Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

First Advisor

Navindra Seeram

Abstract

This study investigated the phytochemical constituents, primarily gallotannins, present in a proprietary extract, namely MaplifaTM, from leaves of the red maple (Acer rubrum L.) species as well as their biological activities and mechanisms of action. Although the red maple species has been traditionally used as folk medicine by Native American Indians for numerous health benefits, the bioactive chemical constituents of the leaves of the red maple still remain unknown. This study carried out the identification of phytochemicals targeting gallotannins, a class of polyphenols, from red maple leaves by using various chromatographic separation techniques and spectroscopic approaches. Furthermore, compounds from the red maple species were evaluated for bioactivities including anti-oxidant, anti-diabetic (α-glucosidase enzyme inhibition and inhibition of advanced glycation end products, AGEs) and skin lightening effects (anti-tyrosinase and anti-melanogenesis) by using a panel of in-house bioassays. In addition, the mechanisms of the inhibitory effects of red maple phytochemicals on α-glucosidase were characterized by using kinetic assay, fluorescence assay, FTIR and CD (circular dichroism) spectra. Moreover, the mechanisms of inhibition of the gallotannins on the formation of AGEs were elucidated at each individual stage of AGEs formation by applying MALDI-TOF mass spectroscopy, HPLC-FL analysis, G.K. peptide assays, and assessing the conformational changes induced by protein glycation by using CD. Plant derived natural products have served as an important resource for medicinal compounds over decades. The red maple is an indigenous plant species of eastern North America and was extensively used as an herbal remedy by the indigenous peoples of eastern North American region for many ailments including abdominal pain, diarrhea and eyes diseases.
Recent studies have shown that extracts of red maple plant parts exhibit free radical scavenging capacities and this antioxidant property was associated with its polyphenolic content. Red maple extracts have also been investigated for their antimicrobial and anticancer effects. In addition, our group has recently demonstrated that a red maple bark extract was able to decrease blood glucose levels in mice after sucrose loading indicating the presence of bioactive compounds that could be relevant to diabetes management. Furthermore, our laboratory has recently isolated a number of new gallotannins, named maplexins A-I, from the stem and bark of the red maple species and has shown that these compounds display potent α-glucosidase inhibitory activity in vitro. As the total polyphenolic content level in red maple leaves is higher than in bark, it is logical to investigate the chemical constituents of red maple leaves for gallotannins which could also serve as α-glucosidase inhibitors. Therefore, we propose a phytochemical study to develop a proprietary extract from red maple leaves and to isolate and identify phenolic compounds therein and evaluate these compounds for their in vitro antioxidant and anti-diabetic activities. This study also proposes to elucidate the mechanisms of inhibitory effects of maple gallotannins on the α-glucosidase enzyme by using various biophysical tools. Understanding the mechanisms of inhibitory action of these compounds will be critical for the further development of α-glucosidase inhibitors from maple for diabetes management. Beside the inhibitory effects on the α-glucosidase enzyme, the red maple gallotannins were also evaluated for their inhibitory effects on the formation of advanced glycation end products (AGEs), another therapeutic target of diabetes. AGEs are a heterogeneous group of irreversible adducts from non-enzymatic glucose-protein condensation reactions. It has been reported that AGEs as oxidative derivatives are increasingly being implicated as a potential risk for diabetes. Numerous studies have demonstrated that phenolics with anti-oxidative capacity from fruits or vegetables could effectively interrupt the condensation reaction of glucose and proteins and further decrease the formation of AGEs. Therefore, we hypothesize that gallotannins from red maple species might act as antioxidants and inhibit the formation of AGEs. In addition to anti-diabetic activities, the skin lightening effects of red maple gallotannins, as a treatment strategy for hyperpigmentary disorders, were also evaluated in this study. Hyperpigmentary disorders, such as melasma, age spot, freckles and lentigies, are caused by the accumulation of abnormal melanin. In the melanin biosynthetic pathway, tyrosinase is an exclusive enzyme responsible for the formation of melanins. Emerging data suggests that several plant-derived constituents may act as natural tyrosinase inhibitors which are of interest to the food (as anti-browning agents) and cosmetic (as skin lightening agents) industries. Therefore, we also evaluated the anti-tyrosinase and anti-melanogenic properties of gallotannins isolated from red maple leaves.

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