Investigation of the Fate of Proteins and Hydrophilicity/Hydrophobicity of Lycopodium clavatum Spores after Organic Solvent-Base-Acid Treatment.

Microcapsules extracted from lycopodium ( Lycopodium clavatum) spores have been increasingly used as an oral therapeutic carrier. A series of sequential treatments involving acetone, KOH, and H₃PO₄ are used to extract a protein-free hollow microcapsule. This study focuses on two critical aspects of lycopodium spores: the fate of native proteins and the wettability of the spores after a chemical treatment. Protein-free spores are desired to prevent an allergic reaction, whereas the wettability is critical for the formulation development. Although the chemically treated lycopodium spores are generally regarded as protein free, the studies that have reported this have not gone into significant depths to understand the nature of residual nitrogen observed even in spores thought to be protein free. Wettability of spores has not received any significant attention. Accordingly, in this study, we performed a comprehensive analysis of natural spores and spores after each chemical treatment step. We show that natural lycopodium spores are hydrophobic and contain low-molecular-weight proteins (∼10 kD). Acetone treatment partially solubilizes unsaturated phospholipids from the spores. Nevertheless, the acetone-treated spores retain native proteins and are still hydrophobic. KOH treatment, however, removes a significant amount of proteins and partially hydrolyzes esters to carboxylic acid salts and results in a hydrophilic spore with a good wettability. Finally, we show that the H₃PO₄ treatment removes residual proteins, hydrolyzes remaining esters to carboxylic acids, and dissolves carbohydrates. H₃PO₄ treatment temperature controls carbohydrate dissolution, which in turn affects the hydroxyl functional groups and hydrophilicity (wettability) of the treated spores. Spores treated at 60 °C as opposed to 160 °C are amphiphilic in nature due to the abundance of hydroxyl functional groups on the surface. In conclusion, this study confirms the removal of native proteins from treated spores and sheds light on the chemical changes that the spores undergo after chemical treatment and correlates these changes to their wettability.