best way to test flavonoids

[deleted-345229]

I was assigned the topic flavonoids for my 11th grade science research project. I have found a lot of information about their health benefits, the different types and even a little about how their properties change in acidic versus base solutions. I need to create a research question and then implement a test to support my question. I'm having trouble.
1. How do I test for flavonoids (e.g. extract them), do I need to quantify the amount in a certain food such as tea? Or do I just prove flavonoids exist.
2. Just because I can prove flavonoids exist in a certain food, how do I then prove their impact? For example, I don't have the time and resources to prove it's benefits towards cardiovascular disease. What is the simplest way to prove their antioxidant characteristics.
Any help would be appreciated.
Mark

[SciB]

Hi Mark,

Welcome to Scibuddies. Bioflavonoids and polyphenols are a highly interesting and controversial group of chemicals. There's a lot of misinformation about them on the web and overblown advertising claims by companies who sell bioflavonoid supplements. However, there is enough hard scientific evidence to support the idea that they may be beneficial to health in our diet.

As you said, developing a project to study the biological effects of these compounds can require more lab resources than most high schools have access to. Testing them on humans is obviously out of the question, but there are several types of organism that can be used to examine specific properties of bioflavonoids. If you wanted to test their antibacterial capability, you can buy a harmless strain of E. coli bacteria and measure the effect of various supplements or vegetable extracts on bacterial growth. Some flavonoids also can have antifungal affects and you could test this using the common bread yeast that you buy at the grocery store.

If you want to see the effects of bioflavonoids on a simple multicellular organism, I would recommend Daphnia which live in fresh water ponds and are also known as water fleas because their shape looks a little like a flea. Their bodies are transparent and you can see organs like the heart and count the beats. They produce eggs and reproduce in an aquarium so you can also study effects on reproduction. Carolina Biologicals sells them: http://www.carolina.com/living-organism ... Avg-8P8HAQ

There are other small critters that can also be raised fairly easily and used as test subjects like nematode worms, planaria and fruit flies.

As to sources of bioflavonoids, any store that sells vitamins and supplements will have many types that you could test. Green tea extract, grape seed extract, quercetin, citrus bioflavonoids, anthocyanin, and genistein are examples of flavonoids that may be available in tablets or capsules that you could test.

I prefer to get my nutrients from my food and flavones and polyphenols are abundant in fruits and vegies like blueberries, red onions and oranges. You could devise a way to make extracts of various foods and test these on one of the model organisms. You could compare fresh fruit extract with a supplement tablet and see which has more potency.

I hope this gives you some ideas about how to create a project about flavonoids. I'm sure you will have more questions, so let us know what you think and what interests you most and we will try to help you develop a hypothesis and some experiments to test it.

Good luck!

Sybee

[deleted-345229]

Sybee,
Thanks for your reply. I would like to look at how cooking methods can impact flavonoids. I thought it might be interesting to compare microwaving versus boiling a vegetable high in flavonoids such as broccoli, spinach or cabbage. What I don't know is how do I extract the flavonoids from the foods and measure them? Could you provide some guidance?
Thanks, Mark

[SciB]

Hi Mark,

I'm afraid extracting and quantitating flavonoids would require some advanced chemistry methods and expensive instruments only found in a university research lab. My recommendation would be to test the foods using a biological method such as the one with Daphnia. Flavonoids have been shown to have a cardioprotective effect in various animals and this is something you can test with Daphnia because under a low-power microscope you can count their heartbeats.
Here is a paper that does experiments with Daphnia and a polyphenolic plant extract: https://www.google.com/url?sa=t&rct=j&q ... 4w&cad=rja

The researchers stressed the heart by adding lactose to the Daphnia medium and then tested the effects of having the plant extract present before adding the lactose. You could do the same with an 'extract' of broccoli. After you cook it in the different ways, puree it in a blender or food processor with a little distilled water then filter the slurry through a washed coffee filter. I don't know how much to add or even if this will protect the Daphnia heart, but that is why we do experiments.

This is a fairly difficult and lengthy set of experiments but you should get some really interesting results and answer a question that may not have been studied before.

Post again with more questions and we will try to help you design a winning project.

Sybee

[deleted-345229]

I have reviewed the project with my teacher, we are looking at using Thin Layer Chromatography. Can this be used to test flavonoid content? I wanted to test raw, "juiced" and cooked broccoli. I'm not sure what to use as my solvent (was thinking 95% Ethyl alcohol). Also, I'm not sure if what substance is detected in the test is actually flavonoid. I could make an acid mixture mixing ground broccoli with HCl and then testing that liquid with the TLC. Does this sound like a doable experiment? Thanks for your help
Mark

[SciB]

Hi Mark,

Chromatography is a great way to detect and quantitate a chemical substance but there are two things you need. The first is a way to detect the chemical that is specific for it. This is especially important if you are trying to detect it in the presence of a mixture of other compounds.

The second thing you need is a sample of the pure chemical that you are trying to detect and measure.

Do you have a workable method for TLC of flavonoids? You can't guess about solvents and detection methods unless you are an experienced organic chemist. If you have a published protocol for TLC of flavonoids then you know it will work.

The sensitivity of the detection system is something else to consider. If you are going to run an extract of broccoli then you have to do some research and find out approximately how much flavonoid would be in a certain amount of broccoli. I am assuming that this value has been published somewhere online, but it may take you some time to dig it out.

Do you have a source for the pure flavonoid? With the pure compound in hand, you would make a solution of known concentration in the appropriate solvent and run a series of dilutions on a TLC plate. What you are aiming for is to find the lowest concentration that is detectable by your system. The published method should have information about the sensitivity that you can use to set up the dilutions.

Next compare the amount of flavonoid that you expect to load in the extract with the limit of sensitivity of your detection method. If the extract flavonoid concentration is well above the detection limit you are home free. If it is below then you have a problem.

I don't want to make this too technical so I'll stop here and wait for your next post. Let us know more details about your methods and we'll be better able to answer you.

Sybee