Diosgenin and diosgenyl saponins : Investigating the relevance of cholesterol on their membrane behavior
概要
The diosgenyl saponins (DGs) have gained pharmacological importance due to their anti-cancer, neuro- and cardio-protective properties.1 These saponins possess a diosgenin (DGN) aglycone with a spirostanol steroidal structure with different sugar pendants. [Figure 1]
This study aims to understand the bioactivities of the DGs through their specific association with cholesterol (Cho) in the membrane. Moreover, since the biological effects of the saponins have been attributed to their aglycone,2 probing the membrane behavior of DGN, aids in recognizing the structural effect of the aglycone in the membrane activities of DG. To bring to light the exact nature of these interactions, different biophysical techniques such as solid-state NMR, fluorescence spectroscopy and differential scanning calorimetry (DSC) have been employed.
To begin with, the membrane effects of DGN was investigated. DGN has been studied extensively for its pharmacological potential.3 DGN is a sterol with a structural similarity to cholesterol (Cho). In particular, DGN possesses an identical tetracyclic backbone but the spiro-acetal structure of DGN is significantly different from the linear C8 sidechain of Cho.
We examined the effects of the common tetracyclic cores and the different sidechains on the physicochemical properties in phosphatidylcholine (PC) lipid bilayer membranes. In diphenylhexatriene (DPH) anisotropy experiments, DGN up to 30 mol% stably bound to the POPC bilayers and induced a similar ordering effect on POPC to Cho. Fluorescence experiments to evaluate membrane hydration using laurdan and prodan also revealed a similar tendency for Cho and DGN. In 2H NMR, DGN and Cho showed similar quadrupolar coupling values in POPC bilayers, which also suggested that DGN is oriented parallel to the membrane normal like Cho. These results suggest that DGN below 30 mol% has comparable effects with Cho on basic biomembrane properties. It was also suggested that the affinity of DGN-Cho in the membrane is stronger than that of DGN-DGN or Cho-Cho interaction. Equimolar mixtures of DGN and Cho in POPC resulted to an additive effect in membrane ordering and lipid packing in fluorescence studies. Cho influences the behavior of DGN in membranes and these results indicate moderate interaction of Cho and DGN.
For the diosgenyl saponins such as trillin and dioscin, the results of similar biophysical experiments revealed that the amphiphilicity of the saponins enable them to interact with phospholipid model membranes regardless of Cho with no further membrane changes. However, in the presence of Cho, it was observed that the increasing number of sugar moieties of the saponin results in an escalating strength of saponin-cholesterol interaction. Especially for dioscin, having three sugars, the membrane activity was enhanced and led to perturbation and further disruption of the membrane. For trillin, laurdan and prodan fluorescence in membrane and solid state NMR results demonstrate that trillin, with one sugar residue, rigidifies membrane similar to Cho and maintains the integrity of the bilayer. The data suggest that trillin weakly interacts with Cho as well as the membrane lipids, thus favorably incorporating itself into the bilayer.
The rigid and planar aglycone of DGs allow them to penetrate the membrane. Consequently, as the saponins partition in the membrane, they can diffuse and easily gain access to membrane constituents such as membrane receptors and enzymes, and could subsequently exert their biological properties.