Protective Effect of Aquilaria crassna Leaf Extract against Benzo[a]pyrene-Induced Toxicity in Neuronal Cells and Caenorhabditis elegans: Possible Active Constituent Includes Clionasterol

Aquilaria crassna (AC) is a beneficial plant widely used to alleviate various health ailments. Nevertheless, the neuroprotection, antiaging, and xenobiotic detoxification against high benzo[a]pyrene induction have not been investigated. This study aimed to investigate the effects of ethanolic extract of AC leaves (ACEE) in vitro using SH-SY5Y cells and in vivo using Caenorhabditis elegans (C. elegans). Neuroprotective activities and cell cycle progression were studied using SH-SY5Y cells. Additionally, C. elegans was used to determine longevity, health span, and transcriptional analysis. Furthermore, ACEE possible active compounds were analyzed by gas chromatograph–mass spectrometry (GC-MS) analysis and the possible active compounds were evaluated using a molecular docking study. First, ACEE possessed neuroprotective effects by normalizing cell cycle progression via the regulation of AhR/CYP1A1/cyclin D1 pathway. Next, ACEE played a role in xenobiotic detoxification in high B[a]P-induced C. elegans by the amelioration of lifespan reduction, and body length and size decrease through the reduction in gene expression in hexokinase (hxk) and CYP35 pathway. Finally, phytochemicals of ACEE were identified and we uncovered that clionasterol was the possible active constituent in powerfully inhibiting both CYP1A1 and hexokinase II receptor. Essentially, ACEE was recognized as a potential alternative medicine to defend against high B[a]P effects on neurotoxicity and xenobiotic detoxification.


Introduction
Aquilaria crassna Pierre ex Lecomte (AC) is known by the name of agarwood or eagle wood.It is a highly precious fragrant plant and widely used as an ingredient in the traditional Chinese and Korean medicines.Likewise, it has different pharmacological activities, including the attenuation of cigarette smoke-induced inflammation and oxidative stress in airway epithelial cells [1], antiglycation, and antioxidant properties [2].Our previous study has demonstrated that AC exerted neuroprotection against hyperglycemia-associated neurodegenerative diseases, healthspan, and longevity improvement in Caenorhabditis elegans (C.elegans) [3].
Neurodegenerative diseases (NDs) are characterized by neuron loss and the pathophysiological change in brain-associated disorders, including the loss of neuronal function and structure.Alzheimer's disease and Parkinson's disease are the most common NDs in the elderly population [4,5].The interesting risk of NDs is an environmental pollutant: benzo[a]pyrene (B[a]P).B[a]P is one of the representatives of polycyclic aromatic hydrocarbons (PAHs).It is a product of industrial processes and is present in wood fires, vehicle exhaust, cigarette smoke, and food products, especially smoked and grilled foods.B[a]P is classified as a cancer-causing agent in humans [6][7][8][9].The main sources of human B[a]P exposure are inhalation, contaminated food, and skin absorption [10,11].The previous findings suggest that chronic B[a]P exposure could induce behavioral, neuropathological, and chemical change causing NDs [4,5].The xenobiotic process of B[a]P is metabolized by cytochrome P450 (CYP) to a carcinogenic agent causing epigenotoxicity, neurotoxicity, and impairment of animals' fertility [12][13][14].For B[a]P's metabolism in the current study, we investigated two prominent isomers of CYP, namely, CYP1A1 and CY35, in C. elegans lacking the classical CYP1A1 pathway [15,16].
Presently, B[a]P is regarded as a dangerous environmental pollutant that can cause serious health ailments, particularly brain tissue and neuronal damage.Based on previous studies, AC possesses multipotent pharmacological properties for human health.Nonetheless, the protective effect of AC on B[a]P-induced toxicity has not been clarified.Consequently, we have been studying the protective activity and underlying mechanisms of AC extract against B[a]P-induced neuronal cell damage and xenobiotic processing in human neuroblastoma SH-SY5Y cells and nematode C. elegans.Additionally, the investigation of phytochemicals in AC extract was performed, and the ability of those identified phytochemicals against CYP and hexokinase was also evaluated using an in silico approach.

Plant Extraction
Leaves of Aquilaria crassna Pierre ex Lecomte (AC) were collected from HRH Princess Maha Chakri Sirindhorn Herbal Garden, Rayong Province, Thailand.The plant was botanically identified and deposited at the herbarium of Kasin Suvatabhandhu, Department of Botany, Faculty of Science, Chulalongkorn University (Voucher specimen: A17634(BCU)).Plant material was extracted by the maceration method [17].Briefly, the AC leaves were cleaned and dried under shade for several days at room temperature.After they had completely dried, the leaves were ground into a fine powder and then 40 g was extracted with 400 mL of absolute ethanol (ratio 1:10 w/v) at room temperature for 72 h.The resulting supernatant was collected, filtrated, and evaporated to dryness.Finally, the extract was prepared as a stock solution at the concentration of 100 mg/mL (as illustrated in Figure 1).The ethanolic extract of AC leaves was designated as ACEE.

