Bioavailable fraction from the edible leaf of Albizia lebbeck (L.) Benth. inhibits neurotoxicity in human microglial HMC3 cells and promotes lifespan in Caenorhabditis elegans

Abstract

Neurodegeneration is involved in the deterioration and death of cells in the central nervous system. Albizia lebbeck (L.) Benth. has exhibited antioxidant and health benefits. This study focused on the protective effect and underlying mechanism of the bioavailable fraction of A. lebbeck leaf (BAL) against toxicity of glutamate-induced endoplasmic reticulum (ER) stress and cell death in human microglial HMC3 cells, as well as evaluated the longevity and antioxidant effects of BAL in Caenorhabditis elegans. The BAL was obtained from the in vitro digestion of A. lebbeck leaf coupled with Caco-2 cells. Results showed that treating HMC3 cells with BAL attenuated glutamate-induced ER stress and apoptosis by decreasing the protein expressions of calpain1, caspase-12, Bax, cytochrome c, and cleaved caspase-9 while increasing the antiapoptotic Bcl-2. Additionally, LC–MS/MS results showed that BAL contained flavonoids and carotenoids such as quercetin-3β-D-glucoside, robinetin, vitexin, kaempferol, kuromanin, daidzein, tanshinon I, nootkatone, rutin, and luteolin. We further investigated molecular docking to illustrate these bioactive compounds on apoptosis-related mechanisms. The results demonstrated that luteolin, kaempferol, and nootkatone inhibited Bax, cytochrome c, and caspase-9 functions. We found that BAL also extended the lifespan of C. elegans and distinctly increased survival in response to juglone-induced oxidative stress. Interestingly, treating C. elegans with BAL could increase superoxide dismutase 3 expression, relating to the anti-stress response. These findings suggest that BAL possesses beneficial function in neuroprotection and longevity, supporting its potential for preventing age-related neurodegeneration.

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Introduction

Glutamate is a crucial excitatory neurotransmitter and it is vital in controlling several brain functions, including learning, memory, and cognition. However, chronic exposure to glutamate can cause overstimulation of glutamate receptors in neurons as well as glial cells (Al-Nasser et al., 2022; Czapski & Strosznajder, 2021; Glaser et al., 2022; Lewerenz & Maher, 2015). This event is called glutamate excitotoxicity, which can contribute to the accumulation of oxidative stress, endoplasmic reticulum (ER) stress, neuron dysfunction, injury, and death of both neuronal and glial cells in the central nervous system (CNS) (Lewerenz & Maher, 2015; Olloquequi et al., 2018). These lead to the progression of neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD) are prevalent worldwide, particularly among the elderly (Fereshtehnejad et al., 2019; Hsa & Ya, 2014; Lewerenz & Maher, 2015). Ageing refers to the decline in physiological function. Numerous studies have indicated that neurodegenerative diseases are associated with the ageing process. Nevertheless, the molecular mechanisms that link ageing to these diseases remain inadequately understood (Abdolmaleky & Zhou, 2023; Azam et al., 2021).

ER stress triggers the unfolded protein response, which plays a crucial role in maintaining protein balance within the cell. Prolonged ER stress can lead to neuronal death and activate an immune response involving microglial cells, indicating a key feature of neurological diseases (Shi et al., 2022; Sprenkle et al., 2017). The ER stress leads to neurotoxicity and cell death through the upregulation of the targeted proteins such as calpains, C/EBP-homologous protein (CHOP), caspase-12, and mitogen-activated protein kinases (MAPKs) pathways (Chen et al., 2023; Hu et al., 2018; Shi et al., 2022). Eventually, downregulating the anti-apoptotic B-cell lymphoma 2 (Bcl-2) family while simultaneously upregulating pro-apoptotic Bcl-2 members results in the neurons and neuroglia more susceptible to apoptotic cell death (Czapski & Strosznajder, 2021; Dahlmanns et al., 2023; Shi et al., 2022; Sprenkle et al., 2017).

Glial cells are various cells types that are involved in the immune response of the nervous system. Microglia are resident innate immune cells sensitive to and responding to pathogens, toxins, and damaged neurons (Fani Maleki & Rivest, 2019; Gao et al., 2023; Kwon & Koh, 2020; Muzio & Perego, 2024). It is now widely recognized that the activation of innate immune responses is associated to the development and progression of many neurodegenerative disorders (Gao et al., 2023; Nakaso, 2024). Several reports showed that high concentration of glutamate in microglia is involved in an excitotoxic damage and impaired ability of cells, which resulted in damage and dying cells (Belov et al., 2020; Iovino et al., 2020; Kigerl et al., 2012; Phoraksa et al., 2023). Importantly, previous studies have demonstrated that glutamate can promote ER stress and cell death in microglial cells (Persson et al., 2006; Phoraksa et al., 2023; Xie et al., 2024; Zhang et al., 2016, 2023). These damage and death of microglial cells can affect the function of other cells in the brain. Consequently, they promote the development and progression of neurodegenerative diseases (Andreone et al., 2020).

Albizia lebbeck (Linn.) Benth (Fabaceae family) is called lebbeck or Ta-kuk (in Thai), a deciduous tree that grows primarily in subtropical regions such as Australia, India, South Africa, and Thailand. Many phytochemicals such as alkaloids, phenolics, flavonoids, and carotenoids are found in A. lebbeck, of which most plant parts are useful and have been used in traditional approaches (Balkrishna et al., 2022; Ibrahim & Abdul-Hafeez, 2023; Praengam et al., 2017; Shirisha et al., 2013). A. lebbeck exhibits various biological and pharmacological activities including antioxidant, anti-inflammation, anticancer, immunomodulation, and neuroprotection (Kamala & Valarmathi, 2020, Samant et al., 2023, Shirisha et al., 2013). Our previous study indicates that A. lebbeck leaf extract can obviously activate cellular antioxidant enzymes activities and inhibit ER stress and apoptosis signalling pathways in human microglial clone 3 (HMC3) cells caused by glutamate (Phoraksa et al., 2023). Interestingly, the aqueous fraction of A. lebbeck young leaves from in vitro digestion contains major bioactive compounds, including flavonoids and carotenoids. This fraction shows antioxidant and anti-inflammatory activities in Caco-2 cells through activation of glutathione protein content and glutathione peroxidase activity and reduction of IL-8, IL-6, and MCP-1 levels (Praengam et al., 2017). The beneficial activities of this plant are interesting to study in terms of antioxidative stress, neuroprotection, and anti-ageing effects in age-related diseases using mammalian cell cultures, such as microglia, and the nematode Caenorhabditis elegans models. This nematode model is widely used for anti-ageing and age-related diseases (Van Pelt & Truttmann, 2020). However, investigations of the mechanisms of the bioavailable fraction of A. lebbeck leaf (BAL) on neuroprotection in human microglial cells as well as the anti-ageing effect of BAL using the C. elegans model have not been reported. Therefore, the present study aimed to examine the protective effects and mechanisms of the BAL on glutamate-induced ER stress and apoptosis in human microglial HMC3 cells. This BAL was obtained from the in vitro digestion of A. lebbeck leaf coupled with Caco-2 cells. Furthermore, the drug-likeliness and pharmacokinetic properties of bioactive compounds in BAL were predicted. The selected compounds were analyzed for their interactions with proteins by molecular docking study. In addition, we also evaluated the anti-ageing and antioxidant effects of the BAL in vivo using the nematode C. elegans as model organism.

