Dissertations, Theses, and Capstone Projects

Date of Degree

2-2014

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Edward J. Kennelly

Subject Categories

Agricultural Science | Agriculture | Allergy and Immunology | Chemistry | Immunology and Infectious Disease | Medical Immunology | Plant Sciences

Keywords

adaptogen, chemometrics, diterpene, ethnobotany, immunology, mass spectrometry

Abstract

An ethnobotanical, ecological and LC-MS-based chemometric investigation of Phaleria nisidai, a traditional adaptogen containing diterpene esters from Palau, Micronesia

Palau is a country with a rich heritage of traditional medicine still being practiced. One of the most popular and respected remedies in Palau is a tea made from fresh leaves of Phaleria nisidai Kaneh. (Thymelaeaceae). Interviews conducted to determine the use of this plant revealed that it is employed non-specifically to treat a variety of general health concerns. Its use as a prophylactic to keep away sickness, as a 'system cleaner', as well as for strength and energy indicate that it is being used as an adaptogen, a medicine taken routinely to help adapt to external pathogenic, mental or physical stress. A series of in vitro immunological assays were conducted to determine the effect of crude extracts and guide phytochemical fractionation of this plant to identify immunostimulant compounds.

Phaleria nisidai is in the family Thymelaeaceae, one of two plant families known to produce diterpene ester compounds. Multivariate analysis to compare mass spectral data of active and inactive fractions allowed for the identification of several daphnane diterpene esters as tentative active marker compounds. Simplexin, the marker contributing most to differentiation of active and inactive fractions, was active when tested alone for stimulation of cytokine output by peripheral blood mononuclear cells (PBMCs). This demonstrated that a chemometric approach to active compound determination as opposed to traditional time- and resource-intensive bioactivity-guided purification methods to determine active compounds in a plant matrix was successful. This work also confirms a methanol extract of this plant and one of its constituents has activity as an immunostimulant, verifying its traditional use. This is also the first report of diterpene esters in the genus Phaleria.

Simplexin and other diterpene esters have shown cocarcinogenic and irritant activity in cell and mouse models and have caused gastric and pulmonary problems in animals grazing on plants containing these compounds. However, these compounds have also shown anti-HIV and anticancer activity. To determine if Palauans are ingesting diterpene esters in traditional preparations of Phaleria nisidai, simplexin, acetoxyhuratoxin and huratoxin were quantified from methanol and aqueous extracts prepared in the lab and in aqueous infusions prepared by six traditional healers in Palau. Diterpenes were not detected in traditional aqueous extracts prepared by healers in Palau, or in aqueous extracts prepared in our lab, but were detected in the methanol extract. PBMC proliferation as well as their production of IFNγ was measured and it was found that aqueous extracts induce both PBMC proliferation as well as an increase in IFNγ production, although these effects were milder and significantly less than the activity demonstrated by the methanol extract. This further validated the traditional use of P. nisidai as an immunestimulating adaptogen and allayed concerns about public health issues with chronic ingestion of aqueous P. nisidai infusions.

Levels of simplexin, acetoxyhuratoxin, huratoxin and a bioactive xanthone, mangiferin, were analyzed in leaf samples from 227 trees collected from 92 populations of Phaleria nisidai. All Rock Island populations contained minute daphnane concentrations and lower amounts of mangiferin than populations on the largest Palauan island, Babeldaob. Savannah, scrub savanna and mature forest habitats all contained populations with exceptionally high levels of daphnanes, while the mangiferin content across habitats was more homogenous. Analysis of these compounds by geographic variables can be useful to identify high-yielding chemotypes for biomedical or toxicological studies.

These studies demonstrate that Palauan traditional medicine can offer a source of plant metabolites with potent biological activity that corresponds to their traditional use. The discovery of simplexin and other diterpene esters in a plant consumed daily indicates that Palauans have found a way to selectively extract beneficial compounds while minimizing exposure to potentially harmful metabolites. The possibility also exists that Palauans have uncovered a low-dose therapeutic window at which diterpene esters can provide immunostimulant benefits while avoiding toxicological risks. The pharmacological potential of simplexin and related diterpenes in Phaleria nisidai should be reevaluated in light of these findings.

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