Gas Chromatograph-Mass Spectrometry (GC-MS) Analysis
Approximately 10 mg of ACEE extract was dissolved in 1 mL of absolute ethanol.The ethanolic extract was analyzed by GC-MS/MS to identify and estimate the relative abundance of organic substances.First, the chemical components of ACEE extract were separated by the GC (an Agilent 7890 series) technique.Then, GC coupled with an Agilent 7000C MS (Agilent Technologies, Inc., Santa Clara, CA, USA) and a capillary column (HP-5MS) worked very well for identifying unknowns.Finally, MS was used to measure the mass-to-charge ratio (m/z) of charged particles and determine the molecular weight, the chemical structures of molecules, and the elemental composition [17].The obtained spectra were compared with NIST Mass Spectrometry Data Center to identify phytochemical constituents (https://www.sisweb.com/software/ms/wiley.htm) (accessed on 5 July 2023).The extraction process of Aquilaria crassna (AC) leaves.Leaves of AC were collected, dried, grinded, and extracted with absolute ethanol using the maceration method.Ethanol was removed using rotary evaporation.The ethanolic extract of AC (ACEE) stock solution (100 mg/mL in DMSO) was prepared and kept at −20 °C.

Gas Chromatograph-Mass Spectrometry (GC-MS) Analysis
Approximately 10 mg of ACEE extract was dissolved in 1 mL of absolute ethanol.The ethanolic extract was analyzed by GC-MS/MS to identify and estimate the relative abundance of organic substances.First, the chemical components of ACEE extract were separated by the GC (an Agilent 7890 series) technique.Then, GC coupled with an Agilent 7000C MS (Agilent Technologies, Inc., Santa Clara, CA, USA) and a capillary column (HP-5MS) worked very well for identifying unknowns.Finally, MS was used to measure the mass-to-charge ratio (m/z) of charged particles and determine the molecular weight, the chemical structures of molecules, and the elemental composition [17].The obtained spectra were compared with NIST Mass Spectrometry Data Center to identify phytochemical constituents (https://www.sisweb.com/software/ms/wiley.htm) (accessed on 5 July 2023).

Antioxidant Determination
These assays including Folin-Ciocalteu phenol, total flavonoid, and radical scavenging activity were modified for a microplate format as previously described [18].

Folin-Ciocalteu Phenol Assay
The total phenolic content was performed by the combination of (50 µL) ACEE or gallic acid (standard phenolic compound) solution and 10% Folin-Ciocalteau reagent (50 µL).This mixture was incubated in the dark at room temperature (RT) for 30 min.Having been incubated, sodium carbonate solution was added and the combination was left in the dark at (RT) for 20 min.The content of phenolic compounds was analyzed by measuring the reaction absorbance at 760 nm and expressed in a gallic acid equivalent (GE) mg/g of dry weight [19].

Assay for Total Flavonoid Content
First, 50 µL of 1 mg/mL ACEE extract was mixed well with 150 µL of 95% ethanol.Then, the extract mixture was added with the solution of 10 µL of 1 M sodium acetate (NaOAc), and 10 µL of aluminum chloride (AlCl3).The combination was left in the dark

Antioxidant Determination
These assays including Folin-Ciocalteu phenol, total flavonoid, and radical scavenging activity were modified for a microplate format as previously described [18].

Folin-Ciocalteu Phenol Assay
The total phenolic content was performed by the combination of (50 µL) ACEE or gallic acid (standard phenolic compound) solution and 10% Folin-Ciocalteau reagent (50 µL).This mixture was incubated in the dark at room temperature (RT) for 30 min.Having been incubated, sodium carbonate solution was added and the combination was left in the dark at (RT) for 20 min.The content of phenolic compounds was analyzed by measuring the reaction absorbance at 760 nm and expressed in a gallic acid equivalent (GE) mg/g of dry weight [19].

Assay for Total Flavonoid Content
First, 50 µL of 1 mg/mL ACEE extract was mixed well with 150 µL of 95% ethanol.Then, the extract mixture was added with the solution of 10 µL of 1 M sodium acetate (NaOAc), and 10 µL of aluminum chloride (AlCl 3 ).The combination was left in the dark at RT for 40 min and the reaction absorbance was measured at 415 nm.Total flavonoid content was expressed in a quercetin equivalent (QE) mg/g of dry weight [20,21].

