Tions, immunological response and vascular changes associated with 1379592 an HCD in CP21 zebrafish are similar to those seen in mammalian models of atherosclerosis. Besides numerous studies demonstrating that treatment of zebrafish with antihyperlipidemic drugs mirrors the response of humans to those drugs [14], [15], scientists are also beginning to test the ability of natural products to treat hypercholesterolemia. In the adult zebrafish, turmeric, laurel, cinnamon and clove reduced blood serum lipid and cholesterol levels [16], [17]. Additionally, BODIPY- cholesterol (BOD-CH) has been established as a marker of intravascular cholesterol levelsAutomated In Vivo Hypercholesterolemia Screenin the zebrafish and it was demonstrated that ground hawthorn leaves and flowers administered in the diet decrease intravascular BOD-CH fluorescence in zebrafish larvae [18]. Until recently, the ability to test natural product treatments in a food-based treatment paradigm via high-throughput screening has not been possible [2]. Here we develop and test an automated, zebrafish-based hypercholesterolemia treatment Pentagastrin chemical information screen focused on natural product drug discovery and amenable to high-throughput testing, which can also be utilized to test the efficacy of purified molecular pharmaceuticals. We utilize this method to test the ability of a methanolic hawthorn (Crataegus laevigata) leaf and flower extract (MHE) to impact hypercholesterolemia. Analyzing time varying cardiac variables is one of the most valuable assessments of a treatment’ overall physiological effects [19]. A treatment that influences cardiac function impacts flow throughout the entire organism. Manually analyzing and quantifying these data sets is time consuming. Further, making measurements on large numbers of organisms creates a significant amount of data to be analyzed. Depending on the complexity of data analysis, manual techniques can be tedious, do not take into account the entirety of the acquired time varying data, o may be prone to subjective biases. We have developed an automated system for analyzing high-speed confocal data of the zebrafish heartbeat, resulting in rapid analysis. We utilize our method to test the ability of MHE to influence cardiac function in the zebrafish.1b. Preparing Hawthorn ExtractThe leaves and flowers of Crataegus laevigata, obtained from Starwest Botanicals (Rancho Cordova, California), were crushed with mortar and pestle. Plant material was then weighed to 6.5 g and added to a 250 mL round bottom flask with Boileezer. Twohundred mL ofmethanol was added to th flask and refluxed for 
70 minutes. Filtrate was passed through Whatman 1 paper and solution was brought up to 250 mL with 80 methanol. This lead to a methanolic solution equivalent to 26 mg/mL pure plant product. Doses for administration in hypercholesterolemia screen were determined from an LD50 curve.2a. Feeding for Automated Hypercholesterolemia ScreenFor high-throughput analysis, 4 days post-fertilization (dpf) fish were fed a mixture that consisted of 2.5 v/v egg yolk in tank water in a method also described in [18]. After sonicating for 20 minutes at 5 minute intervals, 50 mM ezetimibe (Ryan Scientific) (from a stock concentration of 10 mg/mL in DMSO), or between 3.5?9.5 mg/mL methanolic extract of hawthorn leaves and flowers, combined with 2.5 mg/mL 23- (dipyrrometheneboron difluoride)-24-norcholesterol (BOD-CH. TopFluor, Avanti Polar Lipids) from 8 mL stock at a concentration of 0.3125 mg/mL in DMSO wer.Tions, immunological response and vascular changes associated with 1379592 an HCD in zebrafish are similar to those seen in mammalian models of atherosclerosis. Besides numerous studies demonstrating that treatment of zebrafish with antihyperlipidemic drugs mirrors the response of humans to those drugs [14], [15], scientists are also beginning to test the ability of natural products to treat hypercholesterolemia. In the adult zebrafish, turmeric, laurel, cinnamon and clove reduced blood serum lipid and cholesterol levels [16], [17]. Additionally, BODIPY- cholesterol (BOD-CH) has been established as a marker of intravascular cholesterol levelsAutomated In Vivo Hypercholesterolemia Screenin the zebrafish and it was demonstrated that ground hawthorn leaves and flowers administered in the diet decrease intravascular BOD-CH fluorescence in zebrafish larvae [18]. Until recently, the ability to test natural product treatments in a food-based treatment paradigm via high-throughput screening has not been possible [2]. Here we develop and test an automated, zebrafish-based hypercholesterolemia treatment screen focused on natural product drug discovery and amenable to high-throughput testing, which can also be utilized to test the efficacy of purified molecular pharmaceuticals. We utilize this method to test the ability of a methanolic hawthorn (Crataegus laevigata) leaf and flower extract (MHE) to impact hypercholesterolemia. Analyzing time varying cardiac variables is one of the most valuable assessments of a treatment’ overall physiological effects [19]. A treatment that influences cardiac function impacts flow throughout the entire organism. Manually analyzing and quantifying these data sets is time consuming. Further, making measurements on large numbers of organisms creates a significant amount of data to be analyzed. Depending on the complexity of data analysis, manual techniques can be tedious, do not take into account the entirety of the acquired time varying data, o may be prone to subjective biases. We have developed an automated system for analyzing high-speed confocal data of the zebrafish heartbeat, resulting in rapid analysis. We utilize our method to test the ability of MHE to influence cardiac function in the zebrafish.1b. Preparing Hawthorn ExtractThe leaves and flowers of Crataegus laevigata, obtained from Starwest Botanicals (Rancho Cordova, California), were crushed with mortar and pestle. Plant material was then weighed to 6.5 g and added to a 250 mL round bottom flask with Boileezer. Twohundred mL ofmethanol was added to th flask and refluxed for 70 minutes. Filtrate was passed through Whatman 1 paper and solution was brought up to 250 mL with 80 methanol. This lead to a methanolic solution equivalent to 26 mg/mL pure plant product. Doses for administration in hypercholesterolemia screen were determined from an LD50 curve.2a. Feeding for Automated Hypercholesterolemia ScreenFor high-throughput analysis, 4 days post-fertilization (dpf) fish were fed a mixture that consisted of 2.5 v/v egg yolk in tank water in a method also described in [18]. After sonicating for 20 minutes at 5 minute intervals, 50 mM ezetimibe (Ryan Scientific) (from a stock concentration of 10 mg/mL in DMSO), or between 3.5?9.5 mg/mL methanolic extract of hawthorn leaves and flowers, combined with 2.5 mg/mL 23- 
(dipyrrometheneboron difluoride)-24-norcholesterol (BOD-CH. TopFluor, Avanti Polar Lipids) from 8 mL stock at a concentration of 0.3125 mg/mL in DMSO wer.
