**1. Introduction**

Living organisms use small molecules including membrane targeted drugs to enhance membrane fluidity and permeability to elicit their potency in signal transduction as well as in the treatment of various diseases including cancer, fungal and microbial pathogens. Electrochemical signaling from reactive oxygen species (ROS) is a major mechanism used to regulate different cancer-related processes including cell proliferation, migration, invasion, metastasis and vascularization. The key players in the redox microenvironment of the cancer and neighboring cells are superoxide (O2 ), hydrogen peroxide (H2O2), nitric oxide (NO) and ions which are produced or regulated by membrane bound nicotinamide adenine dinucleotide phosphate (NADPH), oxidases known as NOX and by the dual oxidases (DUOX),

or nitric oxide synthases. For example studies using scanning electrochemical microscopy (SECM) and fluorescence microscopy confirmed the release of ROS from prostate cancer (PC3) cells [1, 2].

Among the ion channels that are membrane bound include voltage-gated Na+ channels that selectively allow the passage of Na+ ions into cells resulting in membrane depolarization leading to generation of action potential in excitable cells including neurons, heart and skeletal tissues. It is known that strongly metastatic prostate cancer cell lines such as PC3 cells demonstrate significantly higher expression of voltage-gated Na+ channels (Nav1.9 alpha subunit). Studies have shown that inhibiting specific voltage-gated Na+ channels activity have helped to reduce cell proliferation and therefore such channels including K+ , Ca2+, and Cl− may emerge as novel biomarkers and therapeutic targets for certain cancer treatments. The concept is to develop small molecular probes or nanoparticles that are either delivery vehicles or the nanoparticle itself having the potential to specifically block a particular ion channel to prevent the movement of ions across the membrane which is key critical step for tumor cell survival.

*Saccharomyces cerevisiae* (SC) cells are single-celled eukaryotes and model organisms for studying cellular mechanisms including DNA damage and repair as well as systematic fungal infections. Additionally, *S. cerevisiae* shares the complex internal cell structure of animals without the high percentage of non-coding DNA that can confound research in higher eukaryotes. Three of the most extensively used antibiotic drugs for studying SC is amphotericin B (Amp B), rifampicin and fluconazole [3, 4]. The antibiotics target different cellular organs to elicit their antimicrobial and antifungal effects. For example, Amp B is membrane mediated thereby increasing the permeability of ions and small molecules by binding more strongly to ergosterol, the principal fungi sterol found in SC [5]. Rifampicin and fluconazole on the other hand have broad antibacterial and antifungal influence with rifampicin targeting different forms of mycobacteria by inhibiting DNAdependent RNA polymerase activities, whereas fluconazole is used for a number of fungal infections including candidiasis as well as other fungal diseases. Since Amp B and rifampicin are redox mediators that can interact with eukaryotic cell membrane thereby increasing redox activity by creating pores or inhibiting the synthesis of ergosterol respectively, it will be interesting to compare their redox activity with fluconazole. These antifungal drugs were used as model drugs because they have been extensively used for various *in vitro* and *in vivo* studies of model cells.

Based on the background outlined, it is obvious that there are essentially two pathways (lipid-mediated and diffusion porins) through which both hydrophobic and hydrophilic plant metabolites or drugs elicit their potency at the cellular level. It is obvious that the degree of permeation of the cell membrane has a major impact on the redox activity. In addition, the presence of a hydrophobic drug within the complex architecture of the membrane, enhances easy access of small ions through pores which can be detected electrochemically. Non-membrane-mediated drugs diffuse freely through the membrane and may not necessarily destabilize the membrane architecture; thereby limiting ionic flow that can be captured by electrochemical detection techniques. In the current work, an entrapment strategy was developed using aluminosilicate minerals to selectively pool plant metabolites using different pH conditions and evaluating the polarization and depolarization of model cells using electrochemical sensing techniques. For membrane targeted bioactive compounds, the fluctuation in the redox signals will reveal important lead bioactive compounds for further investigation.