Cell Culture
A human neuroblastoma cell was used in this study including SH-SY5Y cells (cell line service, Heidelberg, Germany).Then, SHSY5Y cells were cultured in DMEM/high glucose (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum and antibiotics including 100 U/mL penicillin and 100 µg/mL streptomycin (Thermo Scientific HyClone, Logan, UT, USA).Cells were maintained in the humidified incubator with 5% CO 2 at 37 • C. 252.31 g/mol) was purchased from Sigma-Aldrich (St. Louis, MO, USA).In this experiment, B[a]P of 0-40 µM and ACEE of 0-100 µg/mL were used to determine both the toxic concentration of B[a]P and nontoxic concentration of ACEE with SH-SY5Y cells.Then, to find out the protective dose of ACEE against B[a]P-induced cytotoxicity, the cells were cotreated with a toxic concentration (40 µM) of B[a]P and ACEE of 0-25 µg/mL for 48 h.Afterwards, 5 mg/mL of MTT reagent (BioBasic Inc., Markham, ON, Canada) was added to each well and incubated at 37 • C for 4 h to induce formazan product formation.Next, 10% SDS in 0.01 N HCl was added and the cells were incubated at 37 • C overnight.The amount of formazan is proportional to the number of alive cells [24].

Cell Cycle Analysis Assay
Cell cycle was analyzed by the assessment of DNA content distribution.Having been treated with either B[a]P or ACEE and incubated for 48 h, fixed cells were prepared by incubating with 70% ethanol at −20 • C for 2 h.Then, the cells were washed 3 times and stained with propidium iodide containing RNase A for 15 min in the dark.The percentages of cell population were analyzed using a BD FACSCalibur flow cytometer and BD CellQuest TM Pro software version 4.0.2(BD Biosciences, San Jose, CA, USA) [25].

Cultivation of C. elegans
C. elegans (Bristol N2 strain) and Escherichia coli (OP50) were acquired from the Caenorhabditis Genetics Center (University of Minnesota, Twin Cities, MN, USA).C. elegans were cultivated on nematode growth medium (NGM) agar coated with E. coli OP50 with an OD 600 of 1.0 and maintained at 20 • C [26].

C. elegans' Lifespan Assay
L1 larvae were cultured on different NGM coated with 0.1% DMSO, 40 µM B[a]P, or cotreatment of 40 µM B[a]P with 5 µg/mL ACEE.At the L4 stage, new NGM was prepared and L4 worms were transferred to separate them from their progeny and to avoid starvation.The number of dead and live worms was counted and recorded every day until all worms were dead [26].
2.10.Measurement of C. elegans' Body Size and Length N2 synchronized L1 larvae were cultured on different NGM coated with B[a]P at 0, 100, 200, and 300 µM, and were allowed to grow into adult day 1.Later, a 10× objective lens of brightfield microscope was used to image adult day 1 worms in each group.Later, the software Motic Image Plus 3.0.was used to analyze the body length and size of the adult worms [26].

Quantitative Reverse Transcription PCR (RT-qPCR)
This assay was performed to understand the underlying signaling pathways that drive the biological effects of B[a]P and ACEE.For cell line, to identify the pathway by which ACEE ameliorates the effect of B[a]P on the induction of cell cycle arrest in neuronal cells, SH-SY5Y cells were treated with 40 µM B[a]P and 5 µg/mL ACEE and cultured for 48 h.As well, for C. elegans, L1 larvae stage worms were treated with 300 µM B[a]P and 5 µg/mL ACEE for 72 h into adult day 1 to investigate the pathway by which B[a]P stimulated toxicity and metabolism in C. elegans.Additionally, RNA from both SH-SY5Y and the worms was extracted by GENEzol TM reagent (Geneaid Biotech Ltd., New Taipei City, Taiwan) following RNA extraction protocol procedure.Reverse transcription and the qPCR were accomplished in the recommended protocol of iTAQ universal SYBR green supermix (Bio-Rad Laboratories, Hercules, CA, USA) and CFX Real time PCR, respectively.The measured fluorescent signals revealed the PCR results.All primer sequences are shown in Table 1.

Molecular Docking
The X-ray crystallographic structures of cytochrome P450 1A1 (PDB ID: 4I8V) [30] and hexokinase 2 (PDB ID: 5HG1) [31] were obtained from the RCSB Protein Data Bank.The structures of the phytochemicals were retrieved from the PubChem database.Initially, proteins and ligands were prepared as described in our previous reports [32,33].The docking analyses were performed using the Lamarckian Genetic Algorithm with default parameters by AutodockTools 1.5.6 (version 1.5.6)software (The Scripps Research Institute, San Diego, CA, USA).Later, the protein-ligand interaction studies were further visualized using the Discovery Studio Visualizer (BIOVIA, San Diego, CA, USA).

