Bornyl gallate (BG) is a potential drug candidate synthesized by the reaction of two natural products, gallic acid and borneol. Previous studies have strongly suggested that BG is worthy of further investigation due to antioxidant, antiatherosclerosis activities, and obvious activity of stimulating intersegmental vessel growth in zebrafish. This work was designed to elucidate the metabolic profile of BG through analyzing its metabolites in vitro and in vivo by a chromatographic separation coupled with a mass spectrometry. The metabolites of BG were characterized from the rat liver microsome incubation solution, as well as rat urine and plasma after oral administration. Chromatographic separation was performed on an Agilent TC-C18 column (250 mm × 4.6 mm, 5
Traditional Chinese medicine (TCM), which serves as a resource of bioactive compounds for drug discovery, is attracting increasing global attention [
Gallic acid (3,4,5-trihydroxybenzoate) (Figure
Chemical structures of gallic acid (a), borneol (b), and bornyl gallate (c).
Metabolite identification is becoming increasingly important in the early stage of drug discovery as a basis for judging whether or not a drug candidate merits further development [
In order to predict the safety and efficacy of BG, it is extremely important to identify its metabolites and thoroughly understand its metabolic fate. Therefore, we firstly analyzed the in vitro metabolites of BG after incubating with rat liver microsome (RLM), subsequently investigated the metabolic profiles of BG in rat plasma and urine, and tentatively identified in vivo metabolites by comparing MS/MS fragment patterns and change of molecular mass with those of the parent drug.
Bornyl gallate (purity: >99%, HPLC) was synthesized and identified by 1HNMR, IR, and LC/Q-TOF/MS in our laboratory. HPLC grade methanol was purchased from Fisher Chemical Co., Inc. (CA, USA). HPLC grade formic acid was purchased from Kermel Chemical Reagent Co., Ltd. (Tianjin, China).
Male Sprague-Dawley rats (200–220 g) were purchased from the Laboratory Animal Research Center of Xi’an Jiaotong University (Shaanxi, China). The rats were kept in metabolic cages in a breeding room with temperature at
Rats were starved overnight before sacrificed. Minced livers were homogenized in 4 × volume of microsome buffer (pH 7.4) containing 0.1 M potassium phosphate, 10% sucrose, 0.1 mM EDTA, 2 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride prior to be centrifuged at 9,000 ×g for 30 min (4°C). Then, the supernatant was further centrifuged at 100,000 ×g for 60 min (4°C). The resulted microsomal pellet was resuspended in fresh microsome buffer and centrifuged again (100,000 ×g, 60 min, 4°C). The collected RLM was dissolved in fresh microsome buffer and immediately stored at −80°C until next use.
Incubations were performed at 37°C in a system containing 3.06 mg/L BG, 0.5 mg/mL rat liver microsomal protein in 0.5 mL of 0.1 mol/L phosphate buffer (pH 7.4). After preincubation at 37°C for 3 min, the reaction between BG and RLM was started by adding a NADPH-regenerating buffer consisting of 1.3 mM
Five male Sprague-Dawley rats were starved overnight with free access to water. Blank blood and urine from each rat were collected prior to dosing. The BG was suspended in 0.5% CMC-Na and orally administered to rats at a dose of 50 mg/kg. 0.5 mL blood samples were collected through ophthalmic veins using heparinized tubes under anesthesia at 1 h after dose. Plasma was prepared by centrifuging the blood for 10 min at 8,000 ×g. Urine samples were collected individually during the time period 0–12 h. The plasma and urine samples were stored at −20°C before further preparation.
