30 min. The solution was ensured to be clear and colorless. The HFIP was allowed to evaporate from the open tubes overnight into the fume hood. A thin, clear film was formed at the bottom of the tube. The film was initially dissolved in DMSO and subsequently adjusted to 13 μM in DMEM/Hams F-12 media containing 1% Pen/strep and 1% FBS. The solution was incubated at 37 °C under 5% CO₂.

The MTT assay was used to determine the protection of nauclediol toward SH-SY5Y cells against Aβ-(1-42) aggregate toxicity. In a 96-well plate, 25,000 cells/well were seeded and incubated overnight. Cells were treated with nauclediol at concentrations of 0.1, 1, 5, and 10 μM for 24 h. Aβ-(1-42) aggregates were added to the wells and incubated for another 24 h; then, 20 μL of the MTT solution (5 mg/mL) was added to each well and incubated at 37 °C for 4 h. Finally, 100 μL of DMSO was added to lyse the cells after the removal of the supernatant in each well. The amount of formazan generated was quantified using a microplate reader (SpectraMax ID3, Pennsylvania, USA) at an absorbance of 595 nm. All experiments were carried out in triplicate.

2.7. Anti-Neuroinflammatory Effect of Nauclediol on LPS-Induced Neuro-Inflammation in BV2 Cells

2.7.1. Cell Culture and Cell Viability Assay

C57/BL6 microglial cells (BV2) were cultured in a complete culture medium containing DMEM supplemented with 10% fetal calf serum, glutamine, and 1% antibiotics (penicillin–streptomycin), and kept at 37 °C under 5% CO₂.

A cell viability assay was performed to determine the non-cytotoxic concentration of nauclediol in BV2 cells. Cells were seeded with a cell density of 20,000 cells/well in 96-well plates for 24 h. The cells were then treated with nauclediol at concentrations of 0.625, 1.25, 2.5, 5, and 10 μM for 24 h. An MTT assay was used to assess cell viability after drug treatment. In brief, 20 μL of the MTT solution (5 mg/mL) was added to each well and incubated at 37 °C for 4 h; then, 100 μL of DMSO was added to lyse the cells after the removal of the supernatant from each well. The amount of formazan generated was quantified using a microplate reader (SpectraMax ID3, Pennsylvania, USA) at an absorbance of 595 nm. All experiments were carried out in triplicate.

2.7.2. Neuroprotective Assay against LPS-Induced Inflammation

In a 96-well plate, 20,000 cells/well were seeded and incubated overnight. Cells were treated with nauclediol at concentrations of 0.0625, 0.125, 0.25, and 0.5 μM for 24 h. LPS (1 ug/mL) were added to the wells and incubated for another 24 h. A second 96-well plate was used for the measurement of nitride oxide. Sulfanilamide and NED solutions were equilibrated to room temperature for 30 min before use. A 50 μL sample of the solution was transferred from the first plate to the second plate. Then, 50 μL of sulfanilamide solution was added to all wells in the second plate and incubated for 5 min. Subsequently, 50 μL of NED solution was added and incubated for another 5 min. The plate was then read at an absorbance of 540 nm using a microplate reader (SpectraMax ID3, Pennsylvania, USA). All experiments were carried out in triplicate. The absorbance was converted into a percentage of nitrite to determine the anti-inflammatory effects of nauclediol.

2.7.3. Statistical Analysis

For this cell-based study, the results were analyzed using R programming. Data are expressed as mean ± standard deviation. Differences between groups are analyzed using one-way ANOVA followed by Student’s t-test for MTT assay and nitrite production. A p-value less than 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Structural Elucidation of Nauclediol

Nauclediol (Figure 1) was isolated as a brownish amorphous solid. The LCMS-IT-TOF spectrum showed a pseudomolecular ion peak [M+Na]⁺ at m/z 359.1275 (calc. 359.1372) corresponding to the molecular formula of C₂₀H₂₀N₂O₃. The UV spectrum of nauclediol demonstrated strong absorptions at 374, 308, 277, 256, and 220 nm. In the IR spectrum, an absorption band at 1643 cm⁻¹ was observed, indicative of a corynanthe type of indole alkaloid with an N₄-C=O amide group [27,28]. In addition, a broad hydroxyl absorption band appeared at 3401 cm⁻¹, suggesting the presence of a hydrogen-bonded OH group [28].

