Chemical Profiling of Five Canadian Haskap Berry Varieties and the Roles of their Phenolic Extracts in Modulating Cellular Stress in Human Fibroblasts
Date
2020-01-15
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ORCID
Type
Thesis
Degree Level
Masters
Abstract
Interest in plant phenolic structure(s) and their applications has grown due to their potent antioxidant and nutraceutical potential. Many plant species produce phenolics for a variety of functions, with high levels of these compounds often found in berry fruits. When applied to cells and organisms, berry fruit phenolics have shown anti-aging, anticancer, and anti-inflammatory activities. Haskap berries (Lonicera caerulea) are an edible honeysuckle species that contain very high levels of phenolics when compared to other commercial fruits. In this study, the chemical and physicochemical composition of five Canadian haskap berry varieties (Aurora, Blizzard, Honey Bee, Indigo Gem, and Tundra) was determined, with a focus on phenolic analysis and the in vitro health-promoting potential of these compounds. Total phenolic chromatographic indices (TPCI) using high performance liquid chromatography with photodiode array detection (HPLC-PDA) identified anthocyanins as the major phenolic subclass with cyanidin-3-O-glucoside, cyanidin-3,5-O-diglucoside, and cyanidin-3-O-rutinoside being the most prevalent. HPLC-tandem mass spectrometry (HPLC-MS/MS) was also employed to identify haskap phenolic structures including three previously unreported phenolic compounds. In vitro free radical scavenging assays showed that these haskap varieties had higher radical scavenging abilities than many other fruits (e.g. blueberry) with the Tundra variety demonstrating the highest capacity. These experiments led to selection of the Tundra variety for use in tissue culture assays due to its high phenolic content and free radical scavenging abilities. Phenolic extracts and fractions from Tundra haskap berries were produced using solid phase extraction, so as to separate phenolic subclasses for comparative cell line treatments in vitro.
In tissue culture assays, phenolic extracts from the Tundra variety slowed the growth of fibroblasts isolated from healthy donors (2DD) and immortalized (NB1 hTERT) fibroblasts without inducing cell death, indicating potential biological impacts similar to resveratrol, a fruit phenolic known to promote cellular health. Tundra phenolic fractions also increased cellular protein and activity levels of Sirtuin-1 (SIRT1), decreased levels of the pro-inflammatory phospho-nuclear factor kappa B (phospho-NF-κB p65) and the phosphorylated mammalian target of rapamycin (phospho-mTOR), and increased nuclear factor erythroid 2 (Nrf2). These observations are significant as these activities have been linked to slowed aging, improved intracellular radical scavenging (resistance to oxidative stress), and decreased inflammatory responses. Tundra phenolic fraction activities were shown to rely primarily on SIRT1 by employing lentiviral and siRNA SIRT1 knockdown cells, with additional SIRT1-independent mechanism(s) related to Nrf2 upregulation. These fractions also showed the ability to decrease transcript levels of select pro-inflammatory cytokines and antioxidant enzymes, demonstrating further anti-inflammatory activities along with decreased requirements for cellular antioxidant mechanisms. These experimental results indicated that Saskatchewan-bred haskaps have the potential to promote health in vitro by mediating cellular stress responses and may be candidates for use as nutraceutical supplements.
Description
Keywords
haskaps, phenolic, phenolics, antioxidants, free radicals, fibroblasts, stress, SIRT1
Citation
Degree
Master of Science (M.Sc.)
Department
Food and Bioproduct Sciences
Program
Food Science