Statistical Analysis
All data were displayed as the mean ± standard error of the mean (SEM).Means were from at least 3 independent experiments.Prior to conducting the multiple comparison tests, the data were run to check for the normality and the homogeneity of variance.A one-way analysis of variance (ANOVA) followed by a post hoc Tukey test was performed for the data that met the assumptions of both the normal distribution and the homogeneity of variance (p-value ≥ 0.05).In case the assumptions had been violated, the data were then analyzed using a Kruskal-Wallis test followed by a post hoc Mann-Whitney test.The p-value < 0.05 was considered statistically significant.

Antioxidant Properties and Total Phenolic and Flavonoid Contents
The ACEE extraction yield was 8.50% (w/w).Interestingly, ACEE contained 61.4 ± 0.90 mg GAE/g dry weight for total phenolic content and 20.61 ± 2.79 mg QE/g dry weight for total flavonoid content.ACEE had antioxidant capacities of 62.16 ± 2.39 mg of vitamin C equivalent antioxidant capacity (VCEAC)/g dry weight in DPPH and 81.97 ± 0.90 mg VCEAC/g dry weight in ABTS.Furthermore, ACEE at the concentration of 5 µg/mL possessed the free radical scavenging capacities of 49.09 ± 2.24% and 94.91 ± 0.95% against DPPH and ABTS radicals, respectively.

Effects of ACEE and B[a]P on Cell Viability
The nontoxic concentrations on cell viability of ACEE were at 5-25 µg/mL (Figure 2a).Cell viability was significantly decreased when cells were treated with ACEE at the concentration of 50 µg/mL (83.29 ± 1.98%, p value = 0.000) and 100 µg/mL (76.85 ± 2.45%, p value = 0.000) compared to the 0.1% DMSO control group.On the contrary, we found that B[a]P at the concentration of 40 µM could significantly decrease cell viability (60.08 ± 1.52% compared to the control group, p value = 0.000) (Figure 2b).Interestingly, only five µg/mL ACEE could notably exert the therapeutic effect of B[a]P-induced cell toxicity (Figure 2c).The effect of 5 µg/mL ACEE could improve cell viability in 40 µM B[a]P-treated cells from 79.56 ± 1.26% to 99.98 ± 5.36% (25.67% increase compared to B[a]P-exposed cells, p value = 0.007).Therefore, 40 µM B[a]P and 5 µg/mL ACEE were used for the further experiment.

Effect of ACEE on the Progression of Cell Cycle of SH-SY5Y Co-Cultured with B[a]P
To further understand the protection of ACEE extract on B[a]P-induced cell cycle disturbance, a flow cytometer generated the data that showed the percentage of cells in the G0/G1 phase when treated with 40 µM B[a]P was significantly lower than in the 0.1% DMSO control group (a decrease of 18.08% when compared to the 0.1% DMSO control group, p value = 0.015).After treatment combined with 40 µM B[a]P and 5 µg/mL ACEE, the percentage of cells in the G0/G1 phase was significantly higher (an increase of 23.66% when compared to the group treated with 40 µM B[a]P alone, p value = 0.006).The flow cytometer results are shown in Figure 3 and Table 3.

Effect of ACEE on the Progression of Cell Cycle of SH-SY5Y Co-Cultured with B[a]P
To further understand the protection of ACEE extract on B[a]P-induced cell cycle disturbance, a flow cytometer generated the data that showed the percentage of cells in the G0/G1 phase when treated with 40 µM B[a]P was significantly lower than in the 0.1% DMSO control group (a decrease of 18.08% when compared to the 0.1% DMSO control group, p value = 0.015).After treatment combined with 40 µM B[a]P and 5 µg/mL ACEE, the percentage of cells in the G0/G1 phase was significantly higher (an increase of 23.66% when compared to the group treated with 40 µM B[a]P alone, p value = 0.006).The flow cytometer results are shown in Figure 3 and Table 3.

Effects of ACEE on B[a]P Disturbed Cell Cycle-Associated mRNA and Protein Expression
The results in Figure 4   Moreover, Figure 5 shows the protein expression of CYP1A1 and cyclin D1 in SH-SY5Y cells was notably increased when cells were treated with 40 µM B[a]P alone (* p < 0.05 vs. control).The relative protein expression of CYP1A1 and cyclin D1 compared to the control group was 1.13 ± 0.01-fold change (p value = 0.019) and 1.94 ± 0.02-fold change (p value = 0.002), respectively.When treatment was combined with 40 µM B[a]P and 5 µg/mL ACEE, both CYP1A1 and cyclin D1 proteins were significantly decreased but only

Effects of ACEE on B[a]P-Reduced Lifespan of C. elegans
Our study indicates that high B[a]P-fed worms were manifested by shortened lifespan.The results in Table 4 reveal that the mean lifespan of 40 µM B[a]P-fed worms was shorter than that of the 0.1% DMSO control group (approximate 13.23%).Cotreatment

Effects of ACEE on B[a]P-Reduced Lifespan of C. elegans
Our study indicates that high B[a]P-fed worms were manifested by shortened lifespan.The results in Table 4 reveal that the mean lifespan of 40 µM B[a]P-fed worms was shorter than that of the 0.1% DMSO control group (approximate 13.23%).Cotreatment with ACEE extract could also attenuate the B[a]P effect and increase the mean lifespan of high B[a]P-fed worms (13.38% in comparison to the mean lifespan of the B[a]P-treated group).All results are shown in Table 4 and Figure 6.

