Screening and Identification of the Metabolites in Rat Plasma and Urine after Oral Administration of Areca catechu L. Nut Extract by Ultra-High-Pressure Liquid Chromatography Coupled with Linear Ion Trap–Orbitrap Tandem Mass Spectrometry

Areca catechu L. nut, a well-known toxic traditional herbal medicine, has been widely used to treat various diseases in China and many other Asian countries for centuries. However, to date the in vivo absorption and metabolism of its multiple bioactive or toxic components still remain unclear. In this study, liquid chromatography coupled with tandem mass spectrometry was used to analyze the major components and their metabolites in rat plasma and urine after oral administration of Areca catechu L. nut extract (ACNE). A total of 12 compounds, including 6 alkaloids, 3 tannins and 3 amino acids, were confirmed or tentatively identified from ACNE. In vivo, 40 constituents, including 8 prototypes and 32 metabolites were identified in rat plasma and urine samples. In summary, this study showed an insight into the metabolism of ACNE in vivo, which may provide helpful chemical information for better understanding of the toxicological and pharmacological profiles of ACNE.


Introduction
Traditional Chinese medicine (TCM) has played an important role in preventing or treating a variety of complicated diseases over thousands of years [1][2][3].It is extensively accepted that the therapeutic or toxic effect of TCM results from the prototype components and/or their metabolites [4,5].However, profiling the absorbed components and metabolites of an herbal medicine in vivo is always a great challenge [6].Metabolites exist in a variety of forms and are usually present at trace levels; therefore, the signals from the metabolites are often masked by background noise from endogenous interference [7].Owing to its high selectivity and sensitivity, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has been extensively used in drug metabolism studies.LC-MS/MS combined with sophisticated software tools can offer abundant structural information and accurate mass measurement for both precursor and product ions, which represents a powerful and reliable analytical technique to detect and identify unknown metabolites in complex matrixes [8][9][10][11][12].
Areca catechu L. one of most popular medicinal herbs, is widely distributed in the south of China.The nut of Areca catechu L. has been recorded in the pharmacopoeia of China for the treatment of parasitic diseases, dyspepsia, abdominal pain, etc. [13].Modern pharmacological studies and clinical practice revealed that Areca catechu L. nut shows a variety of pharmacological functions, including wound healing [14], anti-migraine effect [15], anti-depressant effect [16], and hypoglycemic [17] and antioxidant effects [18].As a toxic herbal medicine, Areca catechu L. nut also has some potential toxicities, such as decline of sperm count sand motility and induction of substantial abnormalities [19,20].Although many reports regarding the pharmacological activities and potential toxicities have been published as described above, very little is known about its absorbed components and metabolites in vivo.
Hence, the overall objective of this research was to investigate in detail the in vivo absorption and metabolism of Areca catechu L. nut extract (ACNE).As we all know, plasma and urine are recognized as ideal biological matrices in drug absorption and metabolism studies [21,22].In this work, a total of 26 components in the plasma and 31 components in the urine were characterized by ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap). From the results, we can know the probable metabolic pathways of chemical constituents in ACNE.To the best of our knowledge, this is the first study on screening the multiple absorbed and metabolic components of ACNE in vivo, which could provide a scientific basis for explaining the curative and toxicological mechanism of ACNE.

