New perspective on the metabolism of AD-1 in vivo: Characterization of a series of dammarane-type derivatives with novel metabolic sites and anticancer mechanisms of active oleanane-type metabolites
Graphical abstract
Introduction
Biologically active substances sourced from medicinal plants play a key role in the discovery of new therapeutic agents and are hence the focus of considerable research. Ginsenosides are divided into three groups: protopanaxatriol, protopanaxadiol and oleanane types [1]. Almost all ginsenosides have low oral bioavailability. Deglycosylation is the most important mode of gastrointestinal metabolism; for example, ginsenoside Rg3(S) is deglycosylated from ginsenoside Rd and has a broad spectrum of pharmacological properties that are known to inhibit tumour cell invasion and metastasis [2]. Many studies show that ginsenosides can be hydrolysed in the gastrointestinal tract to form sapogenins, which are more easily absorbed in vivo [3]. For instance, 20(R)-25-methoxyl-dammarane-3β,12β,20-triol (AD-1) is a dammarane-type triterpene sapogenin isolated from the hydrolysed saponins of Panax notoginseng [4]. For many years, our research group has focused attention on AD-1 (CN Patent: 201010107476. 7). AD-1 exhibits excellent activity against a variety of cancer cells, such as breast [5], prostate [6], lung [7], [8], gastric [9], and pancreatic [10] cancers. It has shown a 10- to 100-fold cytotoxic activity against several cancer cell lines as compared to Rg3, a cancer therapy drug approved in China [11]. Examination of the mode of action in several lung and prostate cancer cell lines showed that AD-1 could inhibit the proliferation of cancer cells, reduce survival, induce cell apoptosis, reduce the production of reactive oxygen species and cause cycle arrest in the G1 phase [6], [10]. In addition, AD-1 up-regulates the phosphorylation levels of extracellular signal-regulated kinase (ERK) and p38 and dose-dependently down-regulates the expression of cyclin D1, cyclin E, and MDM2, which are associated with the inhibition of proliferation [8]. Furthermore, AD-1 could inhibit the growth of xenografts of prostate and lung cancer cells in a dose-dependent manner without affecting the survival rate of normal cells [8], [10]. The mechanistic examination of AD-1 in colorectal cancer suggests that it plays an anticancer role by targeting β-catenin signalling, a key mediator of the Wnt pathway [12]. AD-1 showed excellent development potential as a new anti-cancer agent. However, pharmacokinetic studies indicated that AD-1 possessed poor absorption and low bioavailability [13], [14]. The compounds responsible for the effects of orally administered ginseng are believed to be metabolites produced in the gastrointestinal tract [15], [16], [17], [18], so understanding the metabolism of the drug candidate can help to optimize its pharmacokinetics [19]. Currently, studies on AD-1 metabolites have stagnated at the conjecture stage [20]. To obtain adequate information regarding metabolism and improve clinical applications, it is critical to understand the metabolic fate of AD-1 (see Table 1).
Metabolite identification is an important part of drug discovery and development. In a majority of cases, the sites of metabolism are unpredictable. In previous studies, metabolites could not be determined using the liquid chromatography mass spectrometry (LC-MSn) method alone [21]. Instead, isolation and further structural confirmation of metabolites relies on nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. To better understand metabolism in vivo, we studied the metabolites present in rat faeces after oral administration of AD-1. This article describes, for the first time, the isolation and identification of the phase Ⅰ metabolites of AD-1. In all, 16 compounds were isolated and their structures were precisely identified through chemical and spectroscopic experiments. Of these 16 compounds, metabolites M6-M9 and M11-M16 are novel. Based on the spectrum of the metabolite, the possible metabolic pathway of AD-1 was proposed, along with a discussion on the mechanism of metabolite formation from the perspective of biosynthesis (see Table 2).