Effect of ACEE on Toxicity and Metabolism of B[a]P in C. elegans
The toxicity of B[a]P was determined by body length assay.We found that all B[ at the concentrations of 100-400 µM could significantly reduce the body length compar to the 0.1% DMSO control group (0.77 ± 0.01-fold change, p = 0.000 for 100 B[a]P-trea group; 0.74 ± 0.01-fold change, p = 0.000 for 200 B[a]P-treated group; 0.77 ± 0.01-f change, p = 0.000 for 300 B[a]P-treated group; 0.75 ± 0.01-fold change, p = 0.000 for 4 B[a]P-treated group).
After the cotreatment of B[a]P and 5 µg/mL ACEE, ACEE could attenuate the to effect of B[a]P and cause the improvement in the body length (0.88 ± 0.01-fold change, 0.000 for 100 B[a]P-treated group; 0.83 ± 0.01-fold change, p = 0.000 for 200 B[a]P-trea group; 0.88 ± 0.01-fold change, p = 0.000 for 300 B[a]P-treated group).In contrast, AC activity on the body length improvement could not be detected in the 400 B[a]P-trea group.
Therefore, the highest dose of B[a]P with which ACEE could exert the protective fect on B[a]P-induced body length reduction was 300 µM B[a]P (Figure 7).Based on t result, we determined to use 300 µM B[a]P as an inducer for toxicity and metabolism teration of B[a]P in C. elegans.

Effect of ACEE on Toxicity and Metabolism of B[a]P in C. elegans
The toxicity of B[a]P was determined by body length assay.We found that all B[a]P at the concentrations of 100-400 µM could significantly reduce the body length compared to the 0.1% DMSO control group (0.77 ± 0.01-fold change, p = 0.000 for 100 B[a]P-treated group; 0.74 ± 0.01-fold change, p = 0.000 for 200 B[a]P-treated group; 0.77 ± 0.01-fold change, p = 0.000 for 300 B[a]P-treated group; 0.75 ± 0.01-fold change, p = 0.000 for 400 B[a]P-treated group).
After the cotreatment of B[a]P and 5 µg/mL ACEE, ACEE could attenuate the toxic effect of B[a]P and cause the improvement in the body length (0.88 ± 0.01-fold change, p = 0.000 for 100 B[a]P-treated group; 0.83 ± 0.01-fold change, p = 0.000 for 200 B[a]P-treated group; 0.88 ± 0.01-fold change, p = 0.000 for 300 B[a]P-treated group).In contrast, ACEE activity on the body length improvement could not be detected in the 400 B[a]P-treated group.
Therefore, the highest dose of B[a]P with which ACEE could exert the protective effect on B[a]P-induced body length reduction was 300 µM B[a]P (Figure 7).Based on this result, we determined to use 300 µM B[a]P as an inducer for toxicity and metabolism alteration of B[a]P in C. elegans.
activity on the body length improvement could not be detected in the 400 B[a]P-treated group.
Therefore, the highest dose of B[a]P with which ACEE could exert the protective effect on B[a]P-induced body length reduction was 300 µM B[a]P (Figure 7).Based on this result, we determined to use 300 µM B[a]P as an inducer for toxicity and metabolism alteration of B[a]P in C. elegans.Not only body length assay, but as seen in Figure 8, we showed that 300 µM B[a] would significantly reduce both the body length and body size of worms in the B[a]Ptreated group (0.78 ± 0.03-fold change (p value = 0.000) and 0.69 ± 0.03-fold change (p value = 0.000), respectively, * p < 0.05 vs. control).After cotreatment 300 B[a]P and 5 µg/mL ACEE, ACEE could attenuate the toxic effect of B[a]P and cause the improvement in the body length (0.89 ± 0.02-fold change (p value = 0.000); Figure 8b) and body size of adult
We found that 300 µM B[a]P could significantly increase the mRNA expression of all

The Ability of ACEE-Derived Phytochemical Constituents as Inhibitors of CYP1A1 and Hexokinase II Using an In Silico Approach
To further identify the competence of ACEE on CYP1A1 and hexokinase II, we used alizarin and purpurin as positive control for CYP1A1 [34], and metrizamide and lonidamine for hexokinase II [35][36][37].As shown in Table 5, for the inhibition of CYP1A1, the binding energy was found to be −10.16,−8.56, and −8.97 kcal/mol for original ligand, alizarin, and purpurin, respectively.
Based on the docking results in Table 5 and Figure 11, we found that only three phytochemicals, including vitamin E (−10.0 kcal/mol), β-amyrone (−10.12 kcal/mol), and clionasterol (−10.45 kcal/mol), exerted the outstanding inhibition against CYP1A1 with higher energy binding compared to the others and both positive controls, alizarin and purpurin.Prominently, clionasterol might be a strong inhibitor against CYP1A1 with the highest binding energy.

