Analytical Chemistry 243 Independent Research Project:
Effect of biotic elicitors on isothiocyanate and other phytochemical levels in broccoli microgreens
Chronic diseases are a major health problem in the U.S. and one approach to combatting this problem is to consume a plant-rich diet. Interest in food and nutrition has evolved from the prevention of deficiencies (vitamins, minerals) to the prevention of excesses (obesity). Although the precise mechanisms for health promotion are unclear, interest in the chemical compositions, growing conditions, and biological efficacies of different plant species are of interest. To aid in this effort, we will be conducting original research to measure the chemical compositions of broccoli microgreens grown under different conditions. Specifically, we are interested in answering the question: What biotic elicitors enhance isothiocyanate (ITC) breakdown product concentrations in these species? Are other phytochemical concentrations affected by these elicitors?
During the course of this project, students will learn to:
- Design and execute statistically relevant experiments for measuring a suite of chemicals from treated plants;
- Collect, analyze, and interpret qualitative and quantitative data to form and support conclusions;
- Troubleshoot and revise experiments to demonstrate accurate scientific results;
- Clearly communicate experimental results within statistical limits;
- Work effectively in teams, including individual and joint accountability; assign roles, responsibilities, and tasks; monitor progress; meet deadlines; and integrate individual contributions into a final deliverable (poster for the Science Symposium).
Microgreens are young vegetables harvested between 1-2 weeks from seeding. They consist of three parts: a central stem, cotyledon leaf or leaves, and pair of true leaves (Fig. 1). They are different than sprouts or baby greens because they are harvested without the roots. Interest in microgreens has increased because they contain greater amounts of nutrients and health-promoting micronutrients compared to their mature counterparts. Because they are so rich in nutrients, smaller amounts may provide similar nutritional benefits as larger quantities of mature vegetables. Additionally, the chemical composition of microgreens differs markedly from their mature form. Because of this, there is limited information on the nutrient content of microgreens.
Consumption of glucosinolates (GLS) from Brassica-rich plant species such as broccoli is associated with a decreased risk from cancer. These sulfur-rich secondary metabolites play a role in the plant’s defense system against biotic stressors, as well as affect the taste and play a role in the plant’s health-promoting properties. When glucosinolates break down, several breakdown products can result (Fig. 2), each of which has different health-promoting properties. Ideally, to obtain the most beneficial health effects from the vegetable, it is advantageous to have more isothiocyanates (ITC) and fewer nitriles (CN) as breakdown products. Several variables affect the amount of glucosinolate a plant contains, including the identity of the microgreen as well as the conditions under which it’s grown.
Biotic elicitors are compounds that can stimulate stress responses in plants and cause up-regulation or down-regulation of secondary metabolites such as glucosinolates. As such, they are useful tools as a means of increasing production of secondary metabolites such as glucosinolates. In this lab, we will examine the effect of exposure to different elicitors on the isothiocyanate concentration in broccoli microgreens. Each student pair will have plants from a different elicitor treatment, and therefore, results will be unique for each group.
Which elicitor (if any) up-regulates the production of isothiocyanates extracted from broccoli microgreens? How do other chemical species respond to exposure to these elicitors?
Each pair of researchers will be provided with a unique frozen (-80°C) lyophilized sample of treated broccoli microgreens, as well as a control sample. Your task over the next 4 weeks will be to measure the composition of the following chemicals within your treated plants:
- Total Flavonoids
- Total Phenolics
- Radical scavenging capacity
- Total sulforaphane
Reference: Lopez-Cervantes et al., 2013, International J of Food Sci and Technol. 48, 2267-2275.
To do this, you will need to come to lab each week with a protocol in hand that you will follow to obtain the information above. The procedure must be sufficiently detailed so that you can follow it without hitting any roadblocks during the lab time. To facilitate this, I will have you upload your typed procedures by 9:00 am the morning of lab. I will examine them and if I find any issues, I will alert you prior to lab. The protocols will be graded as part of the lab write-up. Each group can turn in one protocol for consideration, a product of joint effort.
What should the protocol contain?
Components of Your Experimental Plan
Plan of Analysis
Describe the individual steps of your analysis in detail. Be sure to address the issues of sampling, sample preparation and/or separations, clean-up steps, measurement steps, standardization, and data analysis (with the statistics that will be employed).
Schedule of Laboratory Time (can be in the form of a flow chart)
In this section, you will describe how you expect to use the available laboratory period to carry out the proposed analysis. Each step of the procedure outlined above should be accounted for somewhere in your time schedule. Note that it is impossible to guess how much time it will take to solve unforeseen problems. Therefore, be generous in the amount of time that you allow for each step, and perhaps reserve one lab period to work out any difficulties.
Apparatus and Supplies
List the chemicals, glassware, and instrumentation you expect to need to use for analysis.
Quantitation and Calibration
If you intend to quantify the amount of chemicals in your plant extract, you will need to calibrate the instrument. Include detailed instructions on how you will do this, including the concentrations, and conditions that you will use to run your analyses.
Important: For all the chemicals that you will be using for your experiment you are required to include material safety data sheet (MSDS) information from the manufacture. MSDS are available online from Sigma Aldrich and other chemical companies free of charge. Make sure to carefully read the MSDS for any potential hazards and handling requirements and discuss any of the requirements in your procedure.
Experimental Plan Evaluation Rubric (for each experiment)
Overall Plan of analysis. 35 points
|Schedule of laboratory time. 5 points||_____|
|Apparatus and chemicals list. 5 point||_____|
|Material Safety and Data Sheets Information (MSDS). 5 points||_____|
|GRAND TOTAL (50 points)||_____|