Materials and methods

Materials

Dimethyl sulfoxide (DMSO), L-glutamic acid monosodium salt, pepsin from porcine stomach mucosa, lipase and pancreatin from porcine pancreas, the 2,2-diphenyl-1-picrylhydrazyl (DPPH), the 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), the 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), Ferric chloride, Folin-Ciocalteau reagent, trolox, and gallic acid standard (purity ≥98%) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Bio Basic (Markham, ON, Canada). Minimum essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), trypsin–EDTA, fungizone, and sodium pyruvate were obtained from Gibco (Grand Island, NY, USA). Foetal bovine serum (FBS) was purchased from Merk (Darmstadt, Germany). Penicillin–streptomycin solution and nonessential amino acids were acquired from Cambrex Bio Science (Walkersville, MD, USA) and Caisson Labs (Smithfield, UT, USA), respectively. Primary antibodies were purchased from Abcam (Cambridge, UK) and Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

Plant collection and preparation

The edible young leaves of A. lebbeck were collected from the conservation area of the Electricity Generating Authority of Thailand, Srinakarind Dam, Kanchanaburi province, initiated by the Plant Genetic Conservation Project under the Royal Initiation of Her Royal Highness Princess Maha Chakri Sirindhorn. The plant was authenticated and identified by Assistant Professor Dr. Thaya Jenjittikul (Department of Plant Science, Faculty of Science, Mahidol University, Thailand) and it was deposited at Suan Luang Rama IX Herbarium, Bangkok, Thailand with voucher specimen No. 9429. The procedure of sample preparation was performed according to the method of our previous study (Phoraksa et al., 2023). Briefly, after washing the young leaves with clean water and air-dried, the edible portions were cooked in boiling water for 2 min, according to the consumption of the local Thai people (Praengam et al., 2017; Sridonpai et al., 2022). After that, the samples were lyophilized, and ground to a fine powder. Then, powder samples were kept in laminated aluminium foil bags under a vacuum and kept at −20 °C until analysis.

Evaluation of antioxidant activities

Ferric-reducing antioxidant power assay

The principle of the ferric-reducing antioxidant power is based on the reduction of the ferric-tripyridyltriazine complex (Fe³⁺-TPTZ) to an intense blue ferrous-tripyridyltriazine complex (Fe²⁺-TPTZ). It was performed according to a previous study (Judprasong et al., 2013). Briefly, the ferric-reducing antioxidant power (FRAP) reagent, containing ferric chloride and 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) in acetate buffer (pH 3.6), was added to the ethanolic extract of the sample and then incubated in the dark at 37 °C for 4 min. Finally, the absorbance of the sample and trolox (standard) was measured at 595 nm. The results were reported as μmole trolox equivalent (TE)/100 g sample.

Oxygen radical absorbance capacity assay

The oxygen radical absorbance capacity (ORAC) assay involves antioxidants’ ability to inhibit the peroxyl radicals generated by AAPH. This assay was carried out according to the previously described (Sridonpai et al., 2022). The ethanolic extract of the sample was incubated with the fluorescein solution and AAPH solution at 37 °C. Then, the fluorescence was measured using a spectrofluorometer (Luminescence spectrophotometer, Perkin Elmer LS55, Maryland, USA) with an excitation wavelength of 493 nm and an emission wavelength of 515 nm. The areas under the curve of samples or trolox (standard) were calculated compared to blank areas. Data were presented as μmole TE/100 g sample.

DPPH assay

The radical scavenging activity of the sample was examined by DPPH, which was used as a free radical. The radical is reduced by interaction with antioxidants. This assay was performed following to a previous report (Judprasong et al., 2013). The ethanolic extract of the sample was mixed with the DPPH solution and then incubated at 37 °C in the dark for 30 min. The absorbance of trolox (standard) and the sample was measured at 517 nm. Results were represented as mmole TE/100 g sample.

Total phenolic contents

The Folin-Ciocalteau reagent was used to measure total phenolic contents, and the study followed the methodology described in a previous study (Sridonpai et al., 2022). The Folin-Ciocalteau reagent was mixed with the ethanolic extract of the sample. The absorbance of standard (gallic acid) and the sample was measured at 760 nm. Results were represented as mg gallic acid equivalent (GAE) per 100 g sample.

Cell cultures

Human colon adenocarcinoma Caco-2 cells (ATCC HTB-37) and human microglial clone 3 or HMC3 cells (ATCC CRL-3304 TM) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in a complete medium at 37 °C in a humidified incubator (Thermo Scientific, Marietta, OH, USA) with 5% CO₂. The adhered cells were grown to around 80% confluence for the experiments. For Caco-2 cells, they were cultured and maintained in DMEM supplemented with 15% (vol/vol) FBS for pre-confluence and 7.5% (vol/vol) FBS for post-confluence, 1% (vol/vol) of 200 mM L-glutamine, 1% (vol/vol) of 10 mM nonessential amino acids, 1% (vol/vol) of 100 unit/ml penicillin and 100 μg/ml streptomycin (antibiotic) and 0.2% (vol/vol) fungizone. The Caco-2 cells used were between passage numbers 23–35. For HMC3 cells, the complete medium for the cells culture comprised of 10% (vol/vol) heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin (antibiotic), 1% (vol/vol) non-essential amino acids, and 1 mM sodium pyruvate in basal media-MEM. The HMC3 cells used were between passage numbers 2–12.