In the ¹H-NMR spectrum (

Table 1. ¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz) spectral data of nauclediol in MeOD.

Position	1H, δH (Multiplicity, J in Hz)	13C (δC)
2	-	127.5
3	-	135.6
5	4.33 (t, 6.9)	40.5
6	3.06 (t, 6.9)	19.0
7	-	113.0
8	-	125.7
9	7.53 (d, 7.8)	118.9
10	7.04 (t, 7.8)	119.6
11	7.19 (t, 7.8)	124.0
12	7.37 (d, 7.8)	111.3
13	-	138.6
14	7.00 (s)	95.5
15	-	141.2
16	-	120.0
17	6.46 (q, 6.9)	123.8
18	1.86 (d, 6.9)	12.2
19	4.45 (s)	64.1
20	-	129.2
21	4.55 (s)	64.3
22	-	160.9

), the presence of four aromatic proton signals at the downfield region and a -CH₂-CH₂-N- group were observed, indicating the existence of a tetrahydro-β-carboline skeleton [10,28]. In ring A, 2 of the 4 aromatic protons appeared as doublets at δ_H 7.53 and 7.37, corresponding to H-9 (1H, d, J = 7.8 Hz) and H-12 (1H, d, J = 7.8 Hz), respectively. The other two protons, H-10 (δ_H 7.04, 1H, t, J = 7.8 Hz) and H-11 (δ_H 7.19, 1H, t, J = 7.8 Hz), appeared as triplets. Furthermore, the COSY spectrum also showed the correlations between the four aromatic protons; H-9 with H-10, H-10 with H-11, and H-11 with H-12 (Figure 2). In addition, 2 sets of triplet signals, which appeared at δ_H 4.33 (2H, t, J = 6.9 Hz) and δ_H 3.06 (2H, t, J = 6.9 Hz), were attributable to methylene protons H₂-5 and H₂-6, respectively. H-14, in ring D, appeared as a singlet at δ_H 7.00, indicating the presence of neighboring quaternary carbons C-3 (δ_C 135.6) and C-15 (δ_C 141.2). Furthermore, the presence of a downfield quartet at δ_H 6.46 (1H, q, J = 6.9 Hz, H-17), coupled with an upfield doublet at δ_H 1.86 (3H, d, J = 6.9 Hz, H₃-18), implied the presence of a trisubstituted olefin group. Two downfield singlets were observed at δ_H 4.45 (2H, s, H₂-19) and 4.55 (2H, s, H₂-21) which correlated with the carbon signals at δ_C 64.1 (C-19) and δ_C 64.3 (C-21), respectively, in the HSQC spectrum, suggesting the existence of two hydroxyl groups (a diol).

As indicated in the ¹³C-NMR and DEPT-135 spectra, nauclediol has a total of twenty carbon signals comprising one methyl, one carbonyl, four methylenes, six methines, and eight quaternary carbons. The signal of a carbonyl carbon was observed at δ_C 160.9 (C-22). The HMBC spectrum showed a correlation of H₂-19 and H₂-21 with C-20 (δ_C 129.2), indicating the connectivity of the two methylenes with quaternary carbon C-20, as shown in Figure 2. In addition, HMBC correlations between H₃-18 and C-16 (δ_C 120.0), H-19 and C-15, and H-21 and C-15 were observed, thus supporting the connectivity of the trisubstituted olefin group and propane-1,3-diol group to ring D through C-16 and C-15, respectively. The COSY spectrum revealed correlation peaks between H₂-5 and H₂-6 and H₃-18 and H-17, indicating that the respective protons were coupled with each other (Figure 2).

