Text

theses

Vitis vinifera (grape) is one of the most widely cultivated fruit species in the world and the total production of grapes worldwide is approximately 60 million tons. Grape and grape-derived products including wine, juice, foods and condiments contains a unique mixture of bioactive dietary phenolics, which have long been reported to have antioxidant and positive health promoting effects and associated with the prevention of numerous diseases including cardiovascular and neurodegenerative diseases as well as several forms of cancers. Most of the grape polyphenols can be found in grape juice after extraction through pressing, and grape juice is one of the main processed products of grapes on the US market. Grape seeds are also one of the major industrial byproducts of the grape winemaking process, and more than 70% of grape phenolics are retained in skins and seeds. As such, grape seed extracts (GSE) are another popular and widely used dietary supplement (DS) in the USA. Trans-Resveratrol (RSV, systematically named as trans-3,5,4′-trihydroxystilbene), produced by grape berries of Vitis varieties in response to ultraviolet (UV) -irradiation, is a compound found in the skin of grapes with well recognized antioxidant properties. The potential role of RSV in health promotion, such as the prevention and treatment of diabetes, cancer, obesity, pain, inflammation, tissue damage, and even ‘aging’, has made it increasingly popular in recent years as a DS. The dissertation research conducted was done in support and as part of a larger research project that is seeking to understand the influence of dietary botanical supplements on biological and behavioral resilience as part of the Consortium for Advancing Research on Botanical and Other Natural Products (https://ods.od.nih.gov/Research/Dietary_Supplement_Research_Centers.aspx) Program. More specifically, this research hypothesized that the dietary consumption of grape derived DSs and the major polyphenols in these products would support such resilience if the bioactives would be released at specific concentrations over time thus bridging the link between chemistry, product formulation and delivery with acceptable predictable release times to achieve a more robust quality control. This dissertation research was designed to support testing such a hypothesis by first addressing issues of botanical authentication, the chemical standardization and physio-chemical properties of the botanicals, and the metabolites following consumption. This becomes particularly important given that adulteration of grape-derived botanical products can be a significant problem. Therefore, this research first set out to ensure proper botanical authentication and then developed validated analytical protocols to quantitate the chemical constituents in the botanicals and to then understand and identify the metabolites following consumption particularly those that may be responsible for or associated with the bioactivities of interest. Chapter 1 first briefly reviews the history and uses of grapes and then focuses on reviewing grape phytochemistry to set the context for the natural products and bioactive constituents of interest for this research. In Chapter 2, we first collected three kinds of grape derived DS: the manufactured grape seed polyphenol extract (GSE) and trans-resveratrol (RSV) capsules and Concord Grape Juice (CGJ) along with the corresponding original drug materials, and then analyzed and chemically profiled them using various liquid chromatography coupled with UV and mass spectrometry (LC-UV-MS) methods and associated techniques that were developed during this research. The new and/or improved methods and techniques developed were efficiently applied to the investigation and quality control of the phytochemistry in those grape derived DS. The weight variation of GSE and RSV capsules was also evaluated according to the US Pharmacopeia (USP) tests. Results indicated that the total identified polyphenol content in each grape seed extract (GSE) capsule and CGJ was very similar, and all GSE and RSV capsules ‘passed’ the USP the content/weight uniformity test. Next, we focused on ensuring that the delivery of the botanical GSE and RSV capsules would be effective for use in human clinical trial. Chapter 3 then addressed the next step relative to the formulation and dissolution of GSE and RSV capsules to ensure that the deliverable would be uniform for animal and human clinical trials. Results indicated that the released trans-resveratrol for RSV capsules in both of the dissolution media (pH 1.2 0.1 N HCl media and pH 4.6 acetate buffer) meets the USP standards, and that for the GSE capsules, each of the four main marker compounds passed the dissolution test in the HCl medium (pH 1.2) but did not reach a 75% release within 60 min in the acetate buffer (pH 4.6). The reasons for dissolution test failure can be broadly classified into two categories: dissolution procedure and capsule quality. It is possible that these marker compounds may be extracted less efficiently by the dissolution media than by the sonicated organic solvent mixtures. Also, in this study, dissolution method type I was used where the capsules were placed inside the basket with 40 mesh. The openings of 40 mesh size may be small to allow the release of all the dissolved products. Moreover, previous study reported that some products were sensitive to chosen test conditions, including beaker size and the equipment used in dissolution study. Due to the limitation of our lab and the costs, we did not optimize these parameters, and these aspects might be further improved. These promising results indicated that when our botanical formulated products (RSV and GSE) were subjected to the general USP dissolution screening assays, each were found to be acceptable. That is, we observed the successful release of RSV capsules in HCl medium and acetate buffer and GSE capsules (in HCl medium) but may be inadequate for GSE capsules in acetate buffer.

Chapter 4 shifted the research focus from the requisite foundation needed in chemical characterization and quality control of each botanical to bioanalysis of grape derived DS. Malvidin-3-O-glucoside is a natural anthocyanin, present at different proportions in a variety of purple grapes. In this chapter, a UHPLC-QQQ/MS method was developed to determine malvidin- 3-O-glucoside and its derivative in a mouse model of Chronic Unpredictable Stress (CUS), consisting of a plethora of different stressors. We observed that malvidin-3-O-glucoside can be rapidly degraded through hepatic metabolism via glucuronidation.

In chapter 5, we developed and validated a new high-throughput, sensitive and reproducible analytical method for the simultaneous analysis of 31 grape phenolic compounds in human urine and metabolites using Oasis PRiME HLB cleanup for sample preparation combined with ultra-performance liquid chromatography with triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Using our purposed method, the accuracy achieved 69.3% ~ 134.9% (except for six compounds), the recovery achieved 52.4% ~ 134.7% (except for two very polar compounds). For each of the 31 target analytes, the value of intra-day precision was less than 14.3%. The value of inter-day precision was slightly higher than intra-day precision, with a range of 0.7% ~ 19.1%. Results from analysis of variance (ANOVA) and principal component analysis (PCA) indicates there was no difference in the value of accuracy and recovery between different gender or BMI (>30) using our purposed cleanup and UHPLC-QqQ-MS/MS method, which suggest this newly developed and validated method could serve as a powerful tool for analyzing grape phenolic compounds and metabolites in human urine samples.

http://dissertations.umi.com/gsnb.rutgers:12250

Ph.D.

Includes bibliographical references

doi:10.7282/t3-m5gj-2115

The author owns the copyright to this work.