Identification of prototype compounds and derived metabolites of naoxintong capsule in beagle dog urine and feces by UFLC-Q-TOF-MS/MS
Introduction
Naoxintong Capsule (NXTC) is a modern medication that modified from Traditional Chinese Medicine (TCM) formula Buyang-Huanwu decoction (BYHWD). NXTC has been recorded in the Chinese Pharmacopoeia 2015, as a well-known modern formula for treating cardiovascular and cerebrovascular diseases. Studies and clinical observations had demonstrated a broad range of protective effects of NXTC in treating thrombosis [1], myocardial ischemic-reperfusion injury [2] and hypertrophy [3], diabetic nephropathy [4] as well as ischemic-stroke [5,6], which highlight its potent efficacy and promising clinical application.
NXTC is formulated with 16 TCM ingredients including 13 medicinal herbs and 3 animal ingredients. Unlike traditional decoctions, NXTC is produced by directly pulverizing and mixing 16 crude materials with certain amounts of each. This emphasized its complexity in chemical constituents and marked the difficulty in studying its effects comprehensively. Hence, systematically investigations of the chemical composition as well as their in vivo absorption, distribution, metabolism and excretion (ADME) behaviors are prerequisites for decoding efficacious substrate basis, pharmacological effects and mechanism of NXTC prescription.
Several studies had been conducted to explore the chemical characterization of NXTC. Using different platforms such as Q-TOF-MS/MS [7] and LTQ-Orbitrap [8], several research groups had qualitatively or quantitatively characterized the chemical constituents in NXTC in vitro. However, few studies about NXTC in vivo metabolic investigation were carried out. Thus, in this study, we aimed to fill this gap by evaluating the prototype components in NXTC and elucidating the in vivo metabolic mechanism.
Laboratory animals such as mice, rats, rabbits and beagle dogs are typically model animals for pharmacokinetic studies, among which beagle dogs exhibit much similar pharmacokinetic behaviors with human beings and are more recommended in pre-clinical investigation [9].
Based on this, we employed beagle dogs in our study. We have established a rapid and sensitive method to identify the compounds and their derivatives as well as transformation pathways in beagle dog urine and feces after oral administration of NXTC by ultra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry UFLC-Q-TOF-MS/MS. In addition, we have proposed the in vivo metabolic pathways of chemical components of NXTC. Our study not only shed new lights on the understanding of the in vivo metabolism and excretion mechanisms of NXTC, but also highly contributed to further investigation of the pharmacology and mechanism of NXTC.
Section snippets
Chemical and reagents
The reference standards of chlorogenic acid, amygdalin, protocatechuic acid, hydroxysafflor yellow A, protocatechualdehyde, caffeic acid, paeoniflorin, ecdysterone, cinnamic acid, calycosin-7-O-β-D-glucoside, rosmarinic acid, salvianolic acid B, formononetin, ferulic acid and tanshinone IIA were obtained from the National Institutes for the Control of Pharmaceutical and Biological Products (Beijing, China). Astragaloside II, benzoylpaeoniflorin and calycosin were acquired from Chengdu Pufei De
Optimization of LC and MS conditions
Firstly, in order to achieve optimal peak separations, we have finetuned the experimental conditions including chromatographic columns, mobile phase system and mass detector parameters, respectively. We optimized the conditions including Kinetex C18 column, 0.1% acidic acetonitrile-0.1% aqueous with formic acid and aforementioned mass parameters in our study. In addition, we applied acetonitrile for efficient protein precipitation during sample preparations.
Identification of the prototype constituents and metabolites in beagle dog urine and feces
In accord with literatures, in this
Discussion
In this study, we have evaluated the urine and fecal bio-transformation of NXTC in beagle dogs. In summary, a total of 36 parent compounds, including 4 flavonoids, 12 phenolic acids, 4 saponins, 7 terpenoids and 9 other compounds as well as 52 metabolites were tentatively identified. For flavonoids and phenolic acids, we have proposed the in vivo metabolic pathways, suggesting that flavonoids predominantly went through phase II reaction including sulfation and glucuronidation (Fig. 1) and the
Conclusion
In this study, we have investigated the in vivo biotransformation of NXTC in beagle dog and proposed their potential metabolic pathways. By applying UFLC-Q-TOF-MS/MS, a total of 36 natural compounds as well as 52 metabolites of NXTC were tentatively characterized in beagle dog urine and feces. We also tentatively quantified the excretion behaviors of 9 components of NXTC in dog urine and feces during 48 h after oral administration. The results, when taken together, had comprehensively unsealed
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This study was financially supported by the Guangzhou Major Special Projects of People’s Livehood (Grant No. 201803010082) and High-Level Leading Talent Introduction Program of Guangdong Academic of Sciences (Grant No. 2016GDASRC-0104).
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