Several biopolymers and aluminosilicate minerals derived composite drug delivery carriers have been reported from several laboratories. For example halloysites natural tubules (HNTs) are aluminosilicate minerals composed of

different proportions of aluminum, silicon, hydrogen and oxygen often with the chemical formula Al2Si2O5(OH)4.nH2O [6, 7]. They are empty cylinders with widths of about 100 nanometers and consist of two structures; the anhydrous structure with an interlayer dispersing of approximately 7 Å and the hydrated structure with an augmented interlayer dividing of 10 Å, due to the presence of water in the lamellar spaces [8–10]. In each layer of the halloysite nanotube, the SiOH groups are found on the outer surface whiles the AlOH groups are situated on the inner surfaces making the outer and inner surfaces to have different charges [11–13]. The positive charge of the internal lumen is a consequence of protonation of the AlOH group at low pH whereas the SiOH groups have overall negative charge due to the coordination of the atoms. When the halloysites nanotubes are modified with biopolymer such as chitosan new functional materials with improved physicochemical properties are generated. The composite materials can serve as effective drug carriers.

The charge disparity of halloysites has also drawn interest from the research community whereby overall negatively charged proteins taken above their isoelectric points are mostly loaded into the positively charged nanotube lumen [14]. Therefore, in a pool of organic compounds, halloysites nanotubes can facilitate the formation of a transient bond between selected bioactive compounds and the AlOH or SiOH as a function of pH conditions and can be very effective as a nano drug carrier for different applications [15–19].

Traditional herbal medical practices continue to be part of the healthcare needs of the world especially residents of sub-Sahara Africa (sSA) [20–23]. However, the mechanism of action of the plant metabolites to illicit their potency continues to be a mystery due to the lack of standardized methods. Electrochemical detection of drugs interacting with most biological systems is an important strategy to understand cellular stresses that causes cell death [24–26]. Evidence emanating from previous findings, indicate that there are several membrane redox centers in most eukaryotic cells that can be targeted to monitor redox activities in the presence of certain drugs including plant metabolites [27–29].

The concept is investigated using extracts from *Dioclea reflexa (DR)* hook which belongs to the leguminous family. There are certain class of compounds in *Dioclea reflexa (DR)* that have clinical usefulness in both temperate and tropical regions [30–33]. Extract of *DR* seed has been shown to boost hematological parameters and antioxidant activities which protect the kidney and blood from oxidative and related injuries under acute and chronic toxicological challenges [30, 31, 33–37]. Also, the aqueous extract of the seeds produces 100% mortality in third stage mosquito larvae of *Aedes aegypti*. The seed is a potential food source which contains around 14% protein, 8% fats and 58% carbohydrates [32]. Though these metabolites continue to show promise in disease treatment, there is very limited data in the literature of the properties of single isolates and their medicinal relevance, albeit due to the difficulties in pursuing systematic separation of the complex mixtures in a single separation method. Thus, the current work describes the use of a simplified method to systematically pool bioactive compound mixtures from *DR* and test their inhibitory effects on breast (MCF-7) cancer cells and *Saccharomyces cerevisiae* (SC) cells. The rationale is that the larger surface area coupled with the differential polarity of the lumen and the surface of the halloysites nanotubes will be sufficient to bind selectively with the plant metabolites in the crude extracts of *DR*. The evidence of the entrapped species on the halloysites nanotubes was monitored using X-ray diffractometry (XRD) and Fourier transform infrared spectroscopy (FTIR) to determine the degree of aluminol (AlOH) and the siloxane (SiOH) groups modification since these two functional groups will be key sites for bioactive compounds interaction. pH-dependent eluted samples were then tested on breast (MCF-7)

cancer cell lines to investigate their inhibitory effects and the mechanism of inhibition were determined using cyclic voltammetry and flow cytometry analyses [38–41]. The results are reported here and show evidence of differential inhibitory effects of the bioactive compounds from the various pH conditions.
