Toxicity and Its Mechanism Study of Arecae semen Aqueous Extract in Wistar Rats by UPLC-HDMS-Based Serum Metabolomics

Background Arecae semen (AS) is officially recorded in Chinese Pharmacopoeia and it is known for its multiple functions, including antidepressive, antioxidant, anti-inflammatory, and cholesterol-lowering effects, which have been confirmed by modern pharmacological study. Previous study in our laboratory showed that long-term oral administration of Arecae semen (AS) is officially recorded in Chinese Pharmacopoeia and it is known for its multiple functions, including antidepressive, antioxidant, anti-inflammatory, and cholesterol-lowering effects, which have been confirmed by modern pharmacological study. Previous study in our laboratory showed that long-term oral administration of Hypothesis. The aim of this work was to characterize the metabolome, evaluate the metabolic changes, and study the mechanisms of the toxicity induced by different treatment doses of ASAE via metabolomics. Methods Wistar rats were administered orally two different doses of ASAE (1500 and 4500 mg/kg/d) for 30 days. The investigation was carried out to evaluate the safety of ASAE. And, the UPLC-HDMS-based serum metabolomics in conjunction with multivariate statistical techniques was applied to investigate the serum metabolite profile and potential markers of toxicity induced by different doses of ASAE. Results Coupled with blood biochemistry and histopathology results, the significant difference in metabolic profiling was observed between 1500 and 4500 mg/kg/d dosages of ASAE-treated rats and normal rats by using pattern recognition analysis, indicating that changes in serum metabolites must have occurred. Some significant changed metabolites such as arachidonic acid, linoleic acid, stearic acid, and LPC (18 : 1) have been found and identified. These biochemical changes in serum metabolites are related to the perturbation of linoleic acid metabolism, arachidonic acid metabolism, glycerophospholipid metabolism, and purine metabolism, which may be helpful to further understand the cardiotoxicity and neurotoxicity of ASAE. Conclusion The study shows that the metabolomic method may be a valuable tool for studying the essence of toxicity induced by traditional Chinese medicine.


Introduction
Metabolomics is commonly applied to discover potential biomarkers and biological mechanistic information by charactering the metabolic profiling of low-molecular-weight metabolites in biosamples [1]. It offers an alternative method for monitoring the biochemical changes associated with the generation and development of the influences resulting from endogenous and exogenous factors [2]. e change of all metabolites rapidly determined in biological samples, such as serum, plasma, and urine, can reflect the physiological status of the organism systematically. us, metabolomics approach has been enormously used in toxicology research [3,4], biomedical, as well as nutritional studies.
Toxicity is a major concern in the treatment of TCM. e constituents of TCM are highly complex. And TCM has the feature of integrity and systemic, which is well coincident with the systemic thinking and strategy of the metabolomics [5]. Metabolomics can be used to analyze the differences of the blood and urine metabolites of different TCM syndromes, which can make the diagnosis of TCM syndromes more scientific and quantitative, and the uncertainty of the subjective factors can be avoided. As a convenient and effective tool, metabolomics methods have been used in the evaluation of the toxicology of TCM [2,6,7].
It is really a great challenge for identifying the metabolites in complex mixtures. Advanced analytical technologies play a significant role in comprehensive endogenous metabolite profiling. Due to the high sensitivity and selectivity, UPLC-HDMS has become a rapid and reliable tool for metabolomics studies, which has been widely used in different laboratories around the world [8].
AS, the seed of Areca catechu Linn. (Family: Palmaceae) Palm, has been widely used for medicinal purposes for more than 2000 years in South Asian countries [9]. AS is one of the clinically used common drugs for the treatment of gastrointestinal, edematous, and parasitic diseases [10]. Modern pharmacological studies demonstrated that AS has good antidepressive, antioxidant, anti-inflammatory, antiaging, analgesic, and plasma cholesterol-lowering effects [11][12][13][14][15]. Recent researches indicated that arecoline, the major alkaloid in AS, is an agonist for the muscarinic receptor, and has a stimulating effect on parasympathetic and cardiovascular system [16], which is the major source of toxicity at the same time [17]. e common adverse reactions of preparations of AS included nausea and vomiting, abdominal pain, and dizziness [18]. e LD 50 value of ASAE in mice was 3.67 g/kg (p.o.) [17]. Furthermore, the ASAE (200 μg/mL) and arecoline (10 μg/mL) could significantly upregulate the mRNA expression of the c-jun proto-oncogene in oral mucosal fibroblasts, indicating that persistent induction of c-jun proto-oncogene by the areca nut and arecoline may be one of the mechanisms for oral squamous cell carcinoma [19,20]. In addition, it has been reported that the ASAE (40-80 μg/ mL) causes a significant proliferative effect on oral mucosal fibroblasts (OMF) via upregulation of the expression of ICAM-1 [21,22]. ASAE (5 and 20 mg/kg) and arecoline (5 and 20 mg/kg, p.o.) also increase the serum levels of ALT, AST, and ALP in mice [23,24]. Besides, it has been reported that ASAE resulted in signs of reproductive toxicity including decreases in sperm counts and sperm motility and the induction of substantial abnormalities in sperm morphology [25][26][27]. Our previous study showed that ASAE at the dose of 750 mg/kg/day was safe, while long-term (30 days) oral administration of ASAE at higher dosage levels (1500 mg/kg and 4500 mg/kg) showed dose-dependent toxic effects [28]. Besides, we focused on the toxicity induced by different duration of administration (7-day, 14-day, and 30day) of ASAE at a dose of 4500 mg/kg/d. It indicated that Arecae semen possessed time-dependent cardiotoxicity and the metabolomics approach is a useful tool to study the toxicity in TCM [29].
In this study, UPLC-HDMS-based serum metabolomics combined with multivariate statistical techniques was used to characterize the serum metabolite profile and evaluate the metabolic changes and potential markers of toxicity induced by different doses of of ASAE (1500 and 4500 mg/kg/d). e results may provide valuable and in-depth information about the toxic effects of AS on Wistar rats, which will be beneficial for future safe application of this herb in clinic.

