Recipient Organization
ALABAMA A&M UNIVERSITY
4900 MERIDIAN STREET
NORMAL,AL 35762
Performing Department
Food & Animal Sciences
Non Technical Summary
Solanum alkaloids are found in many species of plants, but are abundant in Solanaceae family, which include the potato. Because of their natural presence in the potato, many questions have been raised about the safety of the potato for consumption. While efforts are made to reduce levels through genetic engineering, the altered hybrids still require continuous screening to ensure they remain below the regulatory allowable limit before releasing to the general public. Thus, a method that maximizes complete extraction, provide better recovery and sensitive identification is required for reliable quantitative evaluation of Solanum alkaloids. Supercritical Fluid Extraction (SFE) method will be developed to extract and purify Solanum alkaloids in selected novelty potato cultivars. Although Solanum alkaloids are known to be toxic to humans, interestingly, these same alkaloids may also have beneficial effects in human health. This study, will aim to provide answers through integrated approaches to elucidate the molecular mechanisms of action of the safety and potential toxicity of Solanum alkaloids, specifically, Calystegine alkaloids, a lesser known, but potentially toxic Solanum alkaloid. The proposal will also train minority students, broaden their perspectives, introduce them to sophisticated techniques, instrumentation, and establish collaborations with scientists at institutional and government facilities that will be beneficial for their future careers.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Goals / Objectives
Solanum alkaloids are found in many species of plants, but are abundant in Solanaceae family, which include the potato. Because of their natural presence in the potato, many questions have been raised about the safety of the potato for consumption. While efforts are made to reduce levels through genetic engineering, the altered hybrids still require continuous screening to ensure they remain below the regulatory allowable limit before releasing to the general public. First, the study will embark on the task to optimize extraction techniques/methods using SFE to provide better extraction, and quantification of Solanum alkaloids. Afterwards, to fully understand the total health impact of Solanum alkaloids the study will aim to provide answers through integrated approaches to elucidate the bioefficacy Solanum alkaloids. Therefore, in objective 1, an SFE method will be developed to extract and purify Solanum alkaloids in selected novelty potato cultivars grown at the Alabama A&M University (AAMU) Winfred Thomas Agriculture Research Station (WTARS) Meridianville, Alabama. Since toxicity and a number of health benefits has been described for Solanum alkaloids, a better understanding on the health impact of these compounds is needed. So, objective 2 will utilize nutrigenomics approaches to elucidate the molecular mechanisms of the toxicity/efficacy of Solanum alkaloids, particularly Calystegine alkaloids, a lesser known, but potentially toxic Solanum alkaloid using an in vitro and in vivo model system. In objective 3, we will determine the extent to which Solanum alkaloids; alter gut barrier function and integrity. This study, will aim to provide answers through integrated approaches to elucidate the molecular mechanisms of action of the safety and potential toxicity of Solanum alkaloids, specifically, Calystegine alkaloids, a lesser known, but potentially toxic Solanum alkaloid.
Project Methods
(Obj.1) Upon harvesting of novelty potatoes from WTARS at AAMU, samples from each cultivar and for each harvest replicate will be obtained and used for analysis. Samples will undergo freeze-drying, and then ground. The ground samples will be enclosed in opaque containers and stored at −20 °C under argon prior to extraction and analysis. Prior to SFE extractions, a portion of the samples will be subjected to traditional extraction procedures and physicochemical analysis will be performed. Supercritical Extraction of Glycoalkaloids: A supercritical extraction unit equipped with a pump will be used to extract glycoalkaloids. Briefly, samples will be placed in an extraction vessel and charged with SC-CO2 at different extraction parameters (operation pressures, temperatures, time and co-solvents. The static and dynamic extraction period will be kept constant at selected time periods. The extracts will be collected and stored at -80C until analysis. SFE procedures will be optimized in order to maximize extraction yield and preserve quality of Solanum alkaloids. The extracted Solanum alkaloids will be quantitatively estimated by reverse-phase (RP) HPLC coupled to diode array detector and also by HPLC-MS. Extracts obtained from SFE will be compared to those obtained by traditional and CO2 extraction methods. (Obj. 2A) To elucidate the bioefficacy of Solanum alkaloids (single and binary) using an in vitro model system: Human cell lines (HepaRG and HepG2) will be cultured in appropriate/recommended medium supplemented with fetal bovine serum (FBS) and antibiotics. 24-48h post seeding, cells will be exposed to doses/concentrations of Solanum alkaloids under investigation. Acute and chronic exposure levels will be measured at selected time periods. Medium containing test compounds will be collected and cells will be harvested and used for the determination of therapeutic and toxicity endpoints. Molecular mechanisms involved in the gene expression of toxicity and therapeutic endpoints will be determined using microarray analysis (up to 10 ten gene lists/ biomarkers will be used for evaluating the endpoints) (Obj. 2B) Toxicity Assessment of Solanum alkaloids and the Underlying Mechanism in C. Elegans Model: The nematode C. elegans will be used to decipher the toxic/therapeutic properties of Calystegine alkaloids. C. elegans strains will be propagated following published procedures. After reaching maturity, C. elegans strains will be exposed to different concentrations of Calystegine alkaloids extracts and an appropriate volume of DMSO as solvent control. Exposed nematodes will be assessment toxicity endpoints, (i.e., lethality, lifespan, growth, brood size, locomotion behavior, and ROS production as the endpoints. Effects of Calystegine alkaloids exposure on alteration of expression patterns of genes related to toxicity will be determined using whole genome DNA-microarrays and microRNA (miRNA) microarray with C. elegans.(Obj. 3) Determine the extent to which Solanum alkaloids alter gut barrier function and integrity in vivo rodent models. F344 male rats will be fed by gavage with extracts of Solanum alkaloids. Histopathological and biochemical studies will be performed. Effect of Solanum alkaloids on inflammation markers such as interleukin (IL)-12, tumor necrosis factor (TNF)-α), IL-10, IL-6, IL-8 will be determined. Determination of Solanum alkaloids on gut barrier function/colon injury will be assessed via mRNA levels of critical tight junction proteins that control the integrity of gut barrier (proteins occludin and zonula occludens 1). Protein expression levels of markers of goblet cell function and assessment of mucins will also be determined.