The Ability of ACEE-Derived Phytochemical Constituents as Inhibitors of CYP1A1 and Hexokinase II Using an In Silico Approach
To further identify the competence of ACEE on CYP1A1 and hexokinase II, we used alizarin and purpurin as positive control for CYP1A1 [34], and metrizamide and lonidamine for hexokinase II [35][36][37].As shown in Table 5, for the inhibition of CYP1A1, the binding energy was found to be −10.16,−8.56, and −8.97 kcal/ mol for original ligand, alizarin, and purpurin, respectively.Additionally, as shown in Table 6 and Figure 12, for the inhibition of hexokinase II, the binding energy was found to be −6.52,−6.53, and −4.58 kcal/mol for original ligand, metrizamide, and lonidamine, respectively.
purpurin.Prominently, clionasterol might be a strong inhibitor against CYP1A1 with the highest binding energy.Additionally, as shown in Table 6 and Figure 12, for the inhibition of hexokinase II, the binding energy was found to be −6.52,−6.53, and −4.58 kcal/ mol for original ligand, metrizamide, and lonidamine, respectively.We found that the other three phytochemicals, including β-amyrin (−7.46 kcal/mol), lupenone (−7.05 kcal/mol), and clionasterol (−6.64 kcal/mol), showed strong inhibition against hexokinase II with the higher binding energy (higher than −6.53 kcal/mol of positive control metrizamide).

Discussion
Benzo[a]pyrene or B[a]P is a carcinogen and mostly discovered in cigarette smoke, vehicle exhausts, crude oil, coal tar, tobacco, and cooked food, especially grilled meats [38].B[a]P may be one of the main causes of neurodegenerative diseases.The neurotoxicity effect of B[a]P on cognition, learning, and memory has been reported [39].The neurotoxicity of B[a]P is linked to several metabolites through the activation of aryl hydrocarbon receptor and cytochrome P450 (CYP) system.Aryl hydrocarbon receptor (AHR) is a receptor frequently found in multiple organs such as the brain, liver, gastrointestinal tract, and lungs [40].This receptor plays an important role in the control of xenobiotic detoxification through inducing CYP.An activated AHR translocates to the nucleus and dimerizes with an aryl hydrocarbon receptor nuclear translocator.Once dimerized, AHR binds to xenobiotic-responsive units to increase expression of CYP, mainly CYP1A1 [40].Likewise, our current report confirmed that the ethanolic extract of Aquilaria crassna (ACEE) exerted the neuroprotective effect against the increase in cyclin D1 in B[a]P-induced neurotoxicity through an AHR-mediated CYP1A1 pathway.Interestingly, cyclin D1 is an influential protein of cell cycle activator that directs the progression of cell cycle and is upregulated in neurotrauma or in traumatic brain injury [41].