AS Aqueous Extract
Preparation. 25 kg AS smashed into coarse particle using hammer mill, was extracted with 150 L distilled water for 1 h, and filtered. en, the supernatant was collected for further processing and the residue was extracted with the same process. e aqueous extract was combined and evaporated under vacuum to yield the dark brown extract (1 g of the extract corresponding to 4.91 g of AS). en, the ASAE was stored in an amber bottle and kept at − 80°C until further use. e ASAE was thawed and freshly prepared with distilled water based on the daily requirement before oral administration. And the quantification of chemical compounds by HPLC analysis was shown in Supplementary Materials.

Animals and Treatment.
Adult male Wistar rats were purchased from SPF (Beijing) Biotechnology Co., Ltd., Beijing, China. e animals were provided with commercial food and water ad libitum at the standard laboratory condition (temperature of 25°C ± 2°C, relative humidity of 55 ± 5% and 12 h light-dark cycle). After 5 days of acclimatization, the rats (180-200 g) were divided into one control group and two treated groups (n � 6 in each group). During the 30-day treatment period, the control group was orally administrated with distilled water, and the treated groups were administrated with ASAE at 1500 and 4500 mg/ kg/day based on body weight. According to the Chinese Pharmacopoeia, the maximal dose of AS is 60 g/day for an adult (70 kg). In view of the results of preliminary experiment and the purpose of exhaustively exploring the toxicity of AS, the dosages of ASAE were set at 1500 and 4500 mg/kg/ day, which were 9 and 27 times of the clinical dosage, respectively. e rats were weighed every 3 days. Mortality and clinical reaction of all the rats from different groups were observed before and after administration including death,

Sample Collection and Preparation.
After 30 days of treatment, the rats were fasted overnight but allowed to access water freely before the autopsy. Finally, the blood samples were obtained from abdominal aorta, and the different tissues were rapidly removed for pathological staining and assay under anesthesia (pentobarbital, 1 mL/kg body weight, i.p.). e serum obtained from blood samples by centrifugation (3000 rpm, 10 min, 4°C) was stored in the refrigerator at − 80°C. e serum sample from each rat was divided into two parts, one for UPLC-HDMS analysis and the other for biochemical analysis.

Serum Biochemistry.
e serum samples for biochemistry test were thawed at room temperature for biochemical examination. ALT and AST activities were measured using the corresponding detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), which were used to determine the effect of ASAE on liver. BUN and Cr activities were inspected by using kits (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), which could reflect the effect of ASAE on nervous system. CK detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to evaluate the effect of ASAE on heart.
2.6. Histopathology. After dissection to remove the fat and connective tissue, the following organs were obtained: liver, kidney (the right), spleen, heart, and lungs. All the organs were visually inspected and then fixed in 10% neutral buffered formalin at room temperature, gradually dehydrated in ethanol, and embedded in paraffin. Paraffin blocks were cut into 4 μm sections, and the sections were stained with hematoxylin and eosin (H&E). e sections were examined under an Olympus PROVIS AX70 light microscope (Tokyo, Japan) to detect any morphological changes in the tissue using a Nikon camera.

