Alternative natural sources of indirubin derivatives: analysis thereof in plasma and tissues: toxicological studies

Abstract

Indirubins are bis-indole alkaloid compounds occurring naturally in various indigocontainingplants and mollusk gastropods. These molecules were initially known fortheir long history in the dye industry. However, during the last two decades, thechemical and biological studies of indirubins have increased tremendously due to thediscovery of their bio-activities against numerous human cancer cell lines mainlythrough the inhibition of protein kinases. Therefore, the aim of this work was firstly toseveral potential new sources of indirubins using a UHPLC-ESI-MS/MS platform.Thus, three marine mollusks (Hexaplex trunculus, Rapana venosa, and Plicopurpurapansa), one plant (Isatis tomentella), and one pharmaceutical product (DangguiLonggui Wan) were analyzed for the identification of the indirubins thereof. Theindirubins and the major components of the mollusk Hexaplex trunculus and the plantIsatis tomentella were further isolated using newly developed FCPC methodology.The glands of R. venosa were studied here for the very first time; several extractswere produced and analyzed. The ethyl acetate extract which was the richest insecondary metabolites revealed in LC-MS-ESI(-) the presence of several brominatedand non-brominated indirubin precursors namely. The milk of P. pansa displayedeleven brominated compounds, among which eight are previously reported and threeare unknown. Indirubin and indigo were detected in I. tomentella which also contained several amino acids, nucleosides, glycosylated and non-glycosylatedflavonoids. A total of forty-six metabolites were detected in this extract and its majorconstituents were isolated using step-gradient CPC technique, and structurallyelucidated using NMR and HRMS technique and comparison to data in the literature. In the third chapter of this thesis, the toxicological studies of 7-Bromo-indirubin-3΄-oxime (7BIO) on Wistar rats are reported. 7BIO is a synthetic derivative of indirubincausing rapid cell death in tumor cells in different tumor cell lines, but with no priordata regarding its in vivo toxicity. Hence, healthy rats were daily administered (i.p)with either vehicle (Control), or 20 mg/kg BW (low dose), 50 mg/kg BW (mediumdose), and 100 mg/kg BW (high dose) of 7BIO for 9 consecutive days. By the end ofthe treatment period, 1/6 and 3/6 animals deceased in the medium dose and high dosegroups, respectively. Regarding the histopathology parameters, no treatment-relatedeffects with regard to any of the toxicological biomarkers considered were observedin kidneys, testis, pancreas, spleen, stomach, and colon. However, granulomas wereprominent throughout the peritoneal cavity in a dose-dependent manner; and lungsand intense liver toxicity signs were evident in high dose group. The safety profile of6-Bromo-indirubin-3΄-oxime (6BIO) was further assessed on Wistar rats followingthe same concept described for 7BIO. 6-bromoindirubin-3′-oxime (6BIO) is a semisyntheticanalogue of 6BI that was developed as a potent and selective GSK-3βinhibitor and is currently considered and commercialized as a prototype inhibitor ofthat mammalian kinase. After a 14-day treatment of rats 6BIO, traditionalhematological and biochemical endpoints were evaluated and revealed no significant signs of toxicity. Furthermore, UHPLC-MS/MS was applied for the analysis ofendogenous metabolites in rat serum. Principal component analysis (PCA), partialleast-squares-discriminant analysis (PLS-DA), and orthogonal partial least-squaresdiscriminantanalysis (OPLS-DA) were adopted to investigate the metabolicdifferences between 6BIO treated and untreated groups. And the potential biomarkerswere identified accordingly. Our LC-MS metabolomics platform was provedsufficiently robust and accurate to identify and exhibit perturbations in the circulating small metabolites. The results revealed that the metabolite profiles of 6BIO-treatedgroups deviated obviously from that of control groups (PCA & PLS-DA). Thepotential biomarkers extracted from the S-plot, were primary involved in themetabolic pathways of phosphatidylcholines and phosphatidylserines, which are bothimportant groups of phospholipids class of cellular components with critical roles incellular membranes. It is therefore speculated that the administration of 6BIO maypertubate cell metabolism, specifically from it surface. Finally, a UHPLC-MS/MSmethod was developed and validated to quantify 6BIO and its more soluble derivative6BIO-pip in mice plasma. The validated method was applied to pharmacokineticstudies of the two molecules after oral administration in mice. The results show that6BIO-pip is highly absorbed > 200-fold and more stable in the circulatory system ofmice as compared to 6BIO.Overvall, the comprehensive metabolites profiling of marine, plant andpharmaceutical sources of indirubins were reported with isolation of somerepresentative major constitutens. UHPLC-HRMS were proved to be an efficient toolin the analysis of these natural products as well as the monitoring of their bioanalysis.