All the samples were thawed at room temperature. 0.2 mL acetonitrile containing 0.5% formic acid was added into 0.2 mL plasma or urine. The mixture was thoroughly swirled for 2 min and then centrifuged at 8,000 ×g for 10 min to remove protein in the sample. The supernatant was filtered by 0.45
Chromatographic experiments were performed on an Agilent 1200 series HPLC system, equipped with binary pump, autosampler, on-line degasser and automatic thermostatic column oven (CA, USA). HPLC separation was achieved on an Agilent TC-C18 column (4.6 mm × 250 mm, 5
To identify the metabolites in the elution, the HPLC system was coupled online to an Agilent 6500 series quadrupole-time of flight mass spectrometer (Q-TOF/MS), equipped with a dual electrospray ionization source (Dual-ESI) (CA, USA). The LC effluent was introduced into the ESI source in a postcolumn splitting ratio of 3 : 1. Mass spectra were acquired in negative ion mode with the mass range set at m/z 100–1000. The conditions used for the ESI source included a capillary voltage of 4000 V, a drying gas temperature of 350°C, a drying gas flow of 10 L/min, and a nebulizer pressure of 35 psi as well as a fragmentor voltage of 125 V. Internal reference masses in negative mode were set at m/z 112.9855 and 966.0007. MassHunter Workstation software from Agilent Technologies (CA, USA) was used for data acquisition and processing in full-scan and targeted MS/MS modes.
The HPLC-MS conditions were optimized to provide a full overview of the pattern of the metabolites in rat plasma and urine after oral administration of BG. Ionization of the parent drug BG was much better in the negative mode than that in the positive mode, and the difference between the chromatograms of blank samples and those of samples after oral dosing was more noticeable in the negative mode. Therefore, metabolite identification was performed in the negative ionization mode. Under the proposed condition, the retention time of BG was determined to be 63.0 min (Figure
MS, MS/MS spectrum, and total ion chromatogram (TIC) of reference BG (a) MS spectrum; (b) MS/MS spectrum, and the predominant fragmentation pattern; (c) TIC of BG. The chromatographic separation is performed on an Agilent TC-C18 column (250 mm × 4.6 mm, 5
Compared with the negative control sample, five new compounds (M1a, M1b, M1c, M1d, and M1e) and the parent drug BG were detected in RLM incubation solution. The full-scan mass spectrum analysis revealed that the five compounds had molecular ions [M−H]− at m/z 321.1339, 321.1341, 321.1340, 321.1338, and 321.1335, respectively. All ions had identical calculated formula of C17H22O6 (masscalc. = 321.1344, error <2.7 ppm), representing the notable difference of a single oxygen atom from BG. This interesting result indicated that the new compounds found in vitro should be isomers of monohydroxylated BG. Further MS/MS spectra of each compound at m/z 321 provided same product ions at m/z 169, 168, 124 and 125, which remains same as the MS2 fragment ions of the parent drug. The full-scan mass spectrum, MS/MS spectrum, and the predominant fragmentation pattern of M1a are shown in Figure
Representative MS, MS/MS spectrum, and extracted ion chromatogram (EIC) of in vitro metabolites of BG in rat liver microsomes (RLM). Incubations were performed at 37°C for 30 min in a system containing 3.06 mg/L BG and 0.5 mg/mL liver microsomal protein in 0.5 mL of 0.1 mol/L phosphate buffer (pH 7.4). (a) MS spectrum; (b) MS/MS spectrum and the predominant fragmentation pattern of M1a are shown as the representative spectra of five metabolites, which exhibited almost identical spectra; (c) EIC of m/z 321.1339 representing 5 isomers of in vitro metabolites with their RTs at 49.0, 51.8, 54.0, 56.8, and 57.5 min, respectively.
The retention times of the five metabolites were determined to be 49.0, 51.8, 54.0, 56.8, and 57.5 min from the extracted ion chromatogram of m/z 321.1339 (Figure
Drug metabolism involves chemical conversion to reduce pharmacological activity of a drug candidate and to facilitate its elimination from the body. Metabolic processes can also produce metabolites that are more pharmacologically active. These metabolic reactions are generally divided into two cases called phase I and phase II reactions. According to the rules of metabolic reactions and the results from in vitro experiments, we predict that the probable metabolic reactions of BG involve in phase I reactions including hydroxylation and hydrolysis as well as phase II reactions such as glucuronidation, O-methylation, and sulfation plus acetylation. The possible structures of metabolites in vivo have been analyzed based on the above theory. Additionally, the calculated molecular formulas and mass values (m/z) of corresponding metabolites have been generated by a tool of Mass Calculator in software Qualitative Analysis B.04.00 (MassHunter Workstation, Agilent, USA).