Chromatographic Separation and Mass Spectrometry.
Prior to analysis, the serum samples were thawed at room temperature. Acetonitrile (600 μL) was added into serum (200 μL) and vortex-mixed for 3 min for protein precipitation. After centrifugation at 14,000 rpm for 10 min at 4°C, the supernatant (600 μL) was pipetted out and dried with nitrogen. e residues were dissolved in 100 μL acetonitrile: water (80 : 20). en, the samples were vortex-mixed for 3 min and the supernatant was collected for UPLC-HDMS analysis after centrifugation (14000 rpm, 10 min, 4°C).
Mass spectrometry was performed on an LTQ-Orbitrap mass spectrometer ( ermo Scientific, Bremen, Germany) connected to the UPLC instrument via ESI interface. Samples were analyzed in both negative mode and positive mode with the tune method set as follows: sheath gas (nitrogen) flow rate of 30 arb, aux gas (nitrogen) flow rate of 5 arb, spray voltage of 4.0 kV, capillary temperature of 350°C, capillary voltage of 25 V, and tube lens voltage of 110 V. e measured masses were within 5 ppm of the theoretical masses. e scan range was from 50 to 1200 m/z.

QC Samples and Method
Validation. An equal volume of serum from all the samples was pooled to get a pooled QC sample, and then the QC sample was processed by the same method as the sample preparation described above. e precision of instrument, method repeatability, and system stability were validated by QC samples to make sure that the method of UPLC-HDMS could meet the requirement of metabolomics. e 5 replicated injections from the same QC sample and 5 injections from the different QC samples were used to evaluate the precision of instrument and the method repeatability, respectively. e system stability was verified by injecting a QC sample every 10 samples during the analysis.

Data Analysis and Statistical Analysis.
e raw UPLC-MS data were imported into XCMS online software and then processed with default settings to complete baseline correction, peak discrimination and alignment, and retention time correction. e ion intensities or peak area for each peak detected was also normalized within each sample to the sum of the peak intensities in that sample. e unsupervised PCA and the supervised OPLS-DA were performed on SIMCA-P 13.0 (Umetrics, Umea, Sweden). e differential metabolites were selected on the basic of two criteria: (a) the value of VIP from the OPLS-DA model should be satisfied with VIP > 1 and (b) the result from the Student's t-test should meet the criteria of P < 0.05 by using SPSS 17.0 (IBM, New York, USA).
Evidence-Based Complementary and Alternative Medicine 3

HPLC Analysis.
e HPLC chromatogram of 1500 mg/ kg/d ASAE sample was included in supplementary materials ( Figure S1). e concentrations of the alkaloids in the ASAE of the 1500 mg/kg/d and 4500 mg/kg/d groups are shown in Table 1.
We used HPLC analysis to determine the contents of four alkaloids, which are the main active ingredients in AS. By doing this, we could determine whether some reactions after administration are caused by a high content of a specific compound. It also provides a preliminary basis for further studies on the four alkaloids.

General Observations.
Compared with the control group, the rats in the treated groups exhibited diarrhea, less physical activity, tremors, and body curl up, which suggested that ASAE have effects on the nervous system.
As shown in Figure 1, compared with drug-treated (1500 mg/kg and 4500 mg/kg) and control groups, a significant dose-dependent decrease was observed in the body weights of male rats, which indicated that ASAE had inhibition effects on the normal growth of rats. Table 2, there were no significant differences (P > 0.05) in the levels of AST, BUN, and Cr between the treated and control groups. e level of ALT in 4500 mg/kg group (48.50 U/L) was significantly decreased (P < 0.05) compared with the control group (67.90 U/L), which suggested that AS was beneficial for liver to some extent. Moreover, the histopathological changes of liver, kidney, lung, and spleen were not significant in the treated and control groups (Figures 2(a), 2(b), 2(d) and 2(e)), which were in accord with the biochemical results and our previous study [28]. However, other researchers had come up with AS extracts that were toxic to hepatocytes in mice [35], which were inconsistent with our study. erefore, further researches are still needed to provide more in-depth insight into the hepatotoxicity of AS. e level of CK in 4500 mg/kg group (1695.20 U/L) was significantly increased (P < 0.05) compared with the control group (996.75 U/L), which suggested that AS might possess cardiotoxicity to some extent (Table 2). However, no significant abnormal structures in heart sections were observed in the treated groups (Figure 2(c)), which may be due to the morphology changes being less sensitive than the biochemical alterations.

