Determination of brusatol in plasma and tissues by LC–MS method and its application to a pharmacokinetic and distribution study in mice
Introduction
Brucea javanica (L.) Merr. belongs to the Simaroubaceae specie and is widely distributed in Southeast Asia and northern Australia [1]. Brucea javanica oil is extracted from the seed of this herb and has been used for treating various diseases including cancer, amoebic dysentery and malaria [2], [3]. In recent decades, phytochemical and biological activities of B.javanica were studied and many classes of constituents were separated, including quassinoids, triterpenoids, alkaloids, lignans and flavonoids. Quassinoids, include bruceine A, B, C, D, E, F, G, H, bruceantin, brusatol, bruceoside, brucamarin, brucedic acid, and etc., are the main anti-tumor ingredients of Brucea javanica [3].
The quassinoid brusatol, first isolated from the seeds of B.javanica in 1968[4], was confirmed to have various pharmacological effects of anticancer, antiprotozoal, anti-inflammatory, antiphytovirus, and antifedant [5], [6], [7], [8], [9]. The mechanism of anticancer effect was well studied, which include inhibition of protein synthesis, activation of NF-kB pathway, down-regulation of C-myc protein level, and inhibition of Nrf2 pathway by promoting ubiquitination [10], [11], [12], [13]. Nrf2 (Nuclear factor-E2-related factor 2) is an important factor in antioxidant response. It is controlled by Keap1 and regulates the expression of antioxidant protein and phase II detoxification enzyme through the antioxidant response element (ARE) [14], [15]. Recently, Nrf2 has been confirmed as a double-edged sword: it can suppress tumorigenesis in normal tissues while also can promotes tumor growth and enhances chemoresistance in tumor tissues [16]. Large numbers of cell researches and animal experiments have revealed that the pro-tumorigenic effect of accumulated Nrf2 in elevating the level of detoxifying enzyme in tumor cells, which enhance their resistance to chemotherapy [17], [18], [19]. Moreover, the latest study has identified brusatol as a unique inhibitor of Nrf2 pathway, which can selectively reduce the protein level of Nrf2 through stimulated ubiquitination and protelysis. Therefore, brusatol shows the activity of reducing tumor burden and ameliorating chemoresistance in both in vitro and in vivo models. Combination of brusatol and cisplatin in lung cancer cell line can significantly reduce cell proliferation and inhibit tumor growth when compared with cisplatin treatment alone. However, no cytotoxicity of brusatol was observed [13], [20]. This result indicates that brusatol has great potential to be developed into novel chemotherapy drug, and makes brusatol a hot topic of research.
To improve the development of brusatol, it is essential to develop a sensitive and reproducible analytical method that can be used in the pharmacokinetics and metabolism study. Early reported methods were rarely and companied with sever disadvantages like long run time, insufficient sensitivity, and not well validated [21], [22]. Moreover, no method for the quantification of brusatol in tissues has been described at present. In current study, a sensitive LC–MS method was established and validated for the quantification of brusatol in both plasma and tissues, and successfully used in pharmacokinetics and distribution studies in mice after three different single doses via the tail vein.
Section snippets
Chemicals and reagents
The reference standard of brusatol (purity >97%) was purchased from Shanghai Tauto Biotech Co., Ltd; ornidazole was purchased from Nanjing Sanhome Pharmaceutical Co., Ltd; methanol and ethyl acetate (HPLC grade) were obtained from J.T.Baker (USA); pure water was from Hangzhou Wahaha Group Co., Ltd.
Animals and drug administration
180 Kunming mice (18–22 g), half male and half female, were supplied by the Center of Laboratory Animal Service, Shandong University. The animal experiment protocols were in accordance with the
Method development
For the separation of brusatol and IS, the mobile phase was optimized. At the beginning, we chose methanol and water at a constant ratio of 53:47. Interferences was not found during the runtime, but appeared in the followed sample. Therefore gradient elution was used for washing out the interference. The mass spectrometric parameters were tuned in both positive and negative ion mode, better responses of brusatol and ornidazole were found in the positive mode. The fragment electric voltage for
Conclusion
Brusatol was identified as a unique inhibitor of the Nrf2 pathway and has great potential to be a novel adjuvant chemotherapeutic drug. In this study, a highly sensitive, accurate and reproducible LC–MS method for simultaneous quantification of brusatol in plasma and tissues was developed and validated. Pharmacokinetic and distribution studies were carried out by using this method. The plasma concentration of brusatol decreased rapidly with the half-life time in 10 min; the distribution of
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgement
This study was financially supported by Scientific Development Plan of Shandong Province (2013GSF11864).
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