Bioactivity of Steroid and Triterpenoid Saponins: Influence on Membrane Permeability and Drug Absorption

Abstract

Saponins are widely distributed among flowering plants and some marine invertebrates and serve in defense for these organisms. Their amphiphilic molecules are composed of a lipophilic aglycone and one or more hydrophilic sugar moieties, giving them a high degree of structural diversity. Their biological and pharmacological activities range from antimicrobial, antifungal, anticancer, to immunomodulatory, etc. The most prominent feature of saponins is linked to their effects on cell membranes; they strongly affect cell membrane structure and integrity by different mechanisms depending on their chemical structure. The ability of saponins to increase membrane permeability can be used to facilitate the passage of drug molecules or other natural products through the cell membrane. The ability of saponins to affect cell membrane structure and integrity makes them interesting natural products in pharmacological and medical research and therapy, in particular as agents for enhancing drug efficacy. Saponins are known to interact with cholesterol in cell membranes by forming complexes. Until recently, there has been limited information on their potential as cytotoxicity-enhancing agents and on their molecular mechanisms of action on the membrane. This study explores the mechanisms of action of saponins on membranes and their effects in enhancing the cytotoxicity of certain anticancer drugs/toxins as applied to various cancer cell lines. Two different kinds of saponins were chosen (digitonin, a steroid saponin, and quillaja extract, a triterpenoid saponin mixture). These were investigated in combination with the anticancer drugs berberine, cisplatin, doxorubicin, dexamethasone, and mitomycin C, as well as with the polar toxin ricin (extracted from castor beans), on HeLa, COS-7, MIA PaCa-2, and PANC-1 cancer cell lines. The associated molecular mechanisms of action on membranes were investigated by employing a series of bioanalytical/ biophysical techniques: 1) MTT assay (a formazan test) which measures cell viability; 2) hemolysis (microscopic screening of erythrocytes) to measure the degree of membrane destruction; 3) dynamic light scattering (DLS) on large unilamellar vesicles (LUVs) to observe and quantify membrane leakage; 4) fluorescene/confocal microscopy of giant unilamellar vesicles (GUVs) to visualize membrane permeability; 5) quartz crystal microbalance with dissipation (QCM-D), dual polarization interferometry (DPI), and high-energy specular X-ray (XRR) showing structural changes in supported lipid bilayers (SLB), and 6) differential scanning calorimetry (DSC) which reveals thermotropic features of membranes resulting from saponin action. Digitonin and quillaja extract both enhance the cytotoxicity of the selected anticancer drugs in several cancer cell lines, the effect being either synergistic or additive. Quillaja saponin exerts a stronger cytotoxicity-enhancing effect than digitonin. The highly toxic monodesmosidic digitonin causes complete disruption of membranes at very low concentrations. The membrane activity of saponins strongly depends on the presence and amount of cholesterol in the membrane. Digitonin destroys GUVs, while quillaja saponin rather leads to pore formation. The relatively stable pores formed by the quillaja saponin-cholesterol complexes have a diameter of about 1 nm, only allowing passage of small-size molecules. Digitonin removes cholesterol from the inner membrane layer with formation of an additional layer on the outside, which eventually leads to membrane disruption. The quillaja saponins penetrate into the inner membrane layer forming complexes with cholesterol. The stabilized complexes form pores, which allow passage of water and other small molecules. Both sugar chains of the bidesmosidic quillaja saponins play an important role in stabilizing the pore formation in the membrane. Here, and for the first time, we report the occurrence of monodesmosides in the employed commercial quillaja saponin extract. The presence of both bidesmosides and monodesmosides in the quillaja saponin extract may be responsible for its bioactivity and pharmacological effects.