Discussion
Benzo[a]pyrene or B[a]P is a carcinogen and mostly discovered in cigarette smoke, vehicle exhausts, crude oil, coal tar, tobacco, and cooked food, especially grilled meats [38].B[a]P may be one of the main causes of neurodegenerative diseases.The neurotoxicity effect of B[a]P on cognition, learning, and memory has been reported [39].The neurotoxicity of B[a]P is linked to several metabolites through the activation of aryl hydrocarbon receptor and cytochrome P450 (CYP) system.Aryl hydrocarbon receptor (AHR) is a receptor frequently found in multiple organs such as the brain, liver, gastrointestinal tract, and lungs [40].This receptor plays an important role in the control of xenobiotic detoxification through inducing CYP.An activated AHR translocates to the nucleus and dimerizes with an aryl hydrocarbon receptor nuclear translocator.Once dimerized, AHR binds to xenobiotic-responsive units to increase expression of CYP, mainly CYP1A1 [40].Likewise, our current report confirmed that the ethanolic extract of Aquilaria crassna (ACEE) exerted the neuroprotective effect against the increase in cyclin D1 in B[a]P-induced neurotoxicity through an AHR-mediated CYP1A1 pathway.Interestingly, cyclin D1 is an influential protein of cell cycle activator that directs the progression of cell cycle and is upregulated in neurotrauma or in traumatic brain injury [41].
To further understand the effect of B[a]P and ACEE on organism survival, C. elegans were used as in vivo model.CYP35 family plays a crucial role in the xenobiotic processing in C. elegans lacking classical CYP1A1 enzyme [15].In this study, the transcriptional analysis revealed that B[a]P could induce the expression of all eight cyp-35 genes.Previous reports have linked cyp-35A1, cyp-35A2, cyp-35A3, and cyp-35A5 to the toxic effect of B[a]P including lipid metabolism and alteration in longevity [42].C. elegans worms lacking either cyp-35A1 or cyp-35A3 were characterized by an absence of a B[a]P-induced increase in toxicity and lipid metabolism [43][44][45].Moreover, C. elegans cyp-35A2, cyp-35A3, or cyp-35A5 mutants had no alteration of lifespan after B[a]P exposure [42].In addition, cyp-35A4, cyp-35B3, and cyp-35C1 genes play an important role in toxicity, change in lifespan, and metabolism in C. elegans after 3-bromopyruvate (3-BrPA) exposure [29].Finally, cyp-35B2 gene is involved in detoxification, the protection of dopaminergic neurodegeneration, and the maintenance of neuronal health in C. elegans after bacterial metabolite exposure [46].These reasons are consistent with our current results.We found that B[a]P could cause changes in lifespan, and body length and size through the increase in cyp-35 expression in B[a]P-fed worms.
Additionally, hexokinase (hxk) is an enzyme that plays a role in glucose homeostasis and organism development.Hxk is an enzyme catalyzing almost all hexasaccharides and producing glucose-6-phosphate (G6P) and energy through glycolysis [47,48].In normal tissue, the activity of hxk is limited by low energy demand and the level of its metabolite G6P [49].In a previous study, the high-level expressions of hxk-1, hxk-2, and hxk-3 were identified in C. elegans exposed to 3-Bromopyruvate (3-BrPA).The high expression of these genes might be responding to the large ATP consumption during 3-BrPA metabolism [29].Likewise, in this study, we found that high B[a]P could enhance the expression of hxk-1, hxk-2, and hxk-3.Conserved from C. elegans to mammals, the previous report indicated that hxk-2 and hxk-3 of C. elegans are closely related to hexokinase II in mammals [29].
Based on our DPPH and ABTS assays, ACEE exhibited free radical scavenging activity which may lead to the potential biological properties in antioxidant and anti-inflammation.In addition, correlating the effects of ACEE with the biological activities of its bioactive compounds revealed by the GC-MS are shown in Table 7.

Compound Bioactivity
β-Amyrone Anti-inflammatory activity [72] β-Amyrin Anti-inflammatory activity [73] Lupenone Anti-inflammatory activity [74,75] Anticancer [76,77] Friedelan-3-one Antimicrobial properties [78] Acetyleburicoic acid Antidiabetic and antihyperlipidemic effects [79] Furthermore, the binding affinity of these phytochemicals of ACEE against CYP1A1 and hexokinase II was analyzed by docking analysis.The results presented that clionasterol might be the most potent inhibitor of both CYP1A1 and hexokinase II with the higher binding energy compared to positive control.In the previous study, clionasterol was one of drug-likeness ingredient found in traditional Chinese medicine called Bushen-Tiansui formula.This formula could improve the cognitive function of Alzheimer's disease through CYP1A1 and CYP3A4 metabolism-related targets [80].
Overall, the findings from this present study support that ACEE could provide a neuroprotective effect against high B[a]P-induced neurotoxicity in human neuronal SH-SY5Y cells by the induction of cell cycle progression.Likewise, ACEE could attenuate the effect of B[a]P on shortening C. elegans lifespan, and body length and size through reducing the mRNA expression of AHR-responsive genes cyp-35 and hxk genes.The current study demonstrates the effects of B[a]P and ACEE in both neuronal cell line and animal models.Further analysis of the dose-response relationship could be performed using the linear-quadratic dose-response model in order to better understand and predict the cellular response to B[a]P or ACEE exposure.Collectively, ACEE might be an effective neuroprotectant and detoxification agent against B[a]P exposure with a possible active constituent including clionasterol.

Conclusions
In summary, the AC leaf is composed of rich bioactive compounds and antioxidant properties.ACEE could represent neuroprotectant from B[a]P-induced neurotoxicity.ACEE can protect neuronal cells from B[a]P-induced cell damage, including the induction of cell cycle normalization through the AHR/CYP1A1/CCND1 signaling pathway.ACEE also utilizes lifespan extension and xenobiotic detoxification via the cyp-35 and hxk pathway.Additionally, clionasterol might be the prominent phytochemical that inhibits CYP-induced B[a]P's metabolism into toxic metabolites.Essentially, ACEE could be advanced as an agent for the protection of B[a]P-induced xenobiotic toxicity in neuronal cells and C. elegans.

Nutrients 2023 , 26 Figure 1 .
Figure1.The extraction process of Aquilaria crassna (AC) leaves.Leaves of AC were collected, dried, grinded, and extracted with absolute ethanol using the maceration method.Ethanol was removed using rotary evaporation.The ethanolic extract of AC (ACEE) stock solution (100 mg/mL in DMSO) was prepared and kept at −20 °C.