In vitro digestion of the A. lebbeck leaf sample

In vitro digestion including the gastric and small intestinal phases of A. lebbeck leaves were carried out according to the previous studies (Ferruzzi et al., 2006; Garrett et al., 1999). As mentioned in the step of plant collection and preparation, the 0.8 g of A. lebbeck leaves sample was homogenized with 5% (wt/wt) soybean oil in 30 ml of buffer salts (5 mM KCl, 6 mM CaCl₂, and 120 mM NaCl) in a beaker by a homogenizer. Samples were transferred to a polypropylene tube and adjusted pH to 2.0 ± 0.1 with 1 M HCl. Next, 2 ml of pepsin (40 mg/ml in 100 mM HCl) was added, and the volume was adjusted to 40 ml with buffer salts. In this step, this tube was blanked with nitrogen gas, tightly caped, and incubated in a shaking incubator at 37 °C, 95 rpm for 1 hr. After completion of the gastric phase, the sample was adjusted pH to 6.0 ± 0.1 with 1 M sodium bicarbonate (NaHCO₃). The 3 ml of porcine bile extract (40 mg/ml in 100 mM NaHCO₃), 2 ml of porcine pancreatin (10 mg/ml in 100 mM NaHCO₃), and porcine lipase (5 mg/ml in 100 mM NaHCO₃) were added to the reaction tube. The pH was adjusted to 6.5 ± 0.1 with 1 M NaOH and added up the volume to 50 ml with buffer salts. The tube was then blanked with nitrogen gas, and incubated in the shaking incubator at 37 °C, 95 rpm for 2 hr. After that, this solution was centrifuged at 7,500 rpm for 1 hr and the supernatant was collected and filtered through a 0.22 μm polytetrafluoroethylene filter membrane. This filtered aqueous fraction was called the bioaccessible fraction. In this experiment, the control digestion was simultaneously conducted followed by the A. lebbeck leaves sample digestion by adding all chemicals of the digestive reaction except the lyophilized sample.

Preparation of bioavailable fraction of the A. lebbeck leaf sample

Briefly, Caco-2 cells were seeded at a density of 3.0 × 10⁴ cells/well on 0.4 μm polyethylene terephthalate membrane transwell cell culture inserts in a 6-well plate. Each well contained 2 ml of complete medium (15% FBS) in the apical and basolateral compartments. The cells reached confluence within approximately 5 days, and further cultured for 21–25 days with the complete medium (7.5% FBS) to obtain a fully differentiated polarized epithelium layer. Next, the differentiated epithelium monolayer was examined for tight junction integrity by evaluating the trans-epithelial electrical resistance (TEER). The Caco-2 cells monolayers with TEER values greater than 500 Ω.cm² were used for the next experiments (Ude et al., 2017). Subsequently, the apical compartment was treated with 2 ml of the bioaccessible fraction (obtained from those mentioned above) and diluted 1:3 (vol/vol) with DMEM, while the basolateral compartment was treated with 2 ml of the medium. Both compartments were then incubated for 4 hr at 37 °C. This basolateral compartment was called the bioavailable fraction of A. lebbeck leaves, hereafter referred to as BAL, was kept at −80 °C for subsequent experiments. A schematic diagram of BAL preparation is shown in Figure 1.

Cell viability assay

The viability of HMC3 cells after treating the BAL sample and glutamate was determined by MTT assay. Firstly, HMC3 cells were seeded in 48-well plates at a density of 7.5 × 10⁴ cells/well overnight. After incubation, the cells were exposed to BAL for 24 hr and treated with glutamate (50 mM) for 24 hr. Based on our previous study indicated that cell viability was significantly reduced by around 50% after treating HMC3 cells with glutamate (50 mM) when compared with the control cells (Phoraksa et al., 2023). Next, the culture media was removed, and cells were washed with phosphate buffered saline (PBS). The MTT reagent (5 mg/ml in PBS) was added and then incubated at 37 °C, 5% CO₂ for 3 hr. After incubation, the supernatants were discarded, and the formazan crystals were dissolved in DMSO. The absorbance was detected at 540 nm using a microplate reader (Bio Tek Instruments, Highland, USA). Data were shown as the cell viability percentage relative to the control cells.

Western blot analysis

HMC3 cells were seeded at a density of 1.5 × 10⁵ cells/well in 6-well plates overnight prior to pretreated cells with the BAL for 24 hr and incubated with 50 mM glutamate for 30 min. Next, the media were removed, and the treated cells were washed with cold PBS. Then, the lysis buffer containing protease and phosphatase inhibitors (Cell signaling, Danvers, MA, USA) was added to lyse the cells. The measurement of protein concentration was performed using bicinchoninic acid method. The proteins (40 μg) were loaded in each lane and separated on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred onto nitrocellulose membranes (Sigma Aldrich, Dorset, UK) and incubated with a blocking buffer at room temperature for 1 hr. Then, Tris-buffered saline containing 0.1% Tween-20 (TBST) was added to wash the membranes and incubated with the primary antibodies, namely CHOP (1:1,000), calpain1 (1:1,000), p-ERK (1:1,000), ERK (1:1,000), p-JNK (1:1,000), JNK (1:1,000), p-p38 (1:1,000), p38 (1:1,000), Bcl-2 (1:1,000), cytochrome c (1:1,000), Bax (1:1,000), cleaved caspase-9 (1:1,000), and β-actin (1:20,000) were obtained from Cell Signaling Technology (Danvers, MA, USA) while cleaved caspase-12 (1:1,000) was obtained from Abcam (Cambridge, UK) at 4 °C overnight. Next, TBST was used to wash these membranes and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; Cell Signaling Technology, Danvers, MA, USA) for 2 hr at room temperature. Then, these membranes were washed, and a chemiluminescence substrate was used to detect the specific protein bands following exposure to the X-ray film. The protein band intensity was analyzed using the Image J program (National Institutes of Health, Bethesda, MD, USA), and then normalized with β-actin.

Phytochemical analysis

The BAL was mixed with methanol and chloroform at a ratio of 1:3:1% (vol/vol/vol). This sample was mixed by vortex at 4 °C for 5 min, and centrifuged at 15,000g (Eppendorf, Centrifuge 5425 R, Hamburg, Germany) for 15 min at 4 °C. Then, this supernatant was transferred to microcentrifuge tube and dried at 60 °C. Then, the mobile phase (100 μl) was added and centrifuged in the same condition above. The supernatant (100 μl) was analyzed with an ultra-high performance liquid chromatography-quadrupole orbitrap mass spectrometry (UHPLC–MS/MS [Q-Orbitrap]) (Exploris 480, Thermo Scientific, MA, USA) using an Accucore C18 column (2.1 × 150 mm, 2.6 μm particle size). The temperature of the column was set to 50 °C. The mobile phase comprised of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) with a gradient elution: 0–8 min, 1% B; 8–40 min, 1% B-99%; 40–48 min, 99% B; 48–48.1 min, 99% B-1%; 48.1–60 min 1% B at a flow rate of 0.6 ml/min. This analysis was performed in both positive- and negative-ion modes. The parameters of mass spectrometry were set as follows: ion source type, H-ESI; spray voltage, static; positive ion, 3,500 V; negative ion, 2,800 V; gas mode, static; ion transfer tube temperature, 263 °C; vaporizer temperature, 425 °C; mass range, 50–1,000 m/z; RF lens, 80%.

