Herbal medicine is mainly prepared from boiling herbal water extracts. Many epoch-making immunosuppressant drugs, such as glycyrrhizic acid (old example) and FTY720 (current example), were developed from herbal secondary metabolites in the boiling water extract by partition with organic solvents. However, few immunostimulants have been discovered by this method. Instead of the usual method, we aimed to find a novel immunostimulant component by two unique methods in the research of herbal medicine: ultracentrifugation and electron microscopy. The immunostimulant was not a secondary metabolite, as expected, but the structure was a nanoparticle formed by a polysaccharide. In addition, we clarified the immune effect of the nanoparticle. Intake of the nanoparticle by phagocytosis resulted in immunostimulant effects by increasing the genes and proteins of inflammatory cytokines in macrophage cells. The immunostimulant effects were inhibited by a phagocytosis inhibitor, cytochalasin D. To the best of our knowledge, this study is the first to describe the discovery of a nanoparticle in boiling herbal water extracts and its immunostimulant properties. This study will provide additional understanding of the efficacy of herbal medicine, in that the immunostimulant nanoparticle universally exists in boiling herbal water extracts. Thus, traditional herbal medicine may be an oldest known nanomedicine. Furthermore, this study suggests that the immunostimulant nanoparticle simply can be obtained from herbal medicine only by ultracentrifugation. We hope that this simple strategy will substantially contribute to drug development, including vaccine adjuvant, in the future.

Breast cancer is the most common malignancy in women. Apoptosis is important for tumor suppression and may delay cancer progression. It was found that shikonin induced apoptosis in 4T1 murine mammary cancer cells and MDA-MB-231 human breast cancer cells in vitro. Total p38 and c-Jun N-terminal kinase (JNK) levels were maintained in 4T1 cells, and p38 phosphorylation, but not JNK phosphorylation, was significantly increased. Caspase-3/7 activity was detected, which suggested that the p38 pathway, but not the JNK signaling pathway, induced apoptosis in 4T1 cells. The anti-tumor effects of shikonin on orthotopic mouse models were also examined. On day 7 after inoculation of 4T1 cells into mice, tumor volumes in the shikonin-treated and the control groups began to differ. On day 13, tumors were weighed, and shikonin was revealed to suppress tumor growth in the orthotopic 4T1 model in vivo. In conclusion, shikonin is a potential anti-tumor drug for breast cancer.

A number of immunostimulant effects of herbal medicines have been reported; however, the underlying mechanisms of their immunostimulatory effects have not been elucidated in detail. Our previous study showed that sugar-based nanoparticles derived from cell walls acted as the immunostimulatory component of boiled Glycyrrhizae radix water extracts. Therefore, the aim of the present study was to clarify the molecular mechanisms by which these cell wall-based nanoparticles functioned as immunostimulants. Mouse macrophage RAW-blue cells were stimulated by these nanoparticles and several immunological effects were investigated. When phosphorylation of nuclear factor-κB (NF)-κB p65 subunit was increased, the expression of the inflammatory cytokines interleukin-6 and tumor necrosis factor-α were induced via NF-κB. On the other hand, Toll-like receptor 4 recognizes cell wall components of bacteria and fungi. In the present study, it was also shown that these cell wall-based nanoparticles serve an immunostimulatory role as ligands of Toll-like receptor 4 by RNA interference experiments. The results of the present study suggested that the signaling pathway of nanoparticles obtained from boiled Glycyrrhizae radix water extracts, at least partially involved TLR4 and downstream signaling from this receptor, resulting in the immunostimulatory effects of these nanoparticles in RAW-blue cells.