Electronic Theses and Dissertations

Date of Award


Document Type


Degree Name

Ph.D. in Pharmaceutical Sciences

First Advisor

Ikhlas A. Khan

Second Advisor

Sameer Ross

Third Advisor

David A. Colby

Relational Format



Natural products have been used as a primary means of treating illness since ancient times, and they remain an important pool of U.S. FDA approved drugs. Approximately 33% of all current pharmaceutical medications discovered between 1981 and 2014 are either natural products or derived from natural products (Newman and Cragg 2016). The process of isolating specialized metabolites from medicinal plants has led to the development of particularly important medicines worldwide (Rakotomalala, Agard et al. 2013) such as Taxol® from Taxus brevifolia Nutt. (Taxaceae) and salicylic acid from the bark of Salix spp. This research is composed of five chapters; the first is focused on drug discovery, whereas the rest are focused on efforts to assess the quality and safety of copaiba oil, which is used as a dietary supplement in various parts of the world, including the United States.Mimosa pudica is marketed as a dietary supplement in the United States with claims that it supports the immune system. It is known that M. pudica grows with related species and has been misidentified with a closely related invasive species, M. pigra. A Brazilian study screened over 60 plants for anti-dermatophyte activity, and dichloromethane fractions from the methanolic extract of M. pigra showed the lowest MIC values (1.9 µg/mL) without DNA destruction at 10 and 50 µg/mL of cell viability of human leukocytes; however no further work was conducted to determine the specialized metabolite responsible for the activity shown. (de Morais, Scopel et al. 2017). Therefore, my research is aimed at isolating specialized metabolites that could be utilized as chemical markers to distinguish between M. pudica and M. pigra and which also have promising antifungal activity without human toxicity concerns.

Powdered M. pigra leaves were processed via percolation in methanol at room temperature to create a crude extract after the removal of solvent under reduced pressure. This extract was fractionated by vacuum liquid chromatography (VLC) using reversed-phase C-18 silica with a gradient elution of methanol and water (0:1 to 1:0), resulting in 10 fractions. The crude fractions were tested against Candida albicans, and several of them showed promising anti-candida activity. The obtained fractions were subjected to repeated column chromatography over Sephadex LH-20, RP-18 silica, and normal phase silica to yield eight phenolic compounds. The isolated specialized metabolites were again tested against C. albicans to determine which were responsible for the anti-candida activity. We suggest that the chemical constituents isolated in this study could be used as chemical markers to differentiate M. pigra-based raw materials in various finished products, including dietary supplements that purportedly contain M. pudica.

My second project focuses on assessing the safety and quality of copaiba essential oils. Copaiba oil use has been reported since the 16th century in Amazon traditional medicine, especially as an anti-inflammatory ingredient and for wound healing (da Silva, Puziol Pde et al. 2012, da Trindade, da Silva et al. 2018). This oil’s use continues today, and it is sold in various parts of the world, including the United States.

Adulteration of copaiba oil or resin might occur because of the time-consuming extraction process and demand exceeding the supply(Barbosa, Yoshida et al. 2009). According to the literature, there are two methodscommonly used for copaiba oil adulteration: incorporating a low-cost vegetable oil such as soybean or mineral oil, or adding another inferior essential oil that has a similar density and flavor (Barbosa, Yoshida et al. 2009). Despite the importance of copaiba oil in folk medicine and its economic significance, limited studies have reported a validated GC/MS method for evaluating the quality of copaiba oil (Sousa, Brancalion et al. 2011). A major limitation of the conventional GC method is that it can evaluate the concentration of volatile organic compounds, but it cannot detect nonvolatile adulterants. In addition, the main chemical constituents of copaiba oleoresin are mixtures of diterpenes and sesquiterpenes (Xavier-Junior, Maciuk et al. 2017), which are popular bioactive compounds due to their beneficial effects on human health. However, the potential toxicity of sesquiterpenes and possible herb–drug interactions are often neglected. A case of liver damage in an elderly patient was reported after the use of Copaifera langsdorffii Desf. (Fabaceae) and Hypericum perforatum L. (Hypericaceae) along with levothyroxine (Agollo, Miszputen et al. 2014). Exploring the safety and quality of herbal supplements is crucial for public health. To our knowledge, there are no studies on the impact of copaiba oleoresin or its chemical constituents on Cytochrome P450 (CYP450) enzymes or on the transcriptional activity of the Pregnane X Receptor (PXR).

We aimed to develop a rapid, simple, and robust method to evaluate and detect adulteration in copaiba oil products. We also aimed to evaluate the potential of copaiba chemical constituents to cause herb–drug interactions by evaluating their impact on CYP3A4, CYP1A2, and PXR enzymes. Several samples of copaiba were subjected to repeated silica gel chromatography to isolate and purify their chemical constituents. The isolated specialized metabolites were used to evaluate the safety and quality of copaiba oil products. A large set of copaiba products were subjected to GC/MS, principal component, and SFC/MS analysis to evaluate their quality. We also assessed the potential of 19 specialized metabolites to cause herb–drug interactions by evaluating their impacts on CYP3A4, CYP1A2, and PXR enzymes.

This isolation work resulted in the separation off 12 sesquiterpenes/sesquiterpenoids and 2 diterpenoids from several samples of copaiba oil. Among them, (E)-2,6,10-trimethyldodec-8-en-2-ol was found to be a novel compound that has not been previously described. The GC/MS, principal component, and SFC/MS analyses revealed that several samples contained abundant triglycerides, which are a major components of vegetable oil. At a concentration of 30 µM, 19 specialized metabolites from copaiba oil revealed mild to moderate activation of the PXR signaling pathway. Even though the tested compounds were able to activate the PXR signaling pathway, only two, (-)-kolavelool and ?11(12)-eremophilen-10?-ol, were able to inhibit CYP3A4 and CYP1A2. (-)-Kolavelool and ?11(12)-eremophilen-10?-ol strongly inhibit CYP3A4 and CYP1A2, with an IC50 value of 3.1 and 7 µM, respectively. This research might shed some light on the quality of marketed copaiba oil products and the potential herb-drug interactions of several sesquiterpenes that can be found in other dietary supplements.



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