Chemical characterization and biological assessment of Euterpe oleracea Mart. (açaí) for in vitro botanical-drug interactions with anticancer drugs

Abstract

Cancer is the leading cause of death worldwide, with an estimated 19.3 million new cases in 2020 and almost 10 million cancer deaths. Botanical dietary supplements (BDS) are consumed more by cancer patients than otherwise healthier patients without cancer to alleviate side effects of chemotherapy drugs and/or increase quality of life; up to 80% of cancer patients have reported using BDS following their initial cancer diagnoses. However, many patients do not know the risks of concomitant use of BDS with anticancer drugs and the possibility for increased AEs, which may be life-threatening. Euterpe oleracea Mart. (Arecaceae), commonly known as açaí, is a fruit that grows on the açaí palm tree native to the Amazon region. Açaí presents many health benefits, the most prominent being antioxidant and anti-inflammatory activities. In recent years, it has been introduced into the BDS market, and its popularity is steadily rising. Açaí is now among the top 40 botanicals used in the U.S., and cancer patients increasingly use açaí BDS to complement their conventional chemotherapeutic agents. This rise in popularity has also led to challenges regarding the quality, safety, and efficacy of açaí fruit products. Our group previously found that both passively and non-passively diffused compounds in MeOH açaí fruit extract displayed significant inhibition of hepatic CYP3A4 – suggesting potential for interactions between açaí BDS and CYP3A4-interactive drugs. Before testing this hypothesis, analytical methods to chemically characterize and standardize açaí products prior to in vitro testing are needed. The first objective of this work was to develop a chemical fingerprinting method for untargeted characterization of açaí samples from a variety of sources, including food products and botanical dietary supplement capsules, made with multiple extraction solvents. An optimized LC-MS method was generated for in-depth untargeted fingerprinting of chemical constituents in açaí extracts. Statistical analysis models were used to describe relationships between the açaí extracts based on molecular features found in both positive and negative mode electrospray ionization modes. To elucidate the differences in metabolites among açaí extracts from different cultivars, we identified or tentatively identified 173 metabolites from the various extracts. Of these compounds, there are 138 reported in açaí for the first time. Statistical models showed similar yet distinct differences between the extracts tested based on the polarity of compounds present and the origin of the source material. A high-resolution mass spectrometry method was generated that allowed us to greatly characterize 16 complex extracts made from different sources of açaí with different extraction solvent polarities. Quantitation of bioactive constituents is a crucial preliminary step before utilizing extracts for biological assays so they may be normalized and administered according to a specific constituent concentration. Açaí has four main anthocyanin analytes: cyanidin 3-O-glucoside, cyanidin 3-O-sambubioside, cyanidin 3-O-rutinoside, and peonidin 3-O-rutinoside. This is the first comparison of açaí anthocyanin profiles between fresh fruits, processed powders, and botanical dietary supplement capsules. The materials examined shared a similar anthocyanin profile, with cyanidin 3-O-rutinoside being the most abundant, followed by cyanidin 3-O-glucoside. Among the botanical dietary supplement capsules, the two formulations varied greatly in anthocyanin concentration despite both being aqueous extracts. Previous LC-MS methods range from 35-120 min per injection, while we report a 10 min quantitative method for analysis of anthocyanins in various açaí materials that is fast, reproducible, and accurate. The method produced is useful to assure the quality, efficacy and safety of food and dietary supplement materials containing açaí. We then tested our chemically standardized açaí BDS extracts for inhibition of hepatic CYP3A4. A parallel artificial membrane permeability assay (PAMPA) was utilized to filter intestinal passive diffusion of the four extract constituents so that compounds could be tested for inhibition of hepatic CYP3A4. Passively and non-passively diffused constituents of extracts from PAMPA assays were injected into the LC-MS and characterized for visualization and comparison of chemical entities. To elucidate the non-passively diffused compounds responsible for significant CYP3A4 inhibition, the Bioactivity – GNPS tool was used to hypothesize which compounds within the formulation extract were potentially active to accelerate their identification. We found no significant inhibition of CYP3A4 by passively diffused constituents for any extract. There was, however, significant inhibition by non-passively diffused constituents of both F1ME and F1AC extracts with 50% CYP3A4 inhibition at the donor compartment concentrations 5.764 and 15.58 ng/mL cyanidin 3-O-glucoside, respectively. Six compounds were predicted to be inhibitors of CYP3A4 from F1ME, including betaine and 5 unknown compounds. This indicates that BDS containing açaí may not inhibit CYP3A4 through passively diffused compounds. Compounds from açaí BDS that inhibit CYP3A4 may be absorbed through other mechanisms, such as transporters.