Furthermore, the anti-cancer activities of 16 compounds in several human cancer cell lines were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. We then studied the cellular and molecular mechanisms of M6 as an antitumor agent. This study not only provides important information that may help improve the clinical application of AD-1 but also supports the development of M6 as a potential drug for cancer chemotherapy that will likely surpass AD-1.
Section snippets
Materials
Notoginsenoside hydrolysate (500 g) was separated on a silica gel column using a petroleum ether-ethyl acetate (PE-EtOAc) solvent system (1:3) to afford crude AD-1. After repeated precipitation in ethyl acetate (EtOAc), AD-1 (15 g) was obtained with a purity of >98%.
Instruments and reagents
Both 1-D and 2-D NMR spectra were recorded on a Bruker AVANCE-400/600 spectrometer (Rhein-stetten, Germany). The HR-ESI-MS data were measured on a Bruker micro-TOF-Q mass spectrometer. Commercial silica gel (200–300 mesh, Qingdao
Structure determination
Using repeated chromatographic methods on columns of silica gel, reverse-phase ODS, and preparative HPLC methods, sixteen metabolites were isolated (Fig. 1) from the faeces samples after oral administration of AD-1. Ten compounds (M6-M9, M11-M16) were newly identified. Among them, M4 (262 mg), M6 (158 mg), and M7 (161 mg) were three major metabolites, while others were only found in trace amounts. Their structures (Fig. 2) were identified by ESI-MS, 1H NMR, 13C NMR and two-dimensional NMR
Conclusions
At present, there are many studies on the anti-tumour activity of AD-1, and the research on the separation and identification of metabolites is still blank. In order to understand the process of AD-1 in vivo, understand its metabolism, and prepare related metabolites for subsequent material balance studies, we isolated a total of 16 phase Ⅰ metabolites of AD-1 along with precise identification of their structures through chemical and spectroscopic experiments. Among the metabolites, M6-M9 and
Acknowledgments
This work was supported by the Natural Science Foundation of China (No. 81273389), National Key R&D Program of China (2017YFC1702302), E&T modern centre for Natural Products of Liaoning Province of China (No. 2008402021), Construction of R&D institute of state original new drugs at Benxi, in Liaoning Province (2009ZX09301-012-105B) and Liaoning (FGW) Engineering Technology Research Centre for industrial chromatographic preparation of natural innovative drugs materials (2017-1007). We are glad
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2024, Bioorganic ChemistryDesign, synthesis, and biological evaluation of novel protopanoxadiol derivatives based PROTACs technology for the treatment of lung cancer
2023, Bioorganic ChemistryCitation Excerpt :These reports have spurred efforts to modify the structures of dammarane triterpenoid parent compounds to enhance their pharmacological potency and yields. We have previously isolated and characterized ginseng-derived ginsenosides, leading to the discovery of the novel protopanaxadiol-type ginsenoside 20(R)-25-methoxyl-dammarane-3β,12β,20-triol (AD-1, Fig. 1), which exhibited potent in vitro and in vivo antitumor activity while causing minimal toxicity [8–11]. When used to treat twelve different cancer types including prostate, breast, pancreatic.
Simultaneous quantification and ADME prediction of AD-1 and its eight metabolites in rat feces, and screening of PARP-1 inhibitors through molecular docking
2021, Journal of Molecular StructureCitation Excerpt :In the initial metabolite research, 25-OH-PPD was considered to be the main phase I metabolite of AD-1 [10,11]. However, by isolating and identifying rat feces, we obtained 14 phase Ⅰ metabolites of AD-1 (purity > 97%) [12]. Despite the fact that some studies reported the metabolism of AD-1, little was known about exposure of AD-1 in rats.
Panaxadiol as a major metabolite of AD-1 can significantly inhibit the proliferation and migration of breast cancer cells: In vitro and in vivo study
2021, Bioorganic ChemistryCitation Excerpt :Studies have shown that AD-1 metabolites have significant anti-cancer effects. For instance, previous study isolated some AD-1 metabolites [23]. However, the metabolic pathway and metabolite mechanism of AD-1 has not been elucidated in detail.