Based on the aforementioned and extensive analysis of all spectral data, the structure of nauclediol was established identified as a new corynanthe type of monoterpenoid indole alkaloid. The biogenetic precursor of nauclediol is strictosidine. Strictosidine may undergo cleavage of glycoside to strictosidine aglycone, followed by cleavage of ring D, to form dialdehyde intermediate. Subsequently, a series of rearrangement could lead to the formation of 21-oxogeissoschizine, followed by dehydration, to form nauclediol, as shown in Figure 3.

3.2. Cholinesterase Inhibition of Nauclediol

Cholinesterase inhibitors (ChEIs) are the first and most commonly recognized treatment strategy for AD. They work by reducing the metabolism of cholinergic neurotransmitters and acetylcholine released from the cholinergic neurons, thus increasing the synaptic levels of ACh and BCh [29]. In this study, nauclediol was tested for its inhibitory potential against AChE and BChE, respectively. Nauclediol showed inhibition on cholinesterase enzymes with IC₅₀ values of 15.429 and 8.756 µM, which was two-fold more selective toward BChE (Figure 4). Furthermore, nauclediol was three times more potent as an inhibitor of BChE compared to galanthamine (standard or drug), with a reported IC₅₀ value of 28.29 µM [19].

The mode of inhibition was determined by means of Michaelis–Menten kinetic. An analysis using Graphpad revealed that the mechanism of action of naucleodiol against AChE and BChE was non-competitive based on the greatest goodness of fit (Figure 5). This result was further supported and strengthened by statistical analysis using Excel, which showed that naucleodiol was best suited to be a noncompetitive inhibitor based on the lowest sum of squared residuals (RSS), relative standard error (RSE) for regression, and the Bayesian information criterion (BIC) (Supplementary Materials). The Ki values of nauclediol were 6.9 and 35.41 µM against AChE and BChE, respectively (Supplementary Materials), indicating a stronger binding affinity of naucleodiol towards AChE. This is in opposition to the IC₅₀ values, as nauclediol possessed better IC₅₀ values for BChE than for AChE.

By comparing with the cholinesterase-inhibitory activities of other indole alkaloids from N. officinalis [19], a brief structure–activity relationship study can be conducted. Angustidine, angustine, angustoline, and naucline are also corynanthe types of indole alkaloids [19], with the same precursors as nauclediol. According to the proposed biogenesis pathway, the difference between nauclediol and the four known indole alkaloids begins to appear after the formation of strictosidine. Rotation at C-14 of strictosidine is needed to form strictosidine lactam, and is followed by the formation of the four known indole alkaloids. On the other hand, formation of nauclediol does not require the rotation at C-14 of strictosidine, but involves a ring opening in strictosidine aglycone. Thus, the four known indole alkaloids possess ring E (pyridine or pyran ring), but nauclediol does not. Based on the previously reported data, angustidine, angustine, angustoline, and naucline exhibited moderate to weak AChE inhibition, with angustidine having the lowest IC₅₀ value (21.72 µM) [19]. In comparison with the four known indole alkaloids, nauclediol possesses a better inhibitory effect towards AChE. It was suggested that the potency of nauclediol in AChE inhibition may be due to the presence of a ring-opened system in ring E and the two hydroxyl groups. For BChE, angustidine and angustine (IC₅₀ = 1.03 and 4.98, respectively) [19] showed better inhibitory effects compared to nauclediol. However, nauclediol was shown to be 2.5 and 4 times more potent than angustoline and naucline, respectively, as angustoline has only one hydroxyl group in its structure, while naucline has a pyran ring instead of a pyridine ring (ring E). Based on structure–relationship studies, its potency in inhibiting BChE may be increased by the presence of a pyridine ring and two hydroxyl groups.

3.3. Molecular Docking of Nauclediol

Since nauclediol is potent in inhibiting both enzymes, molecular docking analysis was carried out in order to understand its binding interaction with the enzymes. The results revealed that nauclediol docked well in the catalytic triad and choline binding site of TcAChE (

Table 2. Binding interaction data for nauclediol with amino acid residues of TcAChE and hBChE.