Figure 1 .
Figure 1.The extraction process of Aquilaria crassna (AC) leaves.Leaves of AC were collected, dried, grinded, and extracted with absolute ethanol using the maceration method.Ethanol was removed using rotary evaporation.The ethanolic extract of AC (ACEE) stock solution (100 mg/mL in DMSO) was prepared and kept at −20 • C.

Figure 3 .
Figure 3.The effect of Aquilaria crassna ethanolic extract (ACEE) on the cell cycle progression.A flow cytometer was used to carry out the quantitative determination based on propidium iodide (PI) staining.

Figure 4 .Figure 5 .
Figure 4.The effect of Aquilaria crassna ethanolic extract (ACEE) on cell cycle-associated gene expression in SH-SY5Y cells.Aryl hydrocarbon receptor (AHR) (a), cytochrome P450 1A1 (CYP1A1) (b), and cyclin D1 (CCND1) (c) relative mRNA expressions were quantified using Real Time qRT-PCR.All data are represented as the mean ± SEM, * p < 0.05 vs. control and # p < 0.05 vs. 40 µM B[a]P-treated group.Moreover, Figure 5 shows the protein expression of CYP1A1 and cyclin D1 in SH-SY5Y cells was notably increased when cells were treated with 40 µM B[a]P alone (* p < 0.05 vs. control).The relative protein expression of CYP1A1 and cyclin D1 compared to the

Figure 6 .
Figure 6.The effect of B[a]P and Aquilaria crassna ethanolic extract (ACEE) on C. elegans lifespan

Figure 7 .
Figure 7.The effect of various doses of B[a]P and Aquilaria crassna ethanolic extract (ACEE) on body length.The mean ± SEM values of body length are shown, * p < 0.05 vs. control; # p < 0.05 vs. B[a]Pfed worms.

Figure 7 .Figure 8 .
Figure 7.The effect of various doses of B[a]P and Aquilaria crassna ethanolic extract (ACEE) on body length.The mean ± SEM values of body length are shown, * p < 0.05 vs. control; # p < 0.05 vs. B[a]P-fed worms.Not only body length assay, but as seen in Figure 8, we showed that 300 µM B[a] would significantly reduce both the body length and body size of worms in the B[a]P-treated group (0.78 ± 0.03-fold change (p value = 0.000) and 0.69 ± 0.03-fold change (p value = 0.000), respectively, * p < 0.05 vs. control).After cotreatment 300 B[a]P and 5 µg/mL ACEE, ACEE could attenuate the toxic effect of B[a]P and cause the improvement in the body length (0.89 ± 0.02-fold change (p value = 0.000); Figure 8b) and body size of adult day 1 worms (0.80 ± 0.04-fold change (p value = 0.000); Figure 8c) compared with B[a]P-treated group ( # p < 0.05 vs. 300 µM B[a]P alone).Nutrients 2023, 15, x FOR PEER REVIEW 13 of 26

Figure 8 .
Figure 8.The effect of the ethanolic extract of Aquilaria crassna on B[a]P induced the reduction in body length and body size.Images were taken using a 10× objective lens of brightfield microscope, and representative images are shown (a) the mean ± SEM values of body length (b), and body size (c) are shown, * p < 0.05 vs. control; # p < 0.05 vs. 300 µM B[a]P-fed worms.

Figure 11 .
Figure 11.The results of the molecular docking study of cytochrome P450 1A1 (CYP1A1) (4I8V) are represented by the 2D diagrams of phytochemical-receptor interactions.

Figure 11 .
Figure 11.The results of the molecular docking study of cytochrome P450 1A1 (CYP1A1) (4I8V) are represented by the 2D diagrams of phytochemical-receptor interactions.

Figure 12 .
Figure 12.The results of the molecular docking study of hexokinase II (5HG1) are represented by the 2D diagrams of phytochemical-receptor interactions.

Figure 12 .
Figure 12.The results of the molecular docking study of hexokinase II (5HG1) are represented by the 2D diagrams of phytochemical-receptor interactions.

Table 1 .
Primer sequences for the quantitative polymerase chain reaction (qPCR).

Table 2 .
Proposed phytochemical constituents in the ethanolic extract of Aquilaria crassna (ACEE) compared with the National Institute of Standards and Technology (NIST) database.

Table 3 .
The percentage of cell numbers measured by analytical flow cytometry.

Table 3 .
The percentage of cell numbers measured by analytical flow cytometry.

Table 4 .
Results and statistical analyses of lifespan of C. elegans treated with high B[a]P and Aquilaria crassna ethanolic extract (ACEE).

Table 6 .
Docking results of the compounds with hexokinase II.

Table 6 .
Docking results of the compounds with hexokinase II.