Date of Degree

2-2018

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Akira Kawamura

Committee Members

David R. Mootoo

Charles M. Drain

George John

Kevin Ryan

Moriya Tsuji

Subject Categories

Lipids | Nanomedicine | Natural Products Chemistry and Pharmacognosy | Other Chemicals and Drugs

Keywords

Lipopolysaccharide, Juzen-taiho-to, Lipid A, Kampo medicine

Abstract

Juzen-taiho-to (JTT) is an immune-boosting herbal formulation with an ideal balance of safety and efficacy. For example, JTT is clinically used in Japan to stimulate the immunological functions of cancer patients undergoing chemotherapy and radiation. Although the clinical effects of JTT are recognized, the active compounds and mechanism of action are unknown. The studies conducted previously in our laboratory associated several phytosterols and plant glycolipids with the potent immunostimulatory activity of JTT: namely, β-sitosteryl β-D-glucoside (BSSG), glucocerebroside (GluCer), digalactosyldiacylglycerol (DGDG) and monogalactosyldiacylglycerol (MGDG). However, these compounds, when further purified, exhibited little or no immunostimulatory activity. This suggested that the immunostimulatory factors in JTT (1) had similar physicochemical properties as phytosterols and glycolipids; (2) existed in very small quantities, and (3) were extremely potent. These attributes led us to the hypothesis that the immunostimulatory factors of JTT stemmed not from plant metabolites but from bacterial glycolipids, some of which were known for potent immunostimulatory activity.

This hypothesis was tested in the first half of my thesis work. Through a series of chemical and biochemical studies, including chemical extraction and high resolution mass spectrometry (MS), the main immunostimulatory factor of JTT was identified as lipopolysaccharide (LPS) of Gram-negative bacteria. Detailed chemical and immunological characterization of LPS in JTT, however, was hampered by their heterogeneity and minuscule quantities. In order to gain insights into the LPS in JTT, we decided to characterize microbial community in JTT by Illumina sequencing. 16S metagenomic sequencing of Angelica sinensis, which is a JTT component with most potent immunostimulatory activity, revealed Rahnella aquatilis as the main Gram-negative species. The finding of R. aquatilis in JTT was surprising because LPS produced by R. aquatilis exhibit endotoxicity, yet JTT does not.

The finding of LPS in JTT opened the possibility that plant metabolites might modulate the activity of LPS. In order to gain insights into this possible interaction between LPS and plant metabolites, the second half of my thesis work focused on chemical and immunological characterization of LPS-plant metabolite mixtures. Since LPS was known to form nanoparticles in aqueous solution, I hypothesized that plant metabolites would affect LPS nanoparticle assembly, which, in turn, could modulate immunostimulatory activity. My thesis work mainly focused on the LPS and BSSG mixture, because BSSG was the most prominent plant metabolite in the immunostimulatory fractions purified from JTT. Cell-based assays showed that BSSG significantly modulated the immunostimulatory activity of LPS. On the other hand, dynamic light scattering and transmission electron microscope also revealed that BSSG significantly modulated the nanoscale assembly of LPS. Taken together, these results suggested that the supramolecular assembly of the BSSG/LPS mixture was a key determinant of its immunomodulatory activity.

Collectively, this thesis work provided a new molecular basis to understand the safe and effective immunostimulatory effects of JTT and other immune-boosting medicinal plants.

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