Ligand/ Compound	Enzyme	Binding Energy (kcal/mol)	Number of Conformations in the Cluster	Interacting Site	Residue	Type of Interaction	Distance (Å)	Ligand Interacting
Nauclediol	TcAChE	−13.01	64	Catalytic triad	His 440	Hydrogen	2.60	Hydroxyl group at C-19
				Choline binding site	Trp 84 Tyr 130	Hydrophobic Hydrogen	- 2.26	Ring B and D Oxygen atom of the carbonyl group C-22
	hBChE	−12.19	60	Catalytic triad	His 438	Hydrophobic	-	Ring A and B
				Choline binding site	Trp 82	Hydrophobic	-	C-17

). The hydroxyl group of C-19 formed a hydrogen bond with His 440 at a distance of 2.60 Å in the catalytic triad (Table 2). The catalytic triad serves as the active site of the enzyme, where acetylcholine, the substrate, is hydrolyzed into choline and acetate [22]. In addition, hydrogen bonds and hydrophobic interactions were observed between the compound and the amino acid residues in the choline binding site, which anchored this compound to the TcAChE active site gorge (Figure 6). An oxygen atom of the carbonyl group C-22 interacted with Tyr 130 through a hydrogen bond at a distance of 2.26 Å, while rings B and D of the compound formed a π–π stacking interaction with Trp 84. For hBChE, nauclediol also bound with amino acid residues of the catalytic triad and choline binding site with a hydrophobic interaction (Table 2). A π–π stacking interaction was observed between the indole ring of nauclediol and His 438 from the active site of hBChE, as shown in Figure 7. In addition, a π–σ interaction was observed between Trp 82 and C-17 of nauclediol.

3.4. Nauclediol Reduces Aβ-(1-42)-Induced Toxicity on SH-SY5Y Cells

As illustrated in Figure 8A, nauclediol had no cytotoxic effect on SH-SY5Y cells from 1 to 10 μM. Further, the cells were pretreated with 1 to 10 μM of nauclediol followed by Aβ-(1-42) aggregates. Figure 8B shows that 13 μM Aβ-(1-42) aggregates significantly reduced cell viability to 54.5 ± 6.52%, compared to the untreated cells. The cell viability of neuroblastoma SH-SY5Y cells was increased by 33.28 to 64.87% at the tested concentrations (0.1 to 10 μM) compared to cells treated with Aβ-(1-42) aggregates alone. Overall, our study showed that nauclediol protected neuroblastoma SH-SY5Y cells against Aβ-(1-42)-induced cytotoxicity.

3.5. Nauclediol Reduces LPS-Induced Neuroinflammation in BV2 Cells

Nauclediol was tested for its cytotoxic effect on BV2 cells at 1.25 to 10 μM. Figure 9 shows the BV2 cell viabilities upon exposure to nauclediol. The treated doses, at 1.25 to 10 μM, were cytotoxic towards BV2 cells from 9.28 to 22.7% when compared to untreated cells. Non-toxic doses, at 0.0625 to 0.5 μM, were used for further experiments. Nauclediol demonstrated a statistically significant reduction in nitrite at doses of 0.25 to 0.5 μM.

In this test, we sought to evaluate whether nauclediol could reduce the elevated levels of nitric oxide production by LPS-induced inflammation in BV2 cells. Several studies have shown the relationship between nitric oxide and the pathogenesis of AD, with effects such as neuronal death and S-nitrosylation of cellular components [30]. In addition, in post mortem brain tissues from AD patients, Aβ plaques are strongly associated with reactive microglia and nitric oxide production from activated microglia [31].

4. Conclusions

In conclusion, the present study has yielded a new monoterpenoid indole alkaloid, nauclediol, from Nauclea officinalis. It is a dual cholinesterase inhibitor and is two-fold more selective against the butyrylcholinesterase enzyme. Upon a molar-based comparison with galantamine, a drug or medication used in the treatment of Alzheimer disease, nauclediol was three times more potent in inhibiting BChE. In addition, Michaelis–Menten kinetics showed that nauclediol is a non-competitive inhibitor with a higher affinity towards AChE. In addition, nauclediol significantly protected cells against beta amyloid-induced toxicity and neuroinflammation at submicromolar levels. Overall, the findings in this study are worth further investigation for nauclediol’s potential in assays related to other neurodegenerative diseases.