Comparative metabolites profiles of osthole in normal and osteoporosis rats using liquid chromatography quadrupole time-of-flight mass spectrometry
Introduction
Osthole is a bioactive coumarin derivative isolated from Cnidium monnieri (L.) Cusson [1]. It has been widely used for the treatment of osteoporosis, skin diseases and gynecopathy [2]. Since osthole has a variety of pharmacological and biological uses, it has been considered as a promising lead compound for drug discovery [3]. However, pharmacokinetics researches indicated that this compound undergoes rapid metabolism and its oral bioavailability was low [4]. The contradiction between the definite biological activity and the poor oral bioavailability promoted the hypothesis that the metabolites of osthole may exert biological activity [5].
Previous study suggested that the major biotransformation of coumarin compounds included hydroxylation, glucuronidation, sulfation, and methylation [6]. There have been several reports about the urinary metabolites of osthole in rats. For example, Lv et al. identified 10 phase I and 3 phase II metabolites in rat urine with an oral administration of osthole [7]. Li et al. found 18 phase I and 2 phase II metabolites of osthole in rat urine [8]. The above researches suggested the major phase I metabolic reactions in-vivo were hydroxylation, demethylation and reduction, with glucuronidation and sulfate conjugations contributing to phase II metabolism. However, plasma metabolism of osthole has not yet been investigated thoroughly.
Besides, estrogen deficiency is the main cause of postmenopausal osteoporosis, and possibly plays an important role in male osteoporosis. It is always associated with metabolic derangements in osteoporosis patients [9]. A better understanding of the metabolite profile of osthole under pathological conditions may offer a useful basis for its clinical application for studying the mechanism of its anti-osteoporosis effect. However, there was no metabolism study of osthole based pathological models, as well as no visualized comparison of osthole metabolites between normal and osteoporosis models. Among all the osteoporosis modules, the ovariectomized (OVX) rat model was used for studying the metabolite profile because of the maximum simulation of primary osteoporosis [10]. Additionally, liquid chromatography quadrupole time of flight mass spectrometry (LC-QTOF/MS) has played a key role in drug metabolism studies [[11], [12]]. Thus, in this study, a reliable method using LC/QTOF-MS system was established and successfully applied to investigate the metabolic profile of osthole in both normal and OVX rats.
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Chemicals and reagents
Osthole standard was obtained from Beijing Aoke Biological Technology Co., LTD (Beijing, China). Acetonitrile and methanol were of chromatographic grade (Merck, Darmstadt, Germany). All water used was distilled by an ultrapure water system (Millipore Corporation, Boston, Massachusetts, USA). The reagent kits for the measurement of serum calcium (Ca), alkaline phosphatase (ALP), and estradiol (E2) in plasma were purchased from Nanjing Jiancheng Bioengineering Institute. Other reagents were
Osteoporosis model
As showed in Fig. 1(I), the decreases in the levels of E2, serum Ca and uterine weight were observed with respect to the significant increased body weight and ALP levels in osteoporosis model rats. Ovariectomy induced bone loss in rats, leading to the decrease in BMD value [15]. Sections of tibia were examined for histological changes. Fig. 1(II) showed that the normal group presented normal compactness of the diaphysis and competent trabeculae. Rats in osteoporosis model group showed sparse,
Conclusions
Natural products have been utilized around the world in the prevention and treatment of diseases and becoming more accepted worldwide. More and more metabolites of natural products were identified in vivo, which gave us an opportunity to utilize these metabolites as a chemical resource library for drug discovery. In this study, we compared the metabolism profiles of osthole in rat plasma between the osteoporosis group and the normal group. The metabolites were identified based on the
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The project was sponsored by the National Natural Science Foundation of China (No. 81603252), Zhejiang Provincial Natural Science Foundation of China (No. LQ17H280002), China Postdoctoral Science Foundation (No. 2016M602987), Zhejiang Provincial Science and Technology Plan (No. 2017F30047, No. 2016F10027, No. 2017C33165), and Science and Technology Innovation Team Project of Ningbo Science and Technology Bureau, China (No. 2015C110027).
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