Lipinski’s rule and pharmacokinetic-adsorption, distribution, metabolism, excretion, and toxicity properties analysis

The drug-likeness of the identified active compounds was predicted by the SwissADME online database (http://www.swissadme.ch accessed on 29 January 2024), following to Lipinski’s rule of five parameters (Daina et al., 2017). Based on Lipinski’s rule, the active compounds that had a number of hydrogen bond acceptors ≤10; molecular weight ≤ 500; a number of hydrogen bond acceptors ≤5; and MlogP ≤ 4.15 were considered to be drug-like compounds (Lipinski et al., 2001). The pkCSM online database was used to investigate the pharmacokinetic properties including, the adsorption, distribution, metabolism, excretion, and toxicity (ADMET) of bioactive compounds (http://biosig.unimelb.edu.au/pkcsm/prediction accessed on 1 February 2024) (Pires et al., 2015).

Molecular docking

Preparation of protein and ligand

The 3D structures of Bax protein (PDB ID: 4S0O, https://www.rcsb.org/structure/4S0O [accessed on 5 February 2024]) (Govindarasu et al., 2017), cytochrome c (PDB ID: 6DUJ, https://www.rcsb.org/structure/6DUJ [accessed on 13 February 2024]), and caspase-9 (PDB ID: 4RHW, https://www.rcsb.org/structure/4RHW) [accessed on 20 February 2024]) (Li et al., 2017) were obtained from the RCSB Protein Data Bank. The preparation of all protein structures was carried out by removing all water molecules and ligands using BIOVIA Discovery Studio 2024. Next, the protein structures were processed by adding polar hydrogen atoms and Kollman charges using AutoDockTools-1.5.6 software and then saved in PBDQT format for molecular docking studies (Sillapachaiyaporn et al., 2021). For ligand preparation, the structure data file (SDF) formats of all ligand structures were obtained from the PubChem online database (https://pubchem.ncbi.nlm.nih.gov [accessed on 5 February 2024]). Then, the clean geometry was performed for all ligands by using BIOVIA Discovery Studio 2020 and they were saved as PDB format. Subsequently, AutoDockTools-1.5.6 was utilized to convert the PDB to PDBQT format.

Method validation

The validation of the docking procedure was performed by removing the original ligand of protein and re-docking it into the binding active site of protein. The root-mean-square deviation (RMSD) of the re-docked co-crystalized compound was considered. The RMSD value with a lower than two angstroms represents a good docking solution (El-Sheref et al., 2020). In this study, the re-docking results of cytochrome c and heme c showed an RMSD value of 0.84 Å, whereas the re-docking results of caspase-9 and sulphate ion showed an RMSD value of 0.989 Å.

Molecular docking of selected ligands

In this procedure, AutoDock 4.2.6 software was used with all default parameters for conducting the molecular docking analysis. The grid boxes for all docking were set in the XYZ-dimension of 50 × 50 × 50, spacing 0.375 Å. For Bax, a centre grid box was set at 23.844 × −3.449 × 16.98 (xyz) (Govindarasu et al., 2017). For cytochrome c, a centre grid box was set at −16.11 × −39.694 × 5.405 (xyz). For caspase-9, a centre grid box was set at −8.915 × −17.597 × 5.252 (xyz). Finally, the conformation that provided the lowest binding energy was chosen to investigate the interaction between protein and ligand using BIOVIA Discovery Studio 2020.

C. elegans model

C. elegans culture conditions

In this study, we used C. elegans strains, namely wild-type N2 strain and transgenic CF1553 strain (mu1s84[pAD76(sod-3::GFP)]. These worms were cultured in nematode growth medium (NGM) agar supplemented with Escherichia coli OP50, which served as their food source, at 20 °C in the incubator. These strains and E. coli OP50 were acquired from Caenorhabditis Genetics Center, University of Minnesota, Minneapolis, MN, USA.

Lifespan assay

This assay is a direct method for the determination of ageing and death. The live and dead worms in synchronized populations are counted based on their responses to platinum wire touching in agar plates. Then, the lifespan curve is created based on the percentage of live worms in populations (Ha et al., 2022; Park et al., 2017). In this study, the L4 larvae stage of wild-type worms were randomly transferred to 35 mm NGM agar plate with E. coli OP50 approximately 30–40 worms/plate which were treated with and without BAL. The worms were transferred to the new NGM medium with E. coli OP50 and daily treated sample until the end of the reproductive period. The number of alive and dead worms was counted every day. Data were expressed as a percentage of survival worms.

Measuring survival worms caused by juglone toxicity

In this experiment, wild-type at L1 larvae were cultured on 35 mm dishes containing S-medium and E. coli OP50 approximately 30–50 worms/dish. Worms were treated with BAL and incubated at 20 °C for 48 hr. Then, prooxidant juglone (80 μM) was added and incubated for 24 hr at 20 °C. Regarding the previously reported, our colleagues have used the concentration of juglone at 80 μM, which produces the oxidative stress and is toxic to worms (Kittimongkolsuk et al., 2021; Rangsinth et al., 2019; Thabit et al., 2018). The number of live worms per total worms was counted, and the results were presented as a percentage of live worms.

Detecting the antioxidant superoxide dismutase 3 expression

In this study, the CF1553 transgenic worms that express the superoxide dismutase 3 (SOD-3) tag with the GFP reporter were synchronized at the L1 stage. The worms were incubated with and without BAL at 20 °C for 48 hr. After that, the worms were paralyzed using 10 mM sodium azide, and mounted on a glass slide. Each group was imaged using a confocal microscope, with at least 30 worms per group. The relative intensity of fluorescence was calculated by using ImageJ software.

Statistical analysis

The data are presented as M ± SD from triplicate in at least three independent experiments using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). One-way ANOVA was performed to assess the statistical analysis, followed by a post hoc Tukey test. The student’s t-test was carried out for comparison of the two groups. Statistical significance was considered to be a p-value less than 0.05. For the lifespan assay, a comparison between different groups was performed by a log-rank test and the results are expressed as M ± standard error of means (SEM).

Results

Antioxidant capabilities of A. lebbeck leaves

In this study, the antioxidant capabilities of the lyophilized A. lebbeck leaves sample were measured by FRAP assay, ORAC method, and the DPPH assay. Our results showed that A. lebbeck leaves exhibited potent scavenging activities against radicals, possessed the ability to reduce ferric to ferrous ions, and also contained a high total phenolic content, demonstrating strong antioxidant power as shown in Table 1.

