Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Navindra P. Seeram

Abstract

The objectives of this work are the isolation, purification, structure elucidation, and biological evaluation of bioactive secondary metabolites, primarily tannins and diterpenoids, of two plant species: silver maple (Acer saccharinum) and Euphorbia saudiarabica, respectively.

Based on the ethnopharmacological literature, Acer saccharinum is a traditional medicinal plant that has been used by the people of North America. According to previous phytopharmacological studies by our group, over 70 phytochemicals, primarily tannins and flavonoids, have been discovered from three different maple species, including sugar (Acer saccharum), red (Acer rubrum), and sycamore (Acer pseudoplatanus) maples. Remarkably, the red maple species yielded very interesting bioactive (α-glucosidase inhibitors) glucitol-core containing gallotannins (GCGs). Therefore, to gain a better understanding of the phytochemical composition of other maple species, such as silver maple, the fresh leaves of silver maple were collected and extracted. Following this, the crude extracts of different polarities were prepared, and then all extracts initially screened using reversed-phase high-performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC), and the Folin-Ciocalteau assay for quantifying polyphenol contents. Then, the isolation procedure was carried out using multiple techniques, including open column chromatography (OCC), TLC, and RP-HPLC on the highest total polyphenol content extract (which was the ethyl acetate [EtOAc] extract). Using high-performance liquid chromatography with diode-array detector (HPLC-DAD) analyses using previously isolated standards, nine compounds, including six GCGs, were identified as ginnalins A–C (1–3), maplexins B, D, and F (4–6), methyl syringate (7), methyl gallate (8), and 3-methoxy-4-hydroxyphenol-1-β-D-(6-galloyl)-glucopyranoside (9). Additionally, pubineroid A (10), a sesquiterpenoid, was isolated and identified (by NMR).

The Euphorbia genus is one of the six largest genera of flowering plants which are known to produce several bioactive secondary metabolites, primarily diterpenes such as jatrophane and lathyrane-type. Euphorbia saudiarabica, which is endemic to Saudi Arabia, is well characterized by the production of a milky irritant latex. It is one of the plant species in the region that has not been studied. To increase the knowledge of the constituents of E. saudiarabica, a comprehensive phytochemical investigation was conducted, leading to the isolation of five new lathyrane-type diterpenoids (1-5) named saudiarabicain A–E. These compounds were evaluated for their cytotoxic activities against three cell lines, including, murine B16F10 melanoma cells, human HaCat keratinocytes, and human SH-SY5Y neuroblastoma cells. In B16F10 melanoma cells, only compounds 2, 3, and 4 induced significant toxicity, with compound 2 having the greatest range of toxicity from 100 μM (49.63% decrease) to 20 μM (14.69% decrease). In human keratinocytes (HaCat), each compound caused a decrease in cell viability with concentrations above 40 μM. At a concentration of 30 μM, compounds 3 and 4 caused reductions in cell viability by approximately 12.42% and 17.92%, respectively. Compound 3 was able to decrease viability by as much as 93.63% at 50 μM. Lastly, in SH-SY5Y cells, all of thecompounds (1–5) induced a cytotoxic effect at concentrations of 50 μM and above.

Lastly, for the first time, a qualitative analysis of the flavonoids present in a methanol extract of the aerial parts of E. saudiarabica using LC-ESI-TOF-MS/MS was conducted. This led to the identification of 36 flavonoids including 7 flavonoids based on the galangin aglycon, 9 flavonoids based on the apigenin aglycon, and 20 flavonoids originated from luteolin.

In conclusion, this dissertation provids insights about the phenolic constituents, primarily tannins, of silver maple (Acer saccharinum), compared to other common maple species, mainly the sugar (A. saccharum), red (A.rubrum), and sycamore (A. pseudoplatanus) maples which were previously investigated by our group. In addition, the endemic Euphorbia saudiarabica species from Saudi Arabia yielded five new lathyrane-type of diterpenoids. All compounds evaluated for their cytotoxicity showed moderate effects. Lastly, a rapid and reliable method employing LC-ESI-TOF-MS/MS was developed for the identification of flavonoids in the aerial parts of E. saudiarabica.

Available for download on Monday, April 20, 2020

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