Identification of the Major Components in ACNE by UHPLC-LTQ-Orbitrap
A total of 12 compounds were separated and identified according to the retention time, obtained mass spectra, and by comparing with data from literature and standard sample.All of them were attributed to three types, including 6 alkaloids, 3 tannins and 3 amino acids.Detailed identified information is listed in Table 1.The detailed extracted ion chromatography of ACNE is shown in Figure 1 and Figure S1 in the Supplemental material.The structures of the 12 compounds are shown in Figure 2.
LC-MS/MS combined with sophisticated software tools can offer abundant structural information and accurate mass measurement for both precursor and product ions, which represents a powerful and reliable analytical technique to detect and identify unknown metabolites in complex matrixes [8][9][10][11][12].
Areca catechu L. one of most popular medicinal herbs, is widely distributed in the south of China.The nut of Areca catechu L. has been recorded in the pharmacopoeia of China for the treatment of parasitic diseases, dyspepsia, abdominal pain, etc. [13].Modern pharmacological studies and clinical practice revealed that Areca catechu L. nut shows a variety of pharmacological functions, including wound healing [14], anti-migraine effect [15], anti-depressant effect [16], and hypoglycemic [17] and antioxidant effects [18].As a toxic herbal medicine, Areca catechu L. nut also has some potential toxicities, such as decline of sperm count sand motility and induction of substantial abnormalities [19,20].Although many reports regarding the pharmacological activities and potential toxicities have been published as described above, very little is known about its absorbed components and metabolites in vivo.
Hence, the overall objective of this research was to investigate in detail the in vivo absorption and metabolism of Areca catechu L. nut extract (ACNE).As we all know, plasma and urine are recognized as ideal biological matrices in drug absorption and metabolism studies [21,22].In this work, a total of 26 components in the plasma and 31 components in the urine were characterized by ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap). From the results, we can know the probable metabolic pathways of chemical constituents in ACNE.To the best of our knowledge, this is the first study on screening the multiple absorbed and metabolic components of ACNE in vivo, which could provide a scientific basis for explaining the curative and toxicological mechanism of ACNE.

Identification of the Major Components in ACNE by UHPLC-LTQ-Orbitrap
A total of 12 compounds were separated and identified according to the retention time, obtained mass spectra, and by comparing with data from literature and standard sample.All of them were attributed to three types, including 6 alkaloids, 3 tannins and 3 amino acids.Detailed identified information is listed in Table 1.The detailed extracted ion chromatography of ACNE is shown in Figure 1 and Figure S1 in the Supplemental material.The structures of the 12 compounds are shown in Figure 2.          + .According to the literature data [27], they were tentatively designated as catechin (C) and epicatechin (EC), respectively.The proposed fragmentation pathway of catechin is shown in Figure 3b.

Identification of the Major Metabolites in ACNE by UHPLC-LTQ-Qrbitrap
The identification of the compounds in rat urine and plasma were also performed by UHPLC-LTQ-Qrbitrap.Altogether, 40 compounds, including 8 prototype and 32 metabolites were identified by comparing the retention time and the mass data (Table 2).Among them, 31 of them were from rat urine while 26 were from rat plasma.Detailed extracted ion chromatograms of the urine sample are shown in Figure 4, and the plasma sample are shown in Figure 5.In addition, the metabolic pathways of catechin (C) and arecoline are summarized in Figure 6.Areca catechu L. nut extract (5 g/mL) was diluted to a concentration of 10 mg/mL with water, and then the solution was filtered through a 0.22 µm membrane (pore size).

Animal and Sample Collection
Sprague-Dawley rats (male, 200-250 g) were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.(Beijing, China).All animals were kept in an environmentally controlled breeding room at 23 • C, 60 ± 5% humidity with room-lights alternated on a 12-h dark-light cycle.Water and standard diet were provided ad libitum.The animal facilities and protocols were approved by the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine.
Rats were randomly separated into two groups of three animals each and were acclimated in metabolic cages for three days.Prior to the experiment, the rats were fasted overnight with unlimited access to water.Then, the treatment group rats were given ACNE intragastrically at a single dose of 50 g/kg (crude drug weight/rat weight).Blank control group rats were orally administered with equal dose of saline at the same time.The animals were continuously administered twice per day.Then, 1.5 h after the seventh drug administration, the animals were anesthetized with chloral hydrate (400 mg/kg) by intraperitoneal injection.Then, blood samples were collected from the abdominal aorta.Urine samples were collected every 12 h after the first drug administration, then, all urine samples from each animal were combined into one sample.After the experiments, all rats were sacrificed by performing a bilateral thoracotomy.

Sample Preparation
Each blood sample was centrifuged (5000 rpm, 4 • C ) for 10 min to obtain the plasma, and then treated by addition of methanol in the ratio of 1:3 (v/v) to precipitate protein.The mixture was performed by vortexing for 10 min and centrifuging (10,000 rpm, 4 • C) for 15 min.The supernatant was transferred to another clean tube, and dried under a gentle flow of nitrogen at 40 • C. Then the dried residue was reconstituted with 200 µL of methanol for UHPLC-LTQ-Orbitrap analysis.
Each urine sample (10 mL) was concentrated to the final volume of 5 mL in a vacuum.Then, 500 microL urine were mixed with 1.5 mL of methanol and sonicated in an ultrasonic bath for 0.5 h, and then centrifuged (10,000 rpm, 4 • C) for 10 min.The supernatant was transferred to another clean tube, and dried under a gentle flow of nitrogen at 40 • C. The dried residue was reconstituted with 200 µL of methanol for UHPLC-LTQ-Orbitrap analysis.