Validation of the UPLC-HDMS Method.
e RSD of t R and peak intensities for the selected ions from QC samples were calculated to evaluate the method performance, and the detailed information of the selected ions is shown in Table S2.

Multivariate Statistical Analysis.
e representative BPI chromatograms of the control and treated groups in both negative and positive modes (Figures 3(a) and 3(b)) were obtained from UPLC-HDMS analysis, respectively. Although some of the differences could be observed directly from the BPI chromatograms, the subtle metabolism variations still required to be further investigated by multivariate statistical analysis, such as PCA and OPLS-DA. e PCA, as an unsupervised analysis, is usually performed to visualize clustering, trends or outliers among the observations at the first step of metabolomics analysis. In Figure 4, the PCA score plot showed that the separation between control and two different doses of ASAE-treated groups was obvious in positive mode and negative mode, respectively. e results indicated that there were different metabolic profilings between the control and two different doses of ASAE-treated groups. Dose (mg/kg/d) Figure 1: Effects of ASAE on the body weight changes of rats during the treatment period study (n � 6). Values are mean ± SD. * * P < 0.01 significant difference compared with the control group (One-way ANOVA followed by Dunnett's test). Values are mean ± SD. * P < 0.05 significant difference compared with the control group (one-way ANOVA followed by Dunnett's test).

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Unlike the PCA approach, the OPLS-DA is a supervised method and frequently employed to increase the separation and search for the potential biomarkers by the contribution of the variables in group separation. As shown in Figure 5, the score plots of OPLS-DA showed that there were clear group discriminations and dose-dependent dynamic changes that gradually moved far away from the control group position, no matter in negative mode or positive mode. e model statistics indicated no signs of overfitting, and the model was valid. e results suggested that compared with the control, the variance of metabolites in treated rats was enlarged over different doses of ASAE. Compared with the control group, 196 and 158 differential metabolisms (VIP > 1 and P < 0.05) were screened from the 1500 mg/kg/d and 4500 mg/kg/d doses of ASAE-induced groups in the ESI negative ion mode, respectively. Besides, 217 and 233 biomarkers were screened from the low and high dose of ASAEinduced groups in the ESI positive ion mode, respectively. Considering the stability of the biomarkers at different doses of ASAE in the treated groups, Venn diagram was used to separately integrate the metabolites information. At last, 63 and 47 metabolites were selected as the candidates in the negative mode and positive mode, respectively (Table S3 and Figure 6).

Identification of Biomarkers.
Metabolite peaks were assigned by MS E analysis or interpreted with available biochemical databases, such as METLIN (https://metlin. scripps.edu/), HMDB (http://www.hmdb.ca/), ChemSpider (http://www.chemspider.com/), and MassBank (https:// massbank.eu/MassBank/). In the Supplementary Materials, a potential biomarker with an m/z of 255.2332 is used as an example to illustrate the identification process ( Figure S2). By using the same method described, 16 metabolites (12 from the negative mode and 4 from the positive mode) were identified and tentatively regarded as the potential biomarkers in the treated groups induced by ASAE (Table 3).

Biochemical Interpretation.
MetPA is a part of many functions in MetaboAnalyst network database. It can visualize the metabolic pathway information of potential biomarkers with the help of METLIN, HMDB, and KEGG database. In this study, sixteen potential biomarkers were identified in the ASAE-treated groups using the UPLC-HDMS based on serum metabolomics. e 16 identified potential biomarkers in this experiment were analyzed by MetPA, and the results are shown in Figure 7 and Table 4.

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And the related pathway of those biomarkers included linoleic acid metabolism, arachidonic acid (AA) metabolism, glycerophospholipid metabolism, and purine metabolism.