BAL suppresses glutamate-induced cytotoxicity in HMC3 cells

To evaluate the effect of BAL against glutamate-induced toxicity in human microglia, we used the HMC3 human microglial cell line as a model. Our previous study found that treatment with 50 mM glutamate resulted in HMC3 cell toxicity and death (Phoraksa et al., 2023). The cells were pre-treated with BAL for 24 hr, followed by 50 mM glutamate for 24 hr, and then cell viability was evaluated by MTT assay. The results exhibited that glutamate-alone treatment significantly decreased the viability of HMC3 cell about 55%. Pretreatment of cells with BAL obviously inhibited glutamate-induced toxicity and, moreover, significantly increased the cell viability compared with the glutamate-alone group (Figure 2A). However, the cell viability of the pretreatment of cells with BAL and exposure to glutamate was significantly decreased when compared with the BAL-alone group. These results were consistent with the cell morphology that we observed under a light microscope. The cells treated with glutamate displayed a round shape and abnormal structure, while pretreatment of cells with BAL was able to maintain their normal cell morphology as presented in Figure 2B. Our results demonstrate that the pretreating cells with BAL could rescue cells and inhibit cytotoxicity in HMC3 cells induced by glutamate.

Inhibitory effect of BAL on glutamate-induced ER stress in HMC3 cells

In the CNS, prolonged excessive glutamate is associated with ER stress in cellular, leading to the signal process of cell apoptosis. The activation of the important ER stress signals comprises the calpain, caspase-12, and CHOP pathways (Kritis et al., 2015; Merighi & Lossi, 2022). To demonstrate the effect of BAL on glutamate-mediated ER stress, the specific ER protein expressions, namely cleaved caspase-12, calpain1, and CHOP, were performed by Western blotting. The HMC3 cells were pretreated with BAL. After incubation for 24 hr, cells were treated with 50 mM glutamate for 30 min. Our results showed that pretreatment of cells with BAL significantly decreased the protein expressions of calpain1 and cleaved caspase-12 compared with glutamate treatment (Figure 3A–C). While the expression of CHOP (Figure 3A and D) did not differ compared to the non-treated control cells or glutamate treatment alone. The original blots were shown in the supplementary data (Figure S1). Therefore, these results reveal that the pretreating cells with BAL suppress glutamate-mediated ER stress by inhibiting the expressions of cleaved caspase-12 and calpain1 proteins in our HMC3 cell model.

Effect of BAL on MAPKs signalling in HMC3 cells

Excessive glutamate accumulation in the CNS is able to stimulate the MAPKs (ERK, p38, and JNK) signalling pathway, which is linked to various processes of cellular proliferation, differentiation, inflammation, and apoptosis (Jeong et al., 2023; Rai et al., 2023). In this study, to evaluate the effect of BAL on MAPKs signalling activation in HMC3 cells caused by glutamate, the cells were pretreated with BAL for 24 hr and then exposed to glutamate (50 mM) for 30 min and the protein expression levels of MAPK markers were investigated by Western blot analysis. The results exhibited that the pretreatment of cells with BAL significantly reduced phosphorylated form of ERK (p-ERK) compared with glutamate treatment (Figure 4A and B). Whereas the expressions of phosphorylated p-38 (p-p38) (Figure 4A and C) and phosphorylated JNK (p-JNK) (Figure 4A and D) were not significantly different from those of the non-treated control cells or glutamate treatment alone. Furthermore, treating cells with BAL alone did not affect the expression of all protein MAPKs compared to non-treated control cells. The original blots were presented in the supplementary data (Figure S2). Therefore, our results demonstrate that the pretreating cells with BAL could inhibit glutamate-induced MAPK activation by reducing ERK phosphorylation.

Protective effect of BAL on glutamate-induced apoptosis in HMC3 cells

High glutamate concentration can lead to the death of nerve cells by elevating the levels of certain proteins involved in apoptosis, such as Bax, cytochrome c, and caspase-9, whereas simultaneously decreasing the levels of anti-apoptotic proteins like Bcl-2 (Al-Nasser et al., 2022; Fan et al., 2023). Hence, to examine the effect of BAL on glutamate-mediated apoptosis in HMC3 cells, the expressions of apoptotic protein markers including Bcl-2, Bax, cytochrome c, and cleaved caspase-9 were investigated using Western blotting. The results exhibited that the pretreating cells with BAL interestingly increased the Bcl-2 expression (Figure 5A and B), meanwhile significantly suppressed the expressions of Bax, cytochrome c, and cleaved caspase9 (Figure 5A, C, E, and F) compared with the glutamate treatment group. In addition, we found that pretreated cells with BAL apparently decreased the Bax/Bcl-2 ratio (Figure 5D). The original blots were shown in the supplementary data (Figure S3). Collectively, these results suggest the potential of BAL to effectively prevent glutamate-induced apoptosis by inhibiting apoptotic proteins, including Bax, cytochrome c, and caspase-9, while concurrently activating the anti-apoptotic Bcl-2 protein in HMC3 cells.

Identification of bioactive compounds in BAL

The bioactive compounds in BAL were identified by UHPLC-Q-Orbitrap-MS/MS analysis in the ion-positive and negative mode. We found that most of them were flavonoids such as quercetin-3β-D-glucoside (0.00797%), robinetin (0.01952%), vitexin (0.00381%), kaempferol (0.01508%), kuromanin (0.02576%), daidzein (0.00186%), rutin (0.01955%), and luteolin (0.02209%) and carotenoids namely tanshinon I (0.00029%) and nootkatone (0.00067%) as presented in Table 2. The chromatograms of these bioactive compounds in BAL were shown in the supplementary data. The results showed that most bioactive compounds within BAL were found in the ion-positive mode except rutin and luteolin.