Instrumentation and Analytical Conditions
Sample analyses were performed using an ultimate 3000 LC system coupled to an LTQ Orbitrapmass spectrometer via an electrospray ionization (ESI) interface.The chromatography system consisted of an autosampler, a diode array detector, a column compartment and two pumps.Xcalibur, Metworks and Mass Frontier 7.0 software packages (Thermo Corporation, Waltham, MA, USA) were employed for data collection and data analysis.
The ESI source parameters were as follows: the capillary temperature was 250 • C, source voltage and the spray voltage were set at 5 kV, and sheath gas (N 2 ) flow was 35 psi.The ESI source was operated in both negative and positive ionization mode.In the Fourier transform (FT) cell, full MS scans were acquired in the range of m/z 50-2000.The MS/MS experiments were set as data-dependent scans.

Figure 2 .
Figure 2. The structures of the 12 compounds.Figure 2. The structures of the 12 compounds.

Figure 2 .
Figure 2. The structures of the 12 compounds.Figure 2. The structures of the 12 compounds.

Figure 3 .
Figure 3.The proposed fragmentation pathways of arecoline (a); catechin (b) and valine (c) in positive ion mode.

Compound 9 ( 8 .
68 min, −1.334 ppm) exhibited the [M + H] + ion at m/z 579.1489, and gave rise to four major product ions at m/z 453.1197, 427.1039, 409.0937 and 291.0874.The fragment ion at m/z 409.0937 was generated through the loss of H 2 O from the ion at m/z 427.1039; the fragment ion at m/z 427.1039 was attributed to the retro Diels-Alder (RDA).The ion at m/z 291.0874 [M + H-C 15 H 13 O 6 ] + was a prototype losing an aggregate unit.According to the literature data[28], component 9 was tentatively characterized as procyanidin B2. 2.1.3.Amino Acid Component 1 (1.36 min, -1.568 ppm) displayed the molecular ion at m/z 118.0861 which led the major product ion at m/z 72.0806 [M + H-HCOOH] + .By comparing with the reference compound, component 1 was identified as valine.The proposed fragmentation pathway of valine is shown in Figure 3c.Compound 5 (3.05 min, −0.822 ppm) exhibited [M + H] + ion at m/z 182.0810 and the main fragment ions of it were at m/z 165.0548 and 136.0760.The former ion at m/z 165.0548 was produced from the neutral loss of NH 3 , and the latter ion at m/z 136.0760 was generated through the loss of HCOOH from the molecular ion.Hence, compound 5 was tentatively identified as tyrosine.Compound 11 (9.53 min, −0.996 ppm) showed the [M + H] + ion at m/z 205.0970 and it yielded major ions at m/z 188.0917 [M + H-NH 3 ] + and 159.9331 [M + H-HCOOH] + .Based on these data, component 11 was tentatively assigned as tryptophan.

Figure 4 .
Figure 4. Extracted ion chromatograms of the urine sample in positive ion mode (a); the blank urine sample in positive ion mode (b); the urine sample in negative ion mode (c); the blank urine sample in negative ion mode (d).

Figure 4 .
Figure 4. Extracted ion chromatograms of the urine sample in positive ion mode (a); the blank urine sample in positive ion mode (b); the urine sample in negative ion mode (c); the blank urine sample in negative ion mode (d).

Figure 5 .
Figure 5. Extracted ion chromatograms of the plasma sample in positive ion mode (a); the blank plasma sample in positive ion mode (b); the plasma sample in negative ion mode (c); the blank plasma sample in negative ion mode (d).

Figure 5 .
Figure 5. Extracted ion chromatograms of the plasma sample in positive ion mode (a); the blank plasma sample in positive ion mode (b); the plasma sample in negative ion mode (c); the blank plasma sample in negative ion mode (d).

Table 1 .
The mass spectral data of components observed by UHPLC-LTQ-Orbitrap.