Discussion
In our study, the techniques of metabolomics were combined with phenotypic responses and biochemical assays to confirm the target organ and understand the toxic effects of AS in rats. e interpretation of the metabolism contributes to further understand the toxicity of ASAE in different doses and explore its underlying mechanism. According to the result of HPLC analysis, the content of guvacine was a lot higher than that of three other alkaloids. Based on the LC 50 of four alkaloids on zebrafish in our laboratory, the toxicity of guvacine is inferior to arecoline, which is the most toxic. ere are few reports about the toxicity of guvacine. But for arecoline, it has been widely studied due to its carcinogenic potential [20,[37][38][39][40][41]. Besides, it has parasympathetic effect, including euphoria, central nervous system stimulation, vertigo, salivation, miosis, tremor, and muscarinic effects [42]. erefore, we deduced that the cardiotoxicity of AS described in this study might be largely attributable to arecoline.
Linoleic acid, an important polyunsaturated fatty acid in brain tissue is related to symptoms of growth retardation [43], cardiac toxicity [44], and neurotoxicity. In this experiment, compared with the control group, the body weight changes of rats in treated groups showed significant dosedependent decreases. During the 30 days of oral administration of ASAE, food and water were supplied sufficiently and all the rats were given free diet. erefore, we could infer that it is the AS inducing the toxicity of growth retardation in  the treated groups, which may cause the difference of endogenous metabolites. In this work, the AS-treated rats exhibited tremors and bodies curled up in a dose-dependent manner. ese neurotoxicity symptoms may be attributed to the reduction of linoleic acid. Moreover, the level of CK increased significantly in the AS-treated groups, suggesting that the AS exhibited cardiotoxicity to some extent. Arachidonic acid, a derivative of linoleic acid, is an essential polyunsaturated fatty acid, which cannot be synthesized in the body, but must be obtained from the daily diet [45]. As a constituent exists mainly in the form of phospholipid in cell membrane, AA is the substrate for the synthesis of a range of biologically active compounds including prostaglandins, which can cause diarrhea by stimulating gastrointestinal smooth muscle. In our study, compared with the control group, the level of AA was significantly downregulated in a dose-dependent manner, which may be the major cause of diarrhea.
Glycerophospholipids (GPs), which are the critical components of the lipid bilayer of cellular membranes, play a key role in cell signaling and metabolism. Depending on the different substituents at the sn-3 position of the glycerol backbone, GPs fall into PC, PE, PG, PS, PI, PA, and cardiolipins [46,47]. LPC is generated by hydrolysis or oxidation of PC [48]. Studies have demonstrated that acceleration of the lipid peroxidation of the cardiac systems may lead to heart arrhythmia [49,50]. LPCs, as biomarkers of lipid peroxidation, were positively related to the level of lipid peroxidation [51,52], which plays an important role in the pathogenesis of cardiac tissue [49]. In our study, compared with the control group, the level of LPCs was significantly upregulated in a dose-dependent manner. is result was consistent with the increased level of CK in the AS-treated groups (Table 2), which confirmed that the AS caused cardiotoxicity to some extent. And this result was in accordance with our previous study [29]. Nevertheless, AS, the seed of Areca catechu Linn, contains lipids as well. Objectively speaking, we could not rule out the possibility that the increased LPC in the blood may be from the absorbed constituents of AS.
Uric acid is the final product of purine metabolism. A growing body of evidence suggested that high levels of serum uric acid serve also as biomarkers for cardiovascular disease morbidity and mortality [53]. In our study, compared with the control group, the level of uric acid was upregulated in AS-treated rats (4500 mg/kg/d).
is result also demonstrated that the AS causes cardiotoxicity to a certain degree. Moreover, it is reported that dysregulation of purine metabolism may be one of the causes of mental retardation or unexpected and often devastating neurological dysfunction [54], leading to tremors and body curl up.

Conclusions
In this study, by using biochemistry and histopathology methods, we found that long-term oral administration of ASAE caused cardiotoxicity and neurotoxicity in rats in a dose-dependent manner. Moreover, 16 potential biomarkers such as arachidonic acid, linoleic acid, stearic acid, LPC (18 : 1), and LPC (18 : 2) were identified in the serum of ASAE-treated rats by using metabolomics studies combined with multivariate data analysis.
ose biomarkers are related to the perturbations of linoleic acid metabolism, arachidonic acid metabolism, glycerophospholipid metabolism, and purine metabolism.
e detailed effect of AS on the mentioned metabolic pathways requires further research, and the insights gained are just preliminary in nature to elucidate the metabolic pathways that should receive enlarged interest in further studies. Genomics, transcriptomics and proteomics are needed to comprehensively study the underlying toxicological mechanisms of ASAE.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest regarding the publication of this article.