Lipinski’s rule and ADMET properties of bioactive compounds in BAL

The bioavailable properties of drug-likeness compounds were examined by Lipinski’s rule. This criterion consists of five parameters: molecular weight less than or equal to 500 Da, hydrogen bond acceptors less than or equal to 10, hydrogen bond donors less than or equal to 5, MlogP less than or equal to 4.15, and the compounds that exhibited less than or equal to 1 Lipinski violation were considered drug-like compounds (Lipinski et al., 2001). The results showed that seven bioactive compounds passed Lipinski’s criteria, except quercetin-3β-D-glucoside, kuromanin, and rutin as shown in Table 3. Quercetin-3β-D-glucoside and kuromanin had an excess number of hydrogen bond acceptors and donors. In addition, rutin was a large molecular weight and an excess number of hydrogen bond acceptors and donors. Furthermore, the examination of pharmacokinetic properties, namely ADMET, of seven bioactive compounds in BAL was carried out. As shown in Table 4, all compounds showed high intestinal absorption with more than 70%, except vitexin, which has low intestinal absorption (46.7%). Interestingly, daidzein, tanshinone I, nootkatone, luteolin, and kaempferol could penetrate the blood–brain barrier (BBB). Besides, all interesting bioactive compounds showed no AMES toxicity and hepatotoxicity except tanshinone I that exhibited AMES toxicity. Based on these data, we therefore selected the candidate six bioactive compounds of BAL, namely robinetin, vitexin, kaempferol, daidzein, nootkatone, and luteolin for studying a molecular docking in the following analysis.

In silico evaluation of binding affinity between identified compounds of BAL and apoptotic proteins

The glutamate at high concentration causes neuronal toxicity by activating the apoptosis signalling pathway (Kritis et al., 2015; Park et al., 2019). Our results indicate that glutamate-mediated apoptosis in HMC3 cells by activating the expression of Bax, cytochrome c, and caspase-9. Thereby, to investigate the potential of six bioactive compounds in BAL, the predictions of selected six compounds on the blocking of Bax, cytochrome c, and caspase-9 were evaluated using molecular docking studies. In this study, 1-(3,6-dibromocarbazol-9-yl)-3-piperazin-1-ylpropan-2-ol (BAI1), methazolamide, and (3S)-5-(2,6-difluorophenoxy)-3-[[(2S)-3-methyl-2-(quinoline-2-carbonylamino)butanoyl]amino]-4-oxopentanoic acid (Q-VD-OPh) were used as native inhibitor for Bax, cytochrome c, and caspase-9. The docking results of Bax with BAI1, a native inhibitor, showed a binding energy at −9.32 (kcal/mol). All compounds had higher binding energies than the native inhibitor (Table 5). However, luteolin (−8.85 kcal/mol) and kaempferol (−8.74 kcal/mol) had closer binding energies than the native inhibitor. Figure 6 presents the interactions between Bax and candidate ligands.

According to the docking results of cytochrome c with methazolamide, a native inhibitor, showed a binding energy of −6.88 (kcal/mol). Interestingly, five compounds showed lower binding energies than the native inhibitor, including luteolin (−8.54 kcal/mol), nootkatone (−8.16 kcal/mol), robinetin (−8.13 kcal/mol), kaempferol (−8.07 kcal/mol), and daidzein (−7.64 kcal/mol) as shown in Table 6. The interactions between cytochrome c and candidate ligands are presented in Figure 7.

Additionally, the docking results of caspase-9 with Q-VD-OPh, a native inhibitor, showed a binding energy at −8.20 (kcal/mol). The results presented that all compounds had higher binding energies than the native inhibitor (Table 7). Among them, nootkatone was the lowest binding energy (−6.96 kcal/mol). The interactions between caspase-9 and candidate ligands are presented in Figure 8.

Effect of BAL on lifespan in C. elegans

Based on our results from UHPLC-Q-Orbitrap-MS/MS analysis and molecular docking, it is revealed that the potential bioactive compounds of BAL are robinetin, vitexin, kaempferol, daidzein, nootkatone, and luteolin. Interestingly, supporting evidences revealed that these compounds can promote lifespan extension in C. elegans (Leonov et al., 2015; Okoro et al., 2021). Therefore, to investigate the longevity effects of bioavailable fraction of A. lebbeck leaves, the C. elegans wild-type N2 strain at the L4 stage was treated with BAL every day. The survival plot of C. elegans is presented in Figure 9. The mean lifespans of the BAL-treated group and the control group were 16.28 and 18.12 days, respectively, showing that BAL treatment increased lifespan by approximately 11.30%, as presented in Table 8. The BAL treatment significantly increased the survival and lifespan of C. elegans compared to the control group. Our results indicate that BAL can extend the lifespan of C. elegans.

Effect of BAL on juglone-induced oxidative toxicity in C. elegans

In the brain, many factors lead to oxidative stress such as excitotoxicity, reduction of cellular antioxidant system, and high oxygen demand. Excessive oxidative damage level is related to the development of many neurological disorders, including AD and PD (Hannan et al., 2020). This study employed juglone as a prooxidant agent widely used for the oxidative stress model in C. elegans (Araújo et al., 2023; Rangsinth et al., 2019; Thabit et al., 2018). The effect of BAL on juglone-induced oxidative toxicity was examined in C. elegans wild-type N2 strain. The worms were synchronized at the L1 stage and treated with BAL in S-medium along with E. coli OP50 for 48 hr prior to exposure with 80 μM juglone for 24 hr. These results indicated that juglone treatment significantly decreased survival compared with the control (Figure 10). The survival rate of the juglone treatment group was equal to 34.55 ± 7.67%. Interestingly, the pretreatment of C. elegans with BAL significantly increased the survival rate at 87.47 ± 1.75% compared to the juglone treatment group. Our results indicate that BAL can inhibit juglone-induced oxidative stress in C. elegans.

BAL activates antioxidant SOD3 expression in C. elegans

C. elegans has many genes related to oxidative stress pathways, especially antioxidant superoxide dismutases (Ha et al., 2022; Yang et al., 2022; Zhu et al., 2022). Hence, to examine the effect of BAL on antioxidant defence in C. elegans, we also examined in the CF1553 transgenic worm that expresses the stress-response gene, namely SOD3 fusion with green fluorescence protein (GFP). The worm was age synchronized at the L1 stage and treated with and without BAL in S-medium along with E. coli OP50 for 48 hr. Our results exhibited that the treating worms with BAL significantly increased the fluorescence intensity of SOD3 protein with the relative SOD3 expression at 1.39 ± 0.12 compared to the control group (Figure 11A and B). The results demonstrate that BAL can promote the cellular antioxidant enzyme SOD3 in C. elegans.

Discussion

Glutamate is the major excitatory neurotransmitter found in the mammalian CNS. It controls many functions in the brain such as learning, memory, and cognition. This neurotransmitter is synthesized and recycled in the neurons and neuroglia through the glutamate-glutamine cycle, which is tightly regulated to prevent toxicity in the brain (Lewerenz & Maher, 2015; Olloquequi et al., 2018; Riche & Lenard, 2022). The continuous accumulation of excessive glutamate can lead to overstimulation of glutamate receptors, resulting in increased oxidative stress, ER stress, and ultimately cell injury and death. This event is associated with the development of various neurodegenerative disorders including AD and PD (Al-Nasser et al., 2022; Olloquequi et al., 2018). Several evidences have reported that hyperactive glutamatergic signalling arises in AD pathology at early phases. The reduction of glutamate clearance is associated with the AD and PD pathological mechanisms (Andersen et al., 2021; Fairless et al., 2021; Wang et al., 2020). Furthermore, some studies revealed that the cerebrospinal fluid glutamate levels in patients with probable AD are significantly higher compared to those in healthy controls (Madeira et al., 2018). Therefore, the inhibition of glutamate-induced neurotoxicity in nerve cells may provide a benefit for the treatment of these diseases.