The total ion chromatograms (TICs) of blank samples and samples after oral dosing in negative ion mode are shown in Figure
Retention time (RT), measured mass, calculated formula by elemental compositions, the mass error between the calculated and measured values, MS/MS fragment ions, and metabolic pathway as well as relative peak ratio for each metabolite of BG in rat plasma and urine after oral administration.
Compound | RT |
Measured mass ( |
Mass error (ppm) | Relative peak ratioa in urine | Relative peak ratio in plasma | Formula | MS/MS fragment ions | Metabolic pathway |
---|---|---|---|---|---|---|---|---|
M0 | 63.0 | 305.1400 | −1.8 | 100 | 100 | C17H22O5 | 169, 168, 125, 124 | Parent drug |
M1a | 49.0 | 321.1350 | −2.0 | 0.68 |
|
C17H22O6 | 169, 168, 125, 124 | Hydroxylation |
M1b | 51.8 | 321.1348 | −1.4 | 0.53 |
|
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M1c | 53.9 | 321.1349 | −1.7 | 0.41 |
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M2 | 13.9 | 169.0145 | −1.5 | 0.69 |
|
C7H6O5 | 125, 124, 79, 51 | Hydrolysis |
M3 | 24.4 | 345.0469 | −1.6 | 3.66 | 1.38 | C13H14O11 | 169, 125, 124 | Hydrolysis + glucuronidation |
M4a | 31.8 | 183.0302 | −1.7 | 0.38 |
|
C8H8O5 | 168, 124, 123, 95, 78 | Hydrolysis + O-methylation |
M4b | 32.1 | 183.0304 | −2.8 | 0.32 |
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M5a | 50.5 | 497.1674 | −1.9 | 0.99 | 2.86 | C23H30O12 | 321, 175, 113 | Hydroxylation + glucuronidation |
M5b | 52.9 | 497.1681 | −3.3 | 0.90 |
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M5c | 56.3 | 497.1678 | −2.7 | 2.44 | 2.36 | |||
M6 | 56.4 | 657.2043 | −1.0 | 1.08 | 13.6 | C29H38O17 | 481, 305, 175, 113 | Di-glucuronidation |
M7a | 60.1 | 481.1722 | −1.4 | 24.64 | 105.24 | C23H30O11 | 305, 124, 113 | glucuronidation |
M7b | 67.5 | 481.1728 | −2.6 | 50.96 | 916.65 | |||
M8a | 60.9 | 495.1883 | −2.3 | 95.36 | 31.7 | C24H32O11 | 319, 304, 175, 113 | O-methylation + glucuronidation |
M8b | 62.6 | 495.1879 | −1.4 | 3.66 | 16.95 | |||
M8c | 66.7 | 495.1877 | −1.1 | 2.68 | 2.01 | |||
M9a | 65.5 | 319.1556 | −1.6 | 0.39 | 3.09 | C18H24O5 | 304, 168, 124 | O-methylation |
M9b | 66.0 | 319.1560 | −2.8 | 1.31 | 0.86 |
aRelative peak ratio was calculated on the basis of EIC as follows: (relative peak ratio) = (the peak area of metabolite)/(the peak area of parent, M0) × 100.
b
Total ion chromatograms (TICs) of rat urine and plasma samples by HPLC/Q-TOF/MS. (a) TIC of the urine sample after oral administration at a single dose of 50 mg/kg BG; (b) TIC of blank urine; (c) TIC of the plasma sample after oral administration at a single dose of 50 mg/kg BG; (d) TIC of blank plasma.
The extracted ion chromatograms (EICs) of in vivo metabolites M2 to M9 of BG in rat urine samples.
Representative MS/MS spectra of the in vivo metabolites M2 to M9 of BG in rats.
