Date of Degree

2-2019

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Akira Kawamura

Committee Members

Wayne Harding

Weigang Qiu

Haiping Cheng

Moriya Tsuji

Subject Categories

Alternative and Complementary Medicine | Bacteria | Biochemistry | Environmental Microbiology and Microbial Ecology | Genomics | Immunity | Plants

Keywords

Immunomodulatory herbs, Lipid A, Lipopolysaccharide, Medicinal Plants, Next Generation Sequencing, Probiotics

Abstract

Medicinal plants are one of the biggest sources of natural products with therapeutic importance. There are currently over 28,000 plants with putative medicinal values. Plant-derived compounds have been explored extensively for various biological activities ranging from anti-cancer, immune-boosting to anti-inflammatory and anti-oxidant. Some of the most important therapeutic agents are of plant-origin, such as paclitaxel from Pacific yew (Taxus brevifolia) and artemisinin from qinghao su (a Chinese medicinal herb; a.k.a. Artemisia annua) to name a few.

The study presented in this thesis started out as classical pharmacognosy research, which focused on the identification of immunostimulatory factors in Juzen-taiho-to (JTT), a Japanese herbal formulation with well-established clinical safety and efficacy. The study, however, led to an unexpected discovery that immunostimulatory factors are not of plant-origin; instead, they are from plant-associated bacteria. The ensuing studies presented in this thesis as well as several studies by others gave rise to an emerging new hypothesis that can fundamentally transform the way we understand the immune-boosting activity of medicinal plants.

Chapter 1 gives a brief introduction and history of the most widely accepted herbal medicinal practices around the world. Additionally, some of the well-known bio-active compounds derived from medicinal plants are described. The chapter also highlights the issue of batch-to-batch variability, which is a common issue of herbal medicines. Batch-to-batch variability is a major problem because it raises concerns about quality, safety and efficacy (QSE) of herbal medicines. In order to reduce the batch-to-batch variability, therapeutically relevant compounds have to be identified and used to standardize herbal medicines. However, identification of therapeutically relevant compounds is not a trivial task as exemplified in our quest to identify immunostimulatory factors in JTT.

The focus of Chapter 2 is the identification of immunostimulatory factors in JTT. Although it was relatively easy to purify the fractions enriched with immunostimulatory activity, it was difficult to identify the immunostimulatory factors in the fractions. The readily observable compounds were all plant-derived metabolites with little or no immunostimulatory activity. Ensuing biochemical, chemical, and immunological studies eventually led us to the discovery that immunostimulatory factors arise from lipopolysaccharides (LPSs) of Gram-negative bacteria that are associated with the component herbs of JTT. This finding opened a possibility that medicinal herbs in JTT may contain “immune-boosting” bacteria that can safely stimulate the human immune system.

With this knowledge, we wondered about the identities of these bacteria. It was an opportune time to ask this question as the powerful next-generation sequencing tools, such as Illumina sequencing were becoming widely available at that time. Chapter 3 addresses this question by characterizing the microbial communities in Angelica sinensis, which is the most potent immune-boosting herb in JTT. The sequencing identified several bacterial taxa that are reproducibly observed in multiple samples of A. sinensis. In particular, Rahnella aquatilis was identified as the most abundant bacterial species that is associated with the immunostimulatory activity of A. sinensis. In addition, the sample-to-sample variability of bacterial community provided important new clues to understand the batch-to-batch variability of herbal medicines.

Chapter 4 describes our study to characterize the LPS of R. aquatilis. In particular the study focused on the lipid A (LA) moiety of LPS, because the LA moiety is the so-called pathogen-associated molecular pattern (PAMP) responsible for the activation of innate immune cells, such as monocytes and macrophages. At that time, little was known about the LA variants produced by R. aquatilis. The working hypothesis explored here is that R. aquatilis produces structurally unique variants of LA that give rise to safe and effective immunostimulatory activity as observed for JTT. Fractions enriched with R. aquatilis LAs were purified and evaluated with chromatographic and immunological methods. Although we could not isolate individual R. aquatilis LAs, the study suggested that R. aquatilis LAs were structurally and immunologically different from endotoxic LAs. However, R. aquatilis LAs appear to retain some endotoxicity.

R. aquatilis LAs were not completely devoid of endotoxicity. Yet, A. sinensis is not known for its toxicity; it is in fact quite safe. Other research groups have observed similar endotoxic LAs in Echinacea, another immune-boosting herb. The observation of endotoxic LAs in different immune-boosting herbs raised a possibility that plant metabolites might attenuate undesirable immunological effects of endotoxic LAs, thereby making the immune-boosting herbs safe for human consumption. Chapter 5, therefore, investigates the role of plant compounds from another immunomodulatory medicinal plant, Ashwagandha, in attenuating the adverse effects of endotoxic LAs. The study indicated that plant-derived anti-inflammatory compounds might be responsible for the attenuation of the pro-inflammatory effect of endotoxic LAs.

In conclusion, this thesis work identified bacterial LPSs as the key immune-boosting factors of JTT and Ashwagandha. Our findings, together with the discovery of similar bacterial LPSs in Echinacea, suggest that the immunostimulatory activity of many other medicinal plants might originate from bacterial LPSs as well. Yet, this thesis work also indicated that plant-derived metabolites appear to play an important role to ensure the safety of these herbal medicines. Thus, in order to understand the safe and effective immunostimulatory activity of immune-boosting herbal medicines, it is essential to take a step back and look at the medicinal herb in totality, as a mixture of microbial metabolites and plant-derived compounds.

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