ER is an important organelle that controls many functions in the cellular system, including protein synthesis, lipid synthesis, and homeostasis of calcium ions. Many studies report glutamate-induced toxicity through the stimulation of ER stress (Bhardwaj et al., 2019; Gu et al., 2020; Kang et al., 2020; Shi et al., 2022; Sprenkle et al., 2017; Sukprasansap et al., 2017). When glutamate binds to its receptor, it leads to the release of calcium ions from the ER. The elevated calcium ions level then provokes calcium-dependent enzymes, including calpain1. Once activated, calpain1 triggers the activation of caspase-12. Moreover, CHOP is another ER stress marker that is related to the apoptosis pathway. These stimulations of caspase-12 and CHOP also promote ER stress, eventually resulting in apoptotic cell death. Numerous supporting research demonstrated that ER stress is associated with the progression of neurodegenerative diseases (Kim et al., 2022; Lewerenz & Maher, 2015; Merighi & Lossi, 2022). Likewise, previous study reported that caspase-12 and CHOP were upregulated in the brains of AD patients (Nezhad Salari et al., 2024). In our present study, we found that a high concentration of glutamate-induced ER stress through activating the expression of calpain1 and caspase-12 in human microglia HMC3 cells (Figure 3A–C). However, glutamate treatment did not upregulate the expression of CHOP protein in this study (Figure 3A and D). Previous reports indicate that ER stress can promote the activation of the MAPKs pathway. Moreover, the MAPKs pathway is activated by high glutamate concentration. MAPKs pathways regulate various cellular functions and systems, including proliferation, differentiation, inflammation, and apoptosis (Jeong et al., 2023; Palanivel et al., 2022; Wei et al., 2016). In this report, glutamate upregulates the protein expression of p-ERK, whereas the protein expression of p-p38 and p-JNK did not alter (Figure 4A–D). Several reports reveal that high glutamate concentration promotes cell death in neuronal cells through the activation of ERK (Park et al., 2019; Rakkhittawattana et al., 2022; Song et al., 2017; Subramaniam & Unsicker, 2010; Xiong et al., 2021). Glutamate-induced apoptosis has reported in many neuroglia cells including microglial cells (Belov et al., 2020; Lee et al., 2012; Phoraksa et al., 2023; Vongthip et al., 2024). High concentration of glutamate activates ER stress and MAPK pathway, contributing to the death of cells. This process involves an increase in the apoptotic proteins expression such as Bax, cytochrome c, caspase-9, and caspase-3, while the anti-apoptotic protein Bcl-2 expression is decreased (Afshari et al., 2020; Jeong et al., 2023; Kim et al., 2023; Li et al., 2023; Palanivel et al., 2022; Xie et al., 2023). Our study showed that glutamate upregulated the levels of Bax, cytochrome c, and caspase-9 proteins and repressed Bcl-2 protein (Figure 5).

Consequently, it is interesting to study about the strategy of preventive cell damage and death in nervous cells. From our recent work, we showed that the extracts from an indigenous edible leaf of A. lebbeck (L.) Benth. can prevent apoptosis and also enhance the cellular antioxidant proteins in human microglial cells (Phoraksa et al., 2023). The A. lebbeck leaves consist of many phytochemicals such as alkaloids, phenolics, flavonoids, and carotenoids which have a wide range of biological activities such as anti-inflammatory, antioxidant, anticancer, immunomodulatory, and neuroprotection (Almaamori, 2023; Phoraksa et al., 2023; Praengam et al., 2017; Samant et al., 2023; Shirisha et al., 2013). However, there is no report about the effects and mechanisms of A. lebbeck leaves after digestion and absorption through the human gastrointestinal tract, which refers to the bioavailable fraction of edible plants after eating in the body. Hence, in this present study, we investigated the benefit potential of bioavailable fraction of the A. lebbeck leaf or BAL which obtained from the coupled simulated gastric/small intestinal digestion of lyophilized this leaves sample with the human intestinal epithelial Caco-2 cell model. This model has been developed and applied to study the absorption and bioavailability of nutrients and bioactive compounds, with results comparable to bioavailability data from human studies (Bhagavan et al., 2007). Our findings demonstrate that pretreatment of HMC3 cells with BAL could inhibit glutamate-induced cytotoxicity and apoptosis in our cell model. BAL interestingly suppressed ER stress protein markers, namely calpain1 and cleaved caspase-12 expressions (Figure 3A–C) and repressed ERK phosphorylation (Figure 4A and B). Consequently, apoptotic proteins such as Bax, cytochrome c, and caspase-9 were decreased while the anti-apoptotic Bcl-2 protein was increased by pretreating cells with BAL (Figure 5A, B, C, E, and F). These results are involved in flavonoids, carotenoids, and other phytochemicals found in these leaf extracts which exhibit numerous benefits of biological activities as mentioned above.

Furthermore, in this study, bioactive compounds in BAL were also examined by LC–MS/MS technique. The results showed that most of them are flavonoids and carotenoids such as quercetin-3β-D-glucoside, robinetin, vitexin, kaempferol, kuromanin, daidzein, tanshinon I, nootkatone, rutin, and luteolin (Table 2). Next, we investigated the interactions between apoptotic proteins and the candidate ligands from bioactive compounds of BAL by molecular docking study. In the docking results of Bax protein, we found that luteolin, and kaempferol, their binding energies were closer than those of BAI1 (the native inhibitor), as shown in Table 5. In addition, the docking results of cytochrome c presented that luteolin, nootkatone, robinetin, kaempferol, and daidzein had lower binding energies when compared to methazolamide (the native inhibitor) as presented in Table 6. The docking results of caspase-9 showed that nootkatone had closer binding energy when compared to the native inhibitor (Q-VD-OPh) as exhibited in Table 7. The data indicate that luteolin, kaempferol, and nootkatone are interesting compounds for the inhibition of apoptotic proteins, including Bax, cytochrome c, and caspase-9. Remarkably, Table 4 presents the pharmacokinetic properties of luteolin, kaempferol, and nootkatone which they showed high intestinal absorption more than 70% and all three compounds could penetrate the BBB (Rahul & Siddique, 2021; Xiao et al., 2023; Yang et al., 2020). Some study has reported that luteolin showed antioxidant, anti-apoptotic, and neuroprotective properties (Huang et al., 2023). It prevented glutamate-induced toxicity by reducing reactive oxygen species (ROS) accumulation and apoptosis (Vongthip et al., 2024). Moreover, it inhibited apoptosis by decreasing pro-apoptotic proteins, Bax, cytochrome c, and caspase-3 (Liu et al., 2017) and reduced ER stress in a mouse model of AD (Tana & Nakagawa, 2022). Kaempferol is one of the major flavonoid species that is abundantly found in plants. It increased the anti-apoptotic Bcl-2 protein and decreased pro-apoptotic proteins such as Bax and caspase-3 (Zhang et al., 2021). In addition, kaempferol attenuated glutamate-induced apoptosis by reducing MAPK phosphorylation and pro-apoptotic proteins (Yang et al., 2014). Nootkatone is a sesquiterpene derivative that shows antioxidant and neuroprotection by increasing antioxidant activities such as SOD and catalase (CAT) and decreasing caspase-9 and caspase-3 activities (Dai et al., 2023; Jha et al., 2022). Also, nootkatone could improve behavioural dysfunction and prevented MAPK3 expression in a PD rat model (Yao et al., 2022).

Moreover, we examined the effect of BAL on longevity and oxidative stress in C. elegans model. The nematode C. elegans has applied to in vivo study about stress signalling, apoptosis, ageing, and neurodegenerative diseases because it has small size, short lifespan, and transparency (Ayuda-Durán et al., 2020; Van Pelt & Truttmann, 2020). We found that BAL treatment significantly increased the lifespan in C. elegans (Table 8). Additionally, the antioxidant activity of BAL was examined in C. elegans. The treatment of BAL drastically inhibited juglone-induced oxidative stress (Figure 10). Juglone is found in many parts of walnut trees and it can generate ROS, especially superoxide anion, to cause oxidative stress (Ahmad & Suzuki, 2019). Moreover, BAL increased endogenous antioxidant SOD3 expression in C. elegans (Figure 11). The SOD is the first antioxidant enzyme that catalyzes the superoxide anion to hydrogen peroxide and oxygen in cellular system (Ighodaro & Akinloye, 2018). Additionally, the results from antioxidant activities exhibited that A. lebbeck leaves had high total phenolic content and powerful antioxidant capabilities in both scavenging reactive radicals (ORAC and DPPH assays) and reducing ferric antioxidant power (FRAP assay) (Table 1). Data from these results might support and indicate that this BAL promotes the expression of SOD3, which reduces the production of superoxide anion, inhibiting juglone-induced oxidative stress in C. elegans. Numerous studies demonstrated that polyphenols can promote lifespan extension and stress resistance in C. elegans by reducing ROS levels and increasing gene expression of antioxidant defence (Leonov et al., 2015; Liu et al., 2021; Okoro et al., 2021). Luteolin promotes lifespan and oxidative stress tolerance by upregulating the expressions of gst-4, sod-1, sod-2, and sod-3 (Lashmanova et al., 2017; Yamamoto et al., 2024). Previous reports showed that quercetin enhanced longevity and stress resistance by reducing ROS and increasing antioxidant defence (Kampkötter et al., 2007; Li et al., 2020; Pietsch et al., 2009; Pietsch et al., 2012). Likewise, some study revealed that vitexin could increase survival and stress tolerance by reducing ROS accumulation and increasing expression of sod-3 (Lee et al., 2015). Overall, the BAL showed a protective effects after digestion and absorption through the human gastrointestinal tract. They contain many bioactive compounds which can protect the ageing-associated neurodegenerative diseases. Thus, our findings indicate that the BAL exerts neuroprotective effects that can inhibit cell death caused by glutamate via ER stress and apoptosis signalling mechanisms in HMC3-human microglial cells. Moreover, BAL is also able to extend the lifespan and enhance survival against juglone-induced oxidative stress in C. elegans, which is used as a model organism.

Conclusion

Our research demonstrates the antioxidant, anti-apoptosis, and neuroprotection of the BAL. The BAL suppressed glutamate-mediated cytotoxicity and ER stress in human microglial HMC3 cells by activating anti-apoptotic protein signals and inhibiting apoptotic proteins signals. It also prevents cell death by decreasing the ERK/MAPK phosphorylation, specific ER stress proteins (calpain1 and cleaved caspase-12), and apoptotic proteins signals (Bax, cytochrome c, and caspase-9) and upregulating anti-apoptotic Bcl-2 protein. Remarkably, BAL promotes the survival and lifespan and also activates the cellular antioxidant SOD-3 expression in C. elegans model. Nevertheless, BAL and its interesting compounds are further required to investigate their neuroprotection in other animal models and clinical study. Our findings exhibited the beneficial role and anti-ageing effects of the bioavailable fraction from edible leaf of A. lebbeck, which may encourage people to consume this leaf plant and use it as the nutraceuticals for the prevention of neurodegenerative diseases.

Data availability

Data of this study are contained within the article.

Author contributions

Onuma Phoraksa (Investigation, Formal analysis, Writing—original draft), Wudtipong Vongthip (Investigation, Formal analysis), Pichakorn Juntranggoor (Investigation, Formal analysis), Arnatchai Maiuthed (Conceptualization, Methodology, Data Curation, Writing—review & editing), Siriporn Tuntipopipat (Supervision), Somsri Charoenkiatkul (Supervision), Tewin Tencomnao (Conceptualization, Resources, Methodology, Writing—review & editing), Chawanphat Muangnoi (Conceptualization, Methodology, Validation, Data Curation, Writing—review & editing), and Monruedee Sukprasansap (Conceptualization, Resources, Methodology, Validation, Data Curation, Writing—review & editing, Supervision, Project administration, Funding acquisition).

Funding

This work was supported by the National Research Council of Thailand (889918/NRCT32311/2020) and by the Development and Promotion of Science and Technology Talents Project (DPST)’s scholarship (DPST/SU16/2011).

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors appreciate the Plant Genetic Conservation Project under the Royal Initiation of Her Royal Highness Princess Maha Chakri Sirindhorn and the Electricity Generating Authority of Thailand at Srinakarind Dam for kindly providing A. lebbeck leaves. Special thanks to Assistant Professor Dr. Thaya Jenjittikul for specifying and identifying this plant. The authors thank Napong Lohaphuntrakun for his assistance with the analysis of the UHPLC-Q-Orbitrap-MS/MS technique.

References

Author notes