In the EIC of m/z 321 (M1), three chromatographic peaks of isomers (RT at 49.0, 51.8, and 53.9 min) were observed in urine. M1 showed 16 mass units higher than those of parent drug M0, indicating that they were the monohydroxylated BG. Their retention times and MS2 fragments were the same as those of M1a, M1b, and M1c in RLM incubation solution in section of in vitro investigation.
M2 was eluted at 13.9 min with the [M−H]− ion at m/z 169.0145 (calculated formula = C7H6O5). The MS/MS spectrum of m/z 169 gave abundant daughter ion at m/z 125, which were produced by the loss of CO2 (−44 Da) from precursor ion. Moreover, either retention time or fragment ion of M2 was identical as that of gallic acid by comparing with an authentic standard. Thus, M2 was identified as gallic acid, the hydrolysis product of BG.
M3 and M4 were tentatively assigned as metabolites originating from gallic acid. M3 gave a deprotonated molecule [M−H]− at m/z 345.0469 (calculated formula = C13H14O11). Its MS/MS fragmentation was predominated by the elimination of glucuronide moiety (176 Da) to give product ion at m/z 169. M3 was identified as gallic acid-O-glucuronide. Although baseline separation was not achieved, two peaks were obviously observed in urine containing BG in the EIC of M4. The two peaks represented two isomers M4a and M4b, giving deprotonated molecular ions at m/z 183.0302 and 183.0304 (calculated formula = C8H8O5) and daughter ions at m/z 168 [M−H-CH3]− and m/z 124 [M−H-CH3-COO]−. The two isomers were considered to be O-methylation products of gallic acid, 4-O-methylgallic acid, or 3-O-methylgallic acid. Based on the previous studies on its metabolic fate, gallic acid will be metabolized through decarboxylation, O-methylation, sulfation, and glucuronidation reactions in rats [
Two chromatographic peaks with their RTs at 65.5 (M9a) and 66.0 (M9b) min were detected in the EIC of m/z 319.1556. The MS/MS spectra of m/z 319 showed M9 produced fragment ion at m/z 304 [M−H-CH3]−, indicating that the metabolites M9a and M9b should be O-methyl BG, the O-methylation products of M0.
All the MS/MS fragmentation patterns of M5, M6, M7, and M8 presented neutral loss of 176 Da from precursor ions. Furthermore, the characteristic ions of glucuronide at m/z 175 and m/z 113 were observed in each of their MS/MS spectra, which confirmed the presence of glucuronide according to the reported literature [
Based on the above discussion, the proposed metabolic pathways of BG in rats were presented in Figure
Proposed metabolic pathways of BG in rats.
In this paper, a reliable and sensitive HPLC/Q-TOF/MS method was successfully applied to identify the metabolites of bornyl gallate in vitro and in vivo for the first time. In vitro, BG was metabolized to five isomers of monohydroxylated BG by CYP450 enzymes present in RLM. In vivo, 9 kinds of potential metabolites, altogether 18 compounds including all isomers, were detected and identified. BG is believed to undergo various phases I and II metabolic pathways including hydroxylation, hydrolysis, O-methylation, and glucuronidation, while the conjugation with sulfation or acetylation was not detected. We also proved that BG mainly became products of glucuronidation and O-methylation in vivo, which were identified as BG-O-glucuronide (M7) and O-methyl BG-O-glucuronide (M8).
Traditional Chinese medicine
Bornyl gallate
Rat liver microsome
High performance liquid chromatography/quadrupole time-of-flight mass spectrometry
Electrospray ionization
Total ion chromatogram
Extracted ion chromatograms
Retention time.
There is no conflict of interests.
This work was financially supported by the National Major Scientific and Technological Special Project for “Significant New Drugs Development” (Grant no. 2009ZX09103-121), the National Natural Science Foundation of China (Grant no. 21005060), the Specialized Research Fund for the Doctoral Program of Higher Education on 2011 (Grant no. 20106101110001) and the National Key Technology R&D Program